Structured Review

Upstate Biotechnology Inc histone h4
Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular <t>histone</t> H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.
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1) Product Images from "Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor"

Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

Journal: Molecular and Cellular Biology

doi:

Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.
Figure Legend Snippet: Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

Techniques Used: Over Expression, HAT Assay, Transfection, Mutagenesis

Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.
Figure Legend Snippet: Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

Techniques Used: Immunofluorescence, In Vivo, Staining, Microscopy

Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.
Figure Legend Snippet: Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

Techniques Used: Inhibition, HAT Assay, Activity Assay, Recombinant, Purification, Radioactivity, Labeling, Incubation

Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.
Figure Legend Snippet: Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

Techniques Used: In Vivo, Expressing

2) Product Images from "FoxA Proteins Regulate H19 Endoderm Enhancer E1 and Exhibit Developmental Changes in Enhancer Binding In Vivo"

Article Title: FoxA Proteins Regulate H19 Endoderm Enhancer E1 and Exhibit Developmental Changes in Enhancer Binding In Vivo

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.21.9601-9609.2004

Histone acetylation of the H19 promoter correlates with the H19 expression in the liver development and during F9 cell differentiation. (A) ChIP assays were performed with chromatin immunoprecipitated from e14.5 fetal and adult liver with no antibody (no Ab) or antibodies to acetylated histone H4 proteins (α-Ac-H4). Oligonucleotides from the TTR promoter or H19 promoter were used to amplify the immunoprecipitated DNA. (B) The intensity of PCR-amplified bands was quantitated using ImageQuant 5.0 software. The intensity of input bands for a DNA fragment in both tissues was first normalized to the input DNA; the relative level of DNA-bound protein was then calculated by subtracting the band intensity in the no-antibody sample from that in the sample precipitated with anti-Ac-H4 antiserum. Data from two independent experiments are shown; the data shown in panel A correspond to experiment 1 in panel B. (C) ChIP assays were performed with chromatin immunoprecipitated from undifferentiated F9 cells or cells differentiated into VE or PE for 3, 6, or 8 days, with no antibody (no Ab) or antibodies to acetylated histone H4 proteins. Oligonucleotides from the TTR promoter or H19 promoter were used to amplify the immunoprecipitated DNA.
Figure Legend Snippet: Histone acetylation of the H19 promoter correlates with the H19 expression in the liver development and during F9 cell differentiation. (A) ChIP assays were performed with chromatin immunoprecipitated from e14.5 fetal and adult liver with no antibody (no Ab) or antibodies to acetylated histone H4 proteins (α-Ac-H4). Oligonucleotides from the TTR promoter or H19 promoter were used to amplify the immunoprecipitated DNA. (B) The intensity of PCR-amplified bands was quantitated using ImageQuant 5.0 software. The intensity of input bands for a DNA fragment in both tissues was first normalized to the input DNA; the relative level of DNA-bound protein was then calculated by subtracting the band intensity in the no-antibody sample from that in the sample precipitated with anti-Ac-H4 antiserum. Data from two independent experiments are shown; the data shown in panel A correspond to experiment 1 in panel B. (C) ChIP assays were performed with chromatin immunoprecipitated from undifferentiated F9 cells or cells differentiated into VE or PE for 3, 6, or 8 days, with no antibody (no Ab) or antibodies to acetylated histone H4 proteins. Oligonucleotides from the TTR promoter or H19 promoter were used to amplify the immunoprecipitated DNA.

Techniques Used: Expressing, Cell Differentiation, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Software

3) Product Images from "Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition"

Article Title: Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.21.7719-7731.2003

The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).
Figure Legend Snippet: The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).

Techniques Used: Cell Culture, Isolation, Chromatin Immunoprecipitation, Amplification, Autoradiography, Luciferase, Activity Assay, Reporter Assay

Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.
Figure Legend Snippet: Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.

Techniques Used: Isolation, Cell Culture, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Autoradiography, Immunoprecipitation

4) Product Images from "Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia"

Article Title: Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia

Journal: Journal of Clinical Investigation

doi:

In vivo effects of SAHA, RA, and the combination of both on PLZF-RARα/RARα-PLZF double leukemic TM. ( a ) Western blot analysis of acetylated histone H4 in murine cells upon in vivo administration of SAHA. Wild-type and leukemic TM were given SAHA (20 μg/gbw) by intraperitoneal injection. Histones were acid extracted from murine peripheral blood, BM, and spleen cells in untreated mice and 2 hours after SAHA administration. ( b ) SAHA in combination with RA treatment prolongs survival in PLZF-RARα/RARα-PLZF double TM with leukemia. Upon presentation of leukemia as monitored by automatic and differentiated counts on peripheral blood samples from PLZF-RARα/RARα-PLZF double TM, RA was administered daily at a dose of 1.5 μg/gbw for 2 weeks. The RA-treated mice were then randomly assigned into three groups: RA alone (1.5 μg/gbw), SAHA (50 μg/gbw) alone, and SAHA (50 μg/gbw) in combination with RA (1.5 μg/gbw). The treatment was continued for 4 weeks. During and after the treatment, the mice were bled weekly, and automatic and morphological differential counts were performed on each sample to evaluate response to treatment until each animal died. Kaplan Meier analysis was used to compare the cumulative survival period between SAHA in combination with RA ( n = 11) and RA ( n = 9) or SAHA ( n = 6) alone. The black bar on the abscissa represents the 28-day period of treatment. Survival time reflects the days from the initiation of therapies until death for each mouse. ( c ) Complete remission was induced by SAHA + RA treatment in six of 11 leukemic PLZF-RARα/RARα-PLZF double transgenic mice. Analyses were performed weekly as described above. SAHA + RA causes the same duration of disease-free survival (time in remission) in PLZF-RARα/RARα-PLZF leukemic mice as that achieved in PML-RARα transgenic leukemic mice treated with RA (1.5 μg/gbw).
Figure Legend Snippet: In vivo effects of SAHA, RA, and the combination of both on PLZF-RARα/RARα-PLZF double leukemic TM. ( a ) Western blot analysis of acetylated histone H4 in murine cells upon in vivo administration of SAHA. Wild-type and leukemic TM were given SAHA (20 μg/gbw) by intraperitoneal injection. Histones were acid extracted from murine peripheral blood, BM, and spleen cells in untreated mice and 2 hours after SAHA administration. ( b ) SAHA in combination with RA treatment prolongs survival in PLZF-RARα/RARα-PLZF double TM with leukemia. Upon presentation of leukemia as monitored by automatic and differentiated counts on peripheral blood samples from PLZF-RARα/RARα-PLZF double TM, RA was administered daily at a dose of 1.5 μg/gbw for 2 weeks. The RA-treated mice were then randomly assigned into three groups: RA alone (1.5 μg/gbw), SAHA (50 μg/gbw) alone, and SAHA (50 μg/gbw) in combination with RA (1.5 μg/gbw). The treatment was continued for 4 weeks. During and after the treatment, the mice were bled weekly, and automatic and morphological differential counts were performed on each sample to evaluate response to treatment until each animal died. Kaplan Meier analysis was used to compare the cumulative survival period between SAHA in combination with RA ( n = 11) and RA ( n = 9) or SAHA ( n = 6) alone. The black bar on the abscissa represents the 28-day period of treatment. Survival time reflects the days from the initiation of therapies until death for each mouse. ( c ) Complete remission was induced by SAHA + RA treatment in six of 11 leukemic PLZF-RARα/RARα-PLZF double transgenic mice. Analyses were performed weekly as described above. SAHA + RA causes the same duration of disease-free survival (time in remission) in PLZF-RARα/RARα-PLZF leukemic mice as that achieved in PML-RARα transgenic leukemic mice treated with RA (1.5 μg/gbw).

Techniques Used: In Vivo, Western Blot, Injection, Mouse Assay, Transgenic Assay

5) Product Images from "Antiproliferative effects of TSA, PXD-101 and MS-275 in A2780 and MCF7 cells: Acetylated histone H4 and acetylated tubulin as markers for HDACi potency and selectivity"

Article Title: Antiproliferative effects of TSA, PXD-101 and MS-275 in A2780 and MCF7 cells: Acetylated histone H4 and acetylated tubulin as markers for HDACi potency and selectivity

Journal: Oncology Reports

doi: 10.3892/or.2017.6015

HDACi-induced acetylated histone H4 expression in A2780 and MCF7 cells. (A) Expression profile of HDAC1 and HDAC3 in A2780 and MCF7 cells. Cells were probed with primary anti-HDAC1 and anti-HDAC3 antibodies and the protein expression profile was developed by immunoblotting using ECL reagents as determined in Materials and methods. HDAC1; lanes 1–3: A2780 cells, lanes 4–6: MCF7 cells, HDAC3; lane 1: A2780 cells, lanes 2–3: MCF7 cells. (B) Upregulation of acetylated histone H4 expression in A2780 cells following HDACi treatment was monitored by western blot analysis and flow cytometry as described in Materials and methods. A2780 cells were treated with 0.1% DMSO, 10 µM MS-275 and 5 µM TSA for 24 h. (C) Fold increase of acetylated histone H4 as determined by flow cytometry following a 24-h treatment of 5 µM of HDACi. Left: Flow cytometry histogram, Right: Relative quantification of fluorescence signal. The error bars represent SD for at least n=3 determinations. HDACi, histone deacetylase enzyme inhibitor; TSA, Trichostatin A; SD, standard deviation.
Figure Legend Snippet: HDACi-induced acetylated histone H4 expression in A2780 and MCF7 cells. (A) Expression profile of HDAC1 and HDAC3 in A2780 and MCF7 cells. Cells were probed with primary anti-HDAC1 and anti-HDAC3 antibodies and the protein expression profile was developed by immunoblotting using ECL reagents as determined in Materials and methods. HDAC1; lanes 1–3: A2780 cells, lanes 4–6: MCF7 cells, HDAC3; lane 1: A2780 cells, lanes 2–3: MCF7 cells. (B) Upregulation of acetylated histone H4 expression in A2780 cells following HDACi treatment was monitored by western blot analysis and flow cytometry as described in Materials and methods. A2780 cells were treated with 0.1% DMSO, 10 µM MS-275 and 5 µM TSA for 24 h. (C) Fold increase of acetylated histone H4 as determined by flow cytometry following a 24-h treatment of 5 µM of HDACi. Left: Flow cytometry histogram, Right: Relative quantification of fluorescence signal. The error bars represent SD for at least n=3 determinations. HDACi, histone deacetylase enzyme inhibitor; TSA, Trichostatin A; SD, standard deviation.

Techniques Used: Expressing, Western Blot, Flow Cytometry, Cytometry, Mass Spectrometry, Fluorescence, Histone Deacetylase Assay, Standard Deviation

6) Product Images from "Changes in Histone Acetylation Are Associated with Differences in Accessibility of VH Gene Segments to V-DJ Recombination during B-Cell Ontogeny and Development"

Article Title: Changes in Histone Acetylation Are Associated with Differences in Accessibility of VH Gene Segments to V-DJ Recombination during B-Cell Ontogeny and Development

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.7.2438-2450.2003

Comparison of the histone acetylations associated with the V H and Ig kappa loci following mu expression in B cells from RAG −/− mice. (A) ChIP assays using anti-acetylated H3 and H4 antibodies were performed on pre-B cells from RAG −/− mice expressing the human mu protein as a transgene (TG+), and the results were compared to those with pro-B cells from littermate controls (TG−) following a short culture in the presence of IL-7. Data are representative of results with three to five mice for each genotype. Individual V H gene segments are shown, and the amylase gene was used as a negative control. The kappa primers recognize conserved portions of the V K gene segments. Shown are 1:5 serial dilutions of 1% input and 1:2 serial dilutions of IP fractions for each animal. The increases ( n -fold) of H3 and H4 acetylation relative to that for TG− animals are shown for kappa genes. (B) Graphic representation, using samples which fell into the linear range, of H4 acetylation data from panel A expressed as relative intensity of 0.1% input. The relative intensity of the pancreatic specific gene amylase represents nonspecific levels of histone acetylation.
Figure Legend Snippet: Comparison of the histone acetylations associated with the V H and Ig kappa loci following mu expression in B cells from RAG −/− mice. (A) ChIP assays using anti-acetylated H3 and H4 antibodies were performed on pre-B cells from RAG −/− mice expressing the human mu protein as a transgene (TG+), and the results were compared to those with pro-B cells from littermate controls (TG−) following a short culture in the presence of IL-7. Data are representative of results with three to five mice for each genotype. Individual V H gene segments are shown, and the amylase gene was used as a negative control. The kappa primers recognize conserved portions of the V K gene segments. Shown are 1:5 serial dilutions of 1% input and 1:2 serial dilutions of IP fractions for each animal. The increases ( n -fold) of H3 and H4 acetylation relative to that for TG− animals are shown for kappa genes. (B) Graphic representation, using samples which fell into the linear range, of H4 acetylation data from panel A expressed as relative intensity of 0.1% input. The relative intensity of the pancreatic specific gene amylase represents nonspecific levels of histone acetylation.

Techniques Used: Expressing, Mouse Assay, Chromatin Immunoprecipitation, Negative Control

Histone acetylation associated with intergenic sequences within the V H locus. (A) Schematic diagram showing the primers used to analyze regions directly flanking an individual V H gene segment. Primers were designed to amplify overlapping regions on both the 3′ and 5′ sides of individual gene segments. Each amplified region was ∼300 bp long and overlapped its neighbors by ∼100 bp. (B) ChIP assays were performed using antibodies to acetylated H3 and H4 on pro-B cells from bone marrow of RAG −/− mice following a short culture in IL-7. Shown are 1:5 serial dilutions of 1% input and 1:2 serial dilutions of IP fractions. The amylase promoter is used as a negative control. The data are representative and were repeated in separate preparations. The relative intensity of amylase is also shown. (C) H4 acetylation levels from panel B are graphed as relative intensity of 0.1% input after being quantified by phosphorimager.
Figure Legend Snippet: Histone acetylation associated with intergenic sequences within the V H locus. (A) Schematic diagram showing the primers used to analyze regions directly flanking an individual V H gene segment. Primers were designed to amplify overlapping regions on both the 3′ and 5′ sides of individual gene segments. Each amplified region was ∼300 bp long and overlapped its neighbors by ∼100 bp. (B) ChIP assays were performed using antibodies to acetylated H3 and H4 on pro-B cells from bone marrow of RAG −/− mice following a short culture in IL-7. Shown are 1:5 serial dilutions of 1% input and 1:2 serial dilutions of IP fractions. The amylase promoter is used as a negative control. The data are representative and were repeated in separate preparations. The relative intensity of amylase is also shown. (C) H4 acetylation levels from panel B are graphed as relative intensity of 0.1% input after being quantified by phosphorimager.

Techniques Used: Amplification, Chromatin Immunoprecipitation, Mouse Assay, Negative Control

7) Product Images from "Obatoclax Potentiates the Cytotoxic Effect of Cytarabine on Acute Myeloid Leukemia Cells by Enhancing DNA Damage"

Article Title: Obatoclax Potentiates the Cytotoxic Effect of Cytarabine on Acute Myeloid Leukemia Cells by Enhancing DNA Damage

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2014.09.008

Ectopic overexpression of Mcl‐1 or Bcl‐xL in THP‐1 cells blocks apoptosis induced by cytarabine or cytarabine plus obatoclax. THP‐1 cells were infected with Precision LentiORF Mcl‐1, Bcl‐xL, (THP‐1/Mcl‐1 or THP‐1/Bcl‐xL respectively) or red fluorescent protein control (THP‐1/RFP) lentivirus overnight, washed and then incubated for 48 h before selection drug (Blasticidin) was added to the culture medium. The cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti‐Bcl‐xL, ‐Mcl‐1, ‐PARP, ‐cleaved caspase‐3, or ‐β‐actin antibody (Panels A E). Apoptotic events in the cells post drug treatment were determined by annexin V/7‐AAD staining and flow cytometry analyses (Panels B F). THP‐1/RFP and THP‐1/Bcl‐xL cells were treated with cytarabine and/or obatoclax for 48 h. Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. Due to the extent of overexpression of Bcl‐xL 1/10th of the total protein loaded for the THP‐1/RFP was loaded for the THP‐1/Bcl‐xL samples in order to visualize nuclear Bcl‐xL level changes on the same blot. The membranes were probed with anti‐Bcl‐xL, ‐MEK1/2, or ‐Histone H4 antibody (Panels C D). Experiments were repeated at least 3 independent times with one representative shown. The data are presented as mean ± standard error. *** indicates p
Figure Legend Snippet: Ectopic overexpression of Mcl‐1 or Bcl‐xL in THP‐1 cells blocks apoptosis induced by cytarabine or cytarabine plus obatoclax. THP‐1 cells were infected with Precision LentiORF Mcl‐1, Bcl‐xL, (THP‐1/Mcl‐1 or THP‐1/Bcl‐xL respectively) or red fluorescent protein control (THP‐1/RFP) lentivirus overnight, washed and then incubated for 48 h before selection drug (Blasticidin) was added to the culture medium. The cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti‐Bcl‐xL, ‐Mcl‐1, ‐PARP, ‐cleaved caspase‐3, or ‐β‐actin antibody (Panels A E). Apoptotic events in the cells post drug treatment were determined by annexin V/7‐AAD staining and flow cytometry analyses (Panels B F). THP‐1/RFP and THP‐1/Bcl‐xL cells were treated with cytarabine and/or obatoclax for 48 h. Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. Due to the extent of overexpression of Bcl‐xL 1/10th of the total protein loaded for the THP‐1/RFP was loaded for the THP‐1/Bcl‐xL samples in order to visualize nuclear Bcl‐xL level changes on the same blot. The membranes were probed with anti‐Bcl‐xL, ‐MEK1/2, or ‐Histone H4 antibody (Panels C D). Experiments were repeated at least 3 independent times with one representative shown. The data are presented as mean ± standard error. *** indicates p

Techniques Used: Over Expression, Infection, Incubation, Selection, Western Blot, Staining, Flow Cytometry, Cytometry

The effects of cytarabine and obatoclax treatments on Bcl‐2, Bcl‐xL, and Mcl‐1 expression and subcellular localization in OCI‐AML3 cells. OCI‐AML3 cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting, and then probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, or –β‐actin antibody (Panel A, upper panel). Densitometry for Mcl‐1 expression was measured and graphed as fold change compared to the no drug control (Panel A, lower panel). Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. The membranes were probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, or ‐Histone H4 antibody (Panel B). OCI‐AML3 cells were treated with vehicle control or 2 μM cytarabine plus 175 nM obatoclax for 48 h. Cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panel C). OCI‐AML3 cells were treated with 2 μM cytarabine with or without 175 nM obatoclax for up to 48 h. Nuclear proteins were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1 or ‐Histone H4 antibody (Panel D). The data are presented as mean ± standard error from at least 3 independent experiments. * indicates p
Figure Legend Snippet: The effects of cytarabine and obatoclax treatments on Bcl‐2, Bcl‐xL, and Mcl‐1 expression and subcellular localization in OCI‐AML3 cells. OCI‐AML3 cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting, and then probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, or –β‐actin antibody (Panel A, upper panel). Densitometry for Mcl‐1 expression was measured and graphed as fold change compared to the no drug control (Panel A, lower panel). Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. The membranes were probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, or ‐Histone H4 antibody (Panel B). OCI‐AML3 cells were treated with vehicle control or 2 μM cytarabine plus 175 nM obatoclax for 48 h. Cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panel C). OCI‐AML3 cells were treated with 2 μM cytarabine with or without 175 nM obatoclax for up to 48 h. Nuclear proteins were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1 or ‐Histone H4 antibody (Panel D). The data are presented as mean ± standard error from at least 3 independent experiments. * indicates p

Techniques Used: Expressing, Western Blot, Staining, Confocal Microscopy

8) Product Images from "E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor complex"

Article Title: E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor complex

Journal: Genes & Development

doi: 10.1101/gad.969202

Nucleosome mapping and histone acetylation of the E2F1 promoter. ( A ) Three promoter-proximal nucleosomes were identified by nucleosome mapping. Chromatin from quiescent wild-type 3T3 cells was partially digested with micrococcal nuclease, and primer extension was performed by use of a set of primer pairs spanning the E2F1 promoter region. Extension products were subjected to ligation-mediated PCR (LM–PCR), followed by acrylamide gel electrophoresis (see Materials and Methods). Amplification products were visualized by autoradiography, and approximate nucleosome boundaries were deduced. ( B ) Nucleosome positions were confirmed by PCR of genomic vs. MNase-digested chromatin using primer pairs that are internal to the putative nucleosomes or flank the E2F binding sites. Reduction in band intensity in the MNase-treated lanes indicates lack of nucleosome protection. ( C ) Schematic of nucleosome positioning on the E2F1 promoter. Boxes represent E2F-binding sites, and the arrow corresponds to the major transcription start site. Ovals are enclosed with dotted lines to indicate that nucleosomal positioning is approximate and may be dynamic. ( D ) Acetylation of nucleosomes at the E2F1 promoter. Chromatin from quiescent primary MEFs was digested extensively with micrococcal nuclease (MNase) to produce mononucleosomes. Chromatin immunoprecipitations were then performed using antibodies against acetylated histones H3 and H4, and enrichment was detected by PCR using nucleosome-specific primers (indicated for each of three promoter-proximal nucleosomes) deduced from mapping data in A and B .
Figure Legend Snippet: Nucleosome mapping and histone acetylation of the E2F1 promoter. ( A ) Three promoter-proximal nucleosomes were identified by nucleosome mapping. Chromatin from quiescent wild-type 3T3 cells was partially digested with micrococcal nuclease, and primer extension was performed by use of a set of primer pairs spanning the E2F1 promoter region. Extension products were subjected to ligation-mediated PCR (LM–PCR), followed by acrylamide gel electrophoresis (see Materials and Methods). Amplification products were visualized by autoradiography, and approximate nucleosome boundaries were deduced. ( B ) Nucleosome positions were confirmed by PCR of genomic vs. MNase-digested chromatin using primer pairs that are internal to the putative nucleosomes or flank the E2F binding sites. Reduction in band intensity in the MNase-treated lanes indicates lack of nucleosome protection. ( C ) Schematic of nucleosome positioning on the E2F1 promoter. Boxes represent E2F-binding sites, and the arrow corresponds to the major transcription start site. Ovals are enclosed with dotted lines to indicate that nucleosomal positioning is approximate and may be dynamic. ( D ) Acetylation of nucleosomes at the E2F1 promoter. Chromatin from quiescent primary MEFs was digested extensively with micrococcal nuclease (MNase) to produce mononucleosomes. Chromatin immunoprecipitations were then performed using antibodies against acetylated histones H3 and H4, and enrichment was detected by PCR using nucleosome-specific primers (indicated for each of three promoter-proximal nucleosomes) deduced from mapping data in A and B .

Techniques Used: Ligation, Polymerase Chain Reaction, Acrylamide Gel Assay, Electrophoresis, Amplification, Autoradiography, Binding Assay

9) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.263111

Effects of MS-275 on HepG2 cells. A, HepG2 cells were treated with MS-275 for 24 h, and cell lysates were subjected to Western blot analysis using anti-acetylated histone H3 and H4 antibodies ( Ac-H3 and Ac-H4 ). Total histones H3, H4, and actin were used
Figure Legend Snippet: Effects of MS-275 on HepG2 cells. A, HepG2 cells were treated with MS-275 for 24 h, and cell lysates were subjected to Western blot analysis using anti-acetylated histone H3 and H4 antibodies ( Ac-H3 and Ac-H4 ). Total histones H3, H4, and actin were used

Techniques Used: Mass Spectrometry, Western Blot

10) Product Images from "Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1"

Article Title: Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1

Journal: Biology of Reproduction

doi: 10.1095/biolreprod.110.086397

Effect of Mta2 knockdown on expression of other components of the NuRD complex. A ) One-cell embryos were injected with either Gfp dsRNA (control) or Mta2 dsRNA and then cultured 96 h in vitro. Equal numbers of ds Gfp - and ds Mta2- injected embryos were collected for immunoblot analysis; 100 embryos were loaded per lane, and TUBB was used as a loading control. The experiment was performed twice, and similar results were obtained. B ) ds Gfp - or ds Mta2 -injected embryos were processed for immunocytochemical detection of histone H4 acetylation state. At least 12 control and experimental embryos were analyzed, and the experiment was conducted four times. Shown are representative images. C ) ds Gfp - or ds Mta2 -injected embryos were processed for immunocytochemical detection of histone POU5F1 and NANOG. At least 12 control and experimental embryos were analyzed, and the experiment was conducted four times. Shown are representative images. Original magnification x80.
Figure Legend Snippet: Effect of Mta2 knockdown on expression of other components of the NuRD complex. A ) One-cell embryos were injected with either Gfp dsRNA (control) or Mta2 dsRNA and then cultured 96 h in vitro. Equal numbers of ds Gfp - and ds Mta2- injected embryos were collected for immunoblot analysis; 100 embryos were loaded per lane, and TUBB was used as a loading control. The experiment was performed twice, and similar results were obtained. B ) ds Gfp - or ds Mta2 -injected embryos were processed for immunocytochemical detection of histone H4 acetylation state. At least 12 control and experimental embryos were analyzed, and the experiment was conducted four times. Shown are representative images. C ) ds Gfp - or ds Mta2 -injected embryos were processed for immunocytochemical detection of histone POU5F1 and NANOG. At least 12 control and experimental embryos were analyzed, and the experiment was conducted four times. Shown are representative images. Original magnification x80.

Techniques Used: Expressing, Injection, Cell Culture, In Vitro

11) Product Images from "Identification of a small molecule inhibitor of Sir2p"

Article Title: Identification of a small molecule inhibitor of Sir2p

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.261574398

( A ) Inhibition of NAD-dependent HDA of Sir2p by splitomicin. The effect of splitomicin on NAD + -dependent HDA in wild-type and drug-resistant Sir2p mutants is shown. Chemically [ 3 H]-acetylated histone H4 peptide (40,000 cpm per reaction) was incubated with whole-cell protein extracts (50 μg) prepared from hst2 Δ strain containing overexpressed wild-type SIR2 or two drug-resistant SIR2 alleles ( SIR2 - Y298N and SIR2 - H286Q ), NAD + , and splitomicin at 30°C for 16 h. The assays were done in triplicate. The NAD + -dependent activity in the extract without splitomicin was 1,776 ± 25 cpm for wild-type SIR2 , 1,620 ± 44 cpm for SIR2 - Y298N , 1,795 ± 36 cpm for SIR2 - H286Q , and 28 ± 14 cpm for cells containing the empty vector. ( B ) Immunoblot of Sir2p in whole-cell lysates containing overexpressed wild-type or drug-resistant mutant SIR2 . The whole-cell lysates (25 μg) prepared from hst2 Δ strain containing empty vector or overexpressed wild-type SIR2 and two drug-resistant SIR2 alleles ( SIR2 - Y298N and SIR2 - H286Q ) were probed with an anti-Sir2p antibody (Santa Cruz Biotechnology). ( C ) Telomeric silencing in SIR2 , sir2 Δ, and drug-resistant SIR2 mutants. Cells from a sir2 Δ strain with telomeric URA3 gene containing either empty plasmid ( sir2 Δ), a plasmid with wild-type SIR2 , or drug-resistant alleles SIR2 - H286Q , SIR2 - L287M , and SIR2 - Y298N were replica-plated onto selective medium lacking leucine (for plasmid selection), selective medium lacking uracil (−ura), or selective medium to which 5-flouroorotic acid was added (+5-FOA) with or without 10 μM splitomicin and incubated at 30°C for 2 days. Expression of the telomeric URA3 gene kills cells, because Ura3p converts 5-fluoroorotic acid into a toxic metabolite. ( D ) Sequence alignment between yeast Sir2p and Hst1–4p. The region displayed in the alignment contains the putative substrate-binding site. Arrows indicate the positions of residues that, when mutated in Sir2p, confer splitomicin resistance.
Figure Legend Snippet: ( A ) Inhibition of NAD-dependent HDA of Sir2p by splitomicin. The effect of splitomicin on NAD + -dependent HDA in wild-type and drug-resistant Sir2p mutants is shown. Chemically [ 3 H]-acetylated histone H4 peptide (40,000 cpm per reaction) was incubated with whole-cell protein extracts (50 μg) prepared from hst2 Δ strain containing overexpressed wild-type SIR2 or two drug-resistant SIR2 alleles ( SIR2 - Y298N and SIR2 - H286Q ), NAD + , and splitomicin at 30°C for 16 h. The assays were done in triplicate. The NAD + -dependent activity in the extract without splitomicin was 1,776 ± 25 cpm for wild-type SIR2 , 1,620 ± 44 cpm for SIR2 - Y298N , 1,795 ± 36 cpm for SIR2 - H286Q , and 28 ± 14 cpm for cells containing the empty vector. ( B ) Immunoblot of Sir2p in whole-cell lysates containing overexpressed wild-type or drug-resistant mutant SIR2 . The whole-cell lysates (25 μg) prepared from hst2 Δ strain containing empty vector or overexpressed wild-type SIR2 and two drug-resistant SIR2 alleles ( SIR2 - Y298N and SIR2 - H286Q ) were probed with an anti-Sir2p antibody (Santa Cruz Biotechnology). ( C ) Telomeric silencing in SIR2 , sir2 Δ, and drug-resistant SIR2 mutants. Cells from a sir2 Δ strain with telomeric URA3 gene containing either empty plasmid ( sir2 Δ), a plasmid with wild-type SIR2 , or drug-resistant alleles SIR2 - H286Q , SIR2 - L287M , and SIR2 - Y298N were replica-plated onto selective medium lacking leucine (for plasmid selection), selective medium lacking uracil (−ura), or selective medium to which 5-flouroorotic acid was added (+5-FOA) with or without 10 μM splitomicin and incubated at 30°C for 2 days. Expression of the telomeric URA3 gene kills cells, because Ura3p converts 5-fluoroorotic acid into a toxic metabolite. ( D ) Sequence alignment between yeast Sir2p and Hst1–4p. The region displayed in the alignment contains the putative substrate-binding site. Arrows indicate the positions of residues that, when mutated in Sir2p, confer splitomicin resistance.

Techniques Used: Inhibition, Helicase-dependent Amplification, Incubation, Activity Assay, Plasmid Preparation, Mutagenesis, Selection, Expressing, Sequencing, Binding Assay

12) Product Images from "ATF2 is required for amino acid-regulated transcription by orchestrating specific histone acetylation"

Article Title: ATF2 is required for amino acid-regulated transcription by orchestrating specific histone acetylation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm038

Transcription factor binding and histone acetylation to CHOP AARE in response to leucine starvation. ( A ) Scheme of the human CHOP gene indicating the different amplicons produced for the ChIP analysis: A (−1678 to −1478), B (−472 to −301) and C (+1163 to +1372). The AARE is boxed in grey. ( B ) HeLa cells were incubated 2 h either in control (+leu) or leucine-free medium (−leu) and harvested. ChIP analysis was performed as described under Materials and Methods using antibodies specific for ATF2 and ATF4 and different sets of primers to produce amplicon A, B or C. Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments, and the error bars represent the standard error of the means. ( C ) The experiment described in (B) was also performed using antibodies specific for acetylated H3, acetylated H4 and acetylated H2B.
Figure Legend Snippet: Transcription factor binding and histone acetylation to CHOP AARE in response to leucine starvation. ( A ) Scheme of the human CHOP gene indicating the different amplicons produced for the ChIP analysis: A (−1678 to −1478), B (−472 to −301) and C (+1163 to +1372). The AARE is boxed in grey. ( B ) HeLa cells were incubated 2 h either in control (+leu) or leucine-free medium (−leu) and harvested. ChIP analysis was performed as described under Materials and Methods using antibodies specific for ATF2 and ATF4 and different sets of primers to produce amplicon A, B or C. Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments, and the error bars represent the standard error of the means. ( C ) The experiment described in (B) was also performed using antibodies specific for acetylated H3, acetylated H4 and acetylated H2B.

Techniques Used: Binding Assay, Produced, Chromatin Immunoprecipitation, Incubation, Amplification, Polymerase Chain Reaction

Role of ATF2 in the transcriptional regulation of ATF3 and ASNS in response to amino acid starvation. ( A ) Sequence comparison of the human CHOP AARE (−313 to −298) with the human ATF3 AARE (−12 to −27) and the human ASNS AARE (−57 to −72). The minimum AARE core sequence is boxed. Identical nucleotides in the core are boxed in grey. ATF2 +/+ or ATF2 −/− MEF were incubated either in control (+leu) or leucine-free medium (−leu) and harvested after 4 h. Total RNA was extracted and analysed by real time RT-PCR for ATF3 ( B ) and ASNS ( C ) mRNA content as described in Materials and Methods. ChIP analysis was performed as described under Materials and Methods using antibodies specific for ATF4, ATF2 and acetylated histone H4 and two different sets of primers to amplify ( D ) ATF3 AARE or ( E ) ASNS AARE. Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments, and the error bars represent the standard error of the means.
Figure Legend Snippet: Role of ATF2 in the transcriptional regulation of ATF3 and ASNS in response to amino acid starvation. ( A ) Sequence comparison of the human CHOP AARE (−313 to −298) with the human ATF3 AARE (−12 to −27) and the human ASNS AARE (−57 to −72). The minimum AARE core sequence is boxed. Identical nucleotides in the core are boxed in grey. ATF2 +/+ or ATF2 −/− MEF were incubated either in control (+leu) or leucine-free medium (−leu) and harvested after 4 h. Total RNA was extracted and analysed by real time RT-PCR for ATF3 ( B ) and ASNS ( C ) mRNA content as described in Materials and Methods. ChIP analysis was performed as described under Materials and Methods using antibodies specific for ATF4, ATF2 and acetylated histone H4 and two different sets of primers to amplify ( D ) ATF3 AARE or ( E ) ASNS AARE. Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments, and the error bars represent the standard error of the means.

Techniques Used: Sequencing, Incubation, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

Time course of ATF2 and ATF4 binding and histone acetylation during leucine starvation. ( A ) HeLa cells were incubated either in control (+leu) or leucine-free medium (−leu) and harvested for 0–4 h. ChIP analysis was performed as described under Materials and Methods using antibodies specific for (A) ATF4, ATF2, phospho-ATF2 (Thr-71) and (B) acetylated H3, acetylated H4 and acetylated H2B and a set of primers to amplify amplicon B (see Figure 2 A). Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments and the error bars represent the standard error of the means. The dotted line represents the increase in CHOP mRNA induction level as shown in Figure 1 A.
Figure Legend Snippet: Time course of ATF2 and ATF4 binding and histone acetylation during leucine starvation. ( A ) HeLa cells were incubated either in control (+leu) or leucine-free medium (−leu) and harvested for 0–4 h. ChIP analysis was performed as described under Materials and Methods using antibodies specific for (A) ATF4, ATF2, phospho-ATF2 (Thr-71) and (B) acetylated H3, acetylated H4 and acetylated H2B and a set of primers to amplify amplicon B (see Figure 2 A). Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments and the error bars represent the standard error of the means. The dotted line represents the increase in CHOP mRNA induction level as shown in Figure 1 A.

Techniques Used: Binding Assay, Incubation, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction

Role of ATF2 in histone acetylation in response to leucine deprivation. ( A ) ATF2 +/+ and ATF2 −/− MEF were incubated 2 h either in control (+leu) or leucine-free medium (−leu) and harvested. ChIP analysis was performed as described under Materials and Methods using antibodies specific for ATF4, ATF2, phospho-ATF2 (Thr-71), acetylated H3, acetylated H4 and acetylated H2B and a set of primers to produce amplicon B ( Figure 2 A). Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments and the error bars represent the standard error of the means. ( B ) The same experiment as described in (A) was also performed with wild ATF4 +/+ and ATF4 −/− MEF.
Figure Legend Snippet: Role of ATF2 in histone acetylation in response to leucine deprivation. ( A ) ATF2 +/+ and ATF2 −/− MEF were incubated 2 h either in control (+leu) or leucine-free medium (−leu) and harvested. ChIP analysis was performed as described under Materials and Methods using antibodies specific for ATF4, ATF2, phospho-ATF2 (Thr-71), acetylated H3, acetylated H4 and acetylated H2B and a set of primers to produce amplicon B ( Figure 2 A). Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments and the error bars represent the standard error of the means. ( B ) The same experiment as described in (A) was also performed with wild ATF4 +/+ and ATF4 −/− MEF.

Techniques Used: Incubation, Chromatin Immunoprecipitation, Amplification, Binding Assay, Polymerase Chain Reaction

13) Product Images from "Endoplasmic Reticulum Stress Induction of the Grp78/BiP Promoter: Activating Mechanisms Mediated by YY1 and Its Interactive Chromatin Modifiers"

Article Title: Endoplasmic Reticulum Stress Induction of the Grp78/BiP Promoter: Activating Mechanisms Mediated by YY1 and Its Interactive Chromatin Modifiers

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.11.4529-4540.2005

Model of ER stress-inducible changes in transcription factor occupancy and chromatin remodeling of the Grp78 promoter. In nonstressed cells, NF-Y is in contact with the Grp78 promoter. Upon ER stress, while NF-Y binding remains intact and TFII-I binding is enhanced, ATF6 is cleaved to produce a nuclear form, ATF6(N), which associates with YY1 and enhances its binding to the Grp78 promoter. The YY1-interacting proteins PRMT1 and p300 are also recruited to the Grp78 promoter. Additional chromatin changes of histone H4 include acetylation and arginine 3 methylation.
Figure Legend Snippet: Model of ER stress-inducible changes in transcription factor occupancy and chromatin remodeling of the Grp78 promoter. In nonstressed cells, NF-Y is in contact with the Grp78 promoter. Upon ER stress, while NF-Y binding remains intact and TFII-I binding is enhanced, ATF6 is cleaved to produce a nuclear form, ATF6(N), which associates with YY1 and enhances its binding to the Grp78 promoter. The YY1-interacting proteins PRMT1 and p300 are also recruited to the Grp78 promoter. Additional chromatin changes of histone H4 include acetylation and arginine 3 methylation.

Techniques Used: Binding Assay, Methylation

14) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.263111

Effects of MS-275 on HepG2 cells. A, HepG2 cells were treated with MS-275 for 24 h, and cell lysates were subjected to Western blot analysis using anti-acetylated histone H3 and H4 antibodies ( Ac-H3 and Ac-H4 ). Total histones H3, H4, and actin were used
Figure Legend Snippet: Effects of MS-275 on HepG2 cells. A, HepG2 cells were treated with MS-275 for 24 h, and cell lysates were subjected to Western blot analysis using anti-acetylated histone H3 and H4 antibodies ( Ac-H3 and Ac-H4 ). Total histones H3, H4, and actin were used

Techniques Used: Mass Spectrometry, Western Blot

15) Product Images from "Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor"

Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

Journal: Molecular and Cellular Biology

doi:

Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.
Figure Legend Snippet: Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

Techniques Used: Over Expression, HAT Assay, Transfection, Mutagenesis

Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.
Figure Legend Snippet: Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

Techniques Used: Immunofluorescence, In Vivo, Staining, Microscopy

Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.
Figure Legend Snippet: Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

Techniques Used: Inhibition, HAT Assay, Activity Assay, Recombinant, Purification, Radioactivity, Labeling, Incubation

Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.
Figure Legend Snippet: Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

Techniques Used: In Vivo, Expressing

16) Product Images from "Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling"

Article Title: Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12550

Proteins identified in association with hyperacetylated histone H4 in Bel treated NB4 cells. 2 μM Bel treated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).
Figure Legend Snippet: Proteins identified in association with hyperacetylated histone H4 in Bel treated NB4 cells. 2 μM Bel treated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).

Techniques Used: Chromatin Immunoprecipitation, Mass Spectrometry

Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).
Figure Legend Snippet: Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).

Techniques Used: Chromatin Immunoprecipitation, Mass Spectrometry

Bel effect on acetylated histone H4 association with p27, C/EBPα and C/EBPε promoter regions. ChIP with antibody against hyperacetylated histone H4 was performed with control (C) and NB4 cells treated with 2 μM Bel for 6 hrs (Bel). Specimens were further tested using qPCR analysis. Data are represented as percent input (± SD, n = 2).
Figure Legend Snippet: Bel effect on acetylated histone H4 association with p27, C/EBPα and C/EBPε promoter regions. ChIP with antibody against hyperacetylated histone H4 was performed with control (C) and NB4 cells treated with 2 μM Bel for 6 hrs (Bel). Specimens were further tested using qPCR analysis. Data are represented as percent input (± SD, n = 2).

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

17) Product Images from "Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling"

Article Title: Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12550

Proteins identified in association with hyperacetylated histone H4 in Bel treated NB4 cells. 2 μM Bel treated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).
Figure Legend Snippet: Proteins identified in association with hyperacetylated histone H4 in Bel treated NB4 cells. 2 μM Bel treated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).

Techniques Used: Chromatin Immunoprecipitation, Mass Spectrometry

Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).
Figure Legend Snippet: Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).

Techniques Used: Chromatin Immunoprecipitation, Mass Spectrometry

Bel effect on acetylated histone H4 association with p27, C/EBPα and C/EBPε promoter regions. ChIP with antibody against hyperacetylated histone H4 was performed with control (C) and NB4 cells treated with 2 μM Bel for 6 hrs (Bel). Specimens were further tested using qPCR analysis. Data are represented as percent input (± SD, n = 2).
Figure Legend Snippet: Bel effect on acetylated histone H4 association with p27, C/EBPα and C/EBPε promoter regions. ChIP with antibody against hyperacetylated histone H4 was performed with control (C) and NB4 cells treated with 2 μM Bel for 6 hrs (Bel). Specimens were further tested using qPCR analysis. Data are represented as percent input (± SD, n = 2).

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

18) Product Images from "Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia"

Article Title: Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia

Journal: Journal of Clinical Investigation

doi:

In vivo effects of SAHA, RA, and the combination of both on PLZF-RARα/RARα-PLZF double leukemic TM. ( a ) Western blot analysis of acetylated histone H4 in murine cells upon in vivo administration of SAHA. Wild-type and leukemic TM were given SAHA (20 μg/gbw) by intraperitoneal injection. Histones were acid extracted from murine peripheral blood, BM, and spleen cells in untreated mice and 2 hours after SAHA administration. ( b ) SAHA in combination with RA treatment prolongs survival in PLZF-RARα/RARα-PLZF double TM with leukemia. Upon presentation of leukemia as monitored by automatic and differentiated counts on peripheral blood samples from PLZF-RARα/RARα-PLZF double TM, RA was administered daily at a dose of 1.5 μg/gbw for 2 weeks. The RA-treated mice were then randomly assigned into three groups: RA alone (1.5 μg/gbw), SAHA (50 μg/gbw) alone, and SAHA (50 μg/gbw) in combination with RA (1.5 μg/gbw). The treatment was continued for 4 weeks. During and after the treatment, the mice were bled weekly, and automatic and morphological differential counts were performed on each sample to evaluate response to treatment until each animal died. Kaplan Meier analysis was used to compare the cumulative survival period between SAHA in combination with RA ( n = 11) and RA ( n = 9) or SAHA ( n = 6) alone. The black bar on the abscissa represents the 28-day period of treatment. Survival time reflects the days from the initiation of therapies until death for each mouse. ( c ) Complete remission was induced by SAHA + RA treatment in six of 11 leukemic PLZF-RARα/RARα-PLZF double transgenic mice. Analyses were performed weekly as described above. SAHA + RA causes the same duration of disease-free survival (time in remission) in PLZF-RARα/RARα-PLZF leukemic mice as that achieved in PML-RARα transgenic leukemic mice treated with RA (1.5 μg/gbw).
Figure Legend Snippet: In vivo effects of SAHA, RA, and the combination of both on PLZF-RARα/RARα-PLZF double leukemic TM. ( a ) Western blot analysis of acetylated histone H4 in murine cells upon in vivo administration of SAHA. Wild-type and leukemic TM were given SAHA (20 μg/gbw) by intraperitoneal injection. Histones were acid extracted from murine peripheral blood, BM, and spleen cells in untreated mice and 2 hours after SAHA administration. ( b ) SAHA in combination with RA treatment prolongs survival in PLZF-RARα/RARα-PLZF double TM with leukemia. Upon presentation of leukemia as monitored by automatic and differentiated counts on peripheral blood samples from PLZF-RARα/RARα-PLZF double TM, RA was administered daily at a dose of 1.5 μg/gbw for 2 weeks. The RA-treated mice were then randomly assigned into three groups: RA alone (1.5 μg/gbw), SAHA (50 μg/gbw) alone, and SAHA (50 μg/gbw) in combination with RA (1.5 μg/gbw). The treatment was continued for 4 weeks. During and after the treatment, the mice were bled weekly, and automatic and morphological differential counts were performed on each sample to evaluate response to treatment until each animal died. Kaplan Meier analysis was used to compare the cumulative survival period between SAHA in combination with RA ( n = 11) and RA ( n = 9) or SAHA ( n = 6) alone. The black bar on the abscissa represents the 28-day period of treatment. Survival time reflects the days from the initiation of therapies until death for each mouse. ( c ) Complete remission was induced by SAHA + RA treatment in six of 11 leukemic PLZF-RARα/RARα-PLZF double transgenic mice. Analyses were performed weekly as described above. SAHA + RA causes the same duration of disease-free survival (time in remission) in PLZF-RARα/RARα-PLZF leukemic mice as that achieved in PML-RARα transgenic leukemic mice treated with RA (1.5 μg/gbw).

Techniques Used: In Vivo, Western Blot, Injection, Mouse Assay, Transgenic Assay

19) Product Images from "Retinoic Acid-Induced Chromatin Remodeling of Mouse κ Opioid Receptor Gene"

Article Title: Retinoic Acid-Induced Chromatin Remodeling of Mouse κ Opioid Receptor Gene

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0186-05.2005

ChIP assays to determine binding patterns of chromatin modification and remodeling factors on KOR P1. A , ChIP assays were conducted using antibodies against the proteins (labeled on the left) involved in histone modification regulating promoter activity. The DNA precipitated by anti-HDAC2 was amplified with the primers specific to the NF-κB-binding site of the c- myc promoter as a positive control. B , ChIP assays were conducted using antibodies against the proteins (labeled on the left) that are involved in chromatin remodeling. BRG-1 was detected by Western blot (WB) using anti-BRG-1 (bottom). Experiments were performed at least twice independently.
Figure Legend Snippet: ChIP assays to determine binding patterns of chromatin modification and remodeling factors on KOR P1. A , ChIP assays were conducted using antibodies against the proteins (labeled on the left) involved in histone modification regulating promoter activity. The DNA precipitated by anti-HDAC2 was amplified with the primers specific to the NF-κB-binding site of the c- myc promoter as a positive control. B , ChIP assays were conducted using antibodies against the proteins (labeled on the left) that are involved in chromatin remodeling. BRG-1 was detected by Western blot (WB) using anti-BRG-1 (bottom). Experiments were performed at least twice independently.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Modification, Labeling, Activity Assay, Amplification, Positive Control, Western Blot

20) Product Images from "Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus"

Article Title: Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Cross-modulation between chromatin structure, NM organization, and the acinar phenotype. Confocal fluorescence images (0.2-μm optical sections) of NuMA ( a , e , and i ), collagen IV ( b , f , and j ), β-catenin ( c , g , and k ), and acetylated histone H4 ( d , h , and l ) in control, trichostatin A (TSA)-treated, and NuMA mAb-incubated acini (day 10 of 3D rBM culture). ( a – d ) Nuclear organization and acinar phenotype in controls. Acini exhibit NuMA foci ( a ), an organized endogenous collagen IV-rich BM ( b ), cell–cell localized β-catenin ( c ), and dispersed acetylated H4 histone ( d ). ( e – h ) Effects of TSA on nuclear architecture and acinar phenotype. After 24 hr of TSA treatment (40 nM), > 55% of the cells entered the cell cycle, as indicated by an increase in Ki-67 labeling index ( m ) and the appearance of mitotic cells (arrow in e ). NuMA was uniformly distributed in the nuclei ( e ), collagen IV disappeared ( f ), β-catenin was released from the cell–cell interface ( g ), and the pattern of histone H4 acetylation was altered ( h ). ( i – l ) Effects of mAb-induced NuMA foci disruption on nuclear organization and acinar phenotype. Introduction of a NuMA mAb into the nuclei of the acini by using reversible digitonin permeabilization led to the disruption of NuMA foci ( i ), degradation of the collagen IV-rich BM (arrows in j ), and the nuclear marginalization of acetylated H4 histone ( l ). There was no consistent alteration observed for β-catenin other than increased basal labeling ( k ). These effects were not observed with mock IgGs or mAbs to lamins A/C or B. ( n ) BM degradation after mAb-induced NuMA disruption in acini. Analysis of the percentage of acini with intact collagen IV-rich BMs in relation to control/digitonin-permeabilized (DP) acini ( a ), mock-IgG mAb-treated/DP acini ( b ), NuMA mAb-treated/nonpermeabilized acini ( c ), NuMA mAb-treated/DP acini ( d ), NuMA mAb-treated/DP acini + the metalloproteinase inhibitor GM6001 ( e ), NuMA mAb-treated/DP acini + the inactive metalloproteinase inhibitor GM1210 ( f ), NuMA mAb-treated/DP acini + the uPA inhibitor, aprotinin ( g ), and Lamin B mAb-treated/DP acini ( h ). Acini ( > 35%) degraded their endogenous BMs in response to disruption of NuMA ( d ). The BM loss could be rescued by treatment with the metalloproteinase inhibitor GM6001 ( e ), but not its inactive analogue ( f ) or a uPA protease inhibitor ( g ). (Bar = 10 μm.)
Figure Legend Snippet: Cross-modulation between chromatin structure, NM organization, and the acinar phenotype. Confocal fluorescence images (0.2-μm optical sections) of NuMA ( a , e , and i ), collagen IV ( b , f , and j ), β-catenin ( c , g , and k ), and acetylated histone H4 ( d , h , and l ) in control, trichostatin A (TSA)-treated, and NuMA mAb-incubated acini (day 10 of 3D rBM culture). ( a – d ) Nuclear organization and acinar phenotype in controls. Acini exhibit NuMA foci ( a ), an organized endogenous collagen IV-rich BM ( b ), cell–cell localized β-catenin ( c ), and dispersed acetylated H4 histone ( d ). ( e – h ) Effects of TSA on nuclear architecture and acinar phenotype. After 24 hr of TSA treatment (40 nM), > 55% of the cells entered the cell cycle, as indicated by an increase in Ki-67 labeling index ( m ) and the appearance of mitotic cells (arrow in e ). NuMA was uniformly distributed in the nuclei ( e ), collagen IV disappeared ( f ), β-catenin was released from the cell–cell interface ( g ), and the pattern of histone H4 acetylation was altered ( h ). ( i – l ) Effects of mAb-induced NuMA foci disruption on nuclear organization and acinar phenotype. Introduction of a NuMA mAb into the nuclei of the acini by using reversible digitonin permeabilization led to the disruption of NuMA foci ( i ), degradation of the collagen IV-rich BM (arrows in j ), and the nuclear marginalization of acetylated H4 histone ( l ). There was no consistent alteration observed for β-catenin other than increased basal labeling ( k ). These effects were not observed with mock IgGs or mAbs to lamins A/C or B. ( n ) BM degradation after mAb-induced NuMA disruption in acini. Analysis of the percentage of acini with intact collagen IV-rich BMs in relation to control/digitonin-permeabilized (DP) acini ( a ), mock-IgG mAb-treated/DP acini ( b ), NuMA mAb-treated/nonpermeabilized acini ( c ), NuMA mAb-treated/DP acini ( d ), NuMA mAb-treated/DP acini + the metalloproteinase inhibitor GM6001 ( e ), NuMA mAb-treated/DP acini + the inactive metalloproteinase inhibitor GM1210 ( f ), NuMA mAb-treated/DP acini + the uPA inhibitor, aprotinin ( g ), and Lamin B mAb-treated/DP acini ( h ). Acini ( > 35%) degraded their endogenous BMs in response to disruption of NuMA ( d ). The BM loss could be rescued by treatment with the metalloproteinase inhibitor GM6001 ( e ), but not its inactive analogue ( f ) or a uPA protease inhibitor ( g ). (Bar = 10 μm.)

Techniques Used: Fluorescence, Incubation, Labeling, Protease Inhibitor

21) Product Images from "A Class II Histone Deacetylase Acts on Newly Synthesized Histones in Tetrahymena ▿"

Article Title: A Class II Histone Deacetylase Acts on Newly Synthesized Histones in Tetrahymena ▿

Journal: Eukaryotic Cell

doi: 10.1128/EC.00409-07

Thd2 removes deposition-related acetylation from micronuclear histones. (A) Immunofluorescence using antiserum against acetylated histone H3 (α-H3ac). Cells were counterstained with DAPI to visualize both the macronucleus (M) and the micronucleus (m). Acetylated histone H3 was detected exclusively in the macronuclei of wild-type (WT) cells and additionally in the micronuclei of thd2 Δ cells. (B) Immunofluorescence using antiserum against acetylated histone H4 (α-H4ac). DAPI stain was used to detect both the macronucleus and the micronucleus. Acetylated histone H4 was detected exclusively in the macronucleus of WT cells and additionally in the micronuclei of thd2 Δ cells throughout every stage of the cell cycle. A high proportion of cells contained elongated micronuclei in close proximity to the macronucleus (the phenotype is depicted in the last panel). (C) Immunofluorescence using antiserum against acetylated Lys9 on histone H3 that additionally detects acetylated Lys14 in Tetrahymena (α-H3K9/14ac). Cells were counterstained with DAPI to visualize both the macronucleus and the micronucleus. Histone H3 Lys9/Lys14 acetylation was detected only in the macronuclei of wild-type cells, but also in the micronuclei of thd2 Δ cells. (D) Total nuclear proteins from purified macronuclei and micronuclei were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblot analysis using antiserum against acetylated Lys16 on histone H4 (α-H4K16ac) or against general histone H4 (α-H4). In both wild-type and thd2 Δ cells, H4 Lys16 was acetylated only in macronuclei.
Figure Legend Snippet: Thd2 removes deposition-related acetylation from micronuclear histones. (A) Immunofluorescence using antiserum against acetylated histone H3 (α-H3ac). Cells were counterstained with DAPI to visualize both the macronucleus (M) and the micronucleus (m). Acetylated histone H3 was detected exclusively in the macronuclei of wild-type (WT) cells and additionally in the micronuclei of thd2 Δ cells. (B) Immunofluorescence using antiserum against acetylated histone H4 (α-H4ac). DAPI stain was used to detect both the macronucleus and the micronucleus. Acetylated histone H4 was detected exclusively in the macronucleus of WT cells and additionally in the micronuclei of thd2 Δ cells throughout every stage of the cell cycle. A high proportion of cells contained elongated micronuclei in close proximity to the macronucleus (the phenotype is depicted in the last panel). (C) Immunofluorescence using antiserum against acetylated Lys9 on histone H3 that additionally detects acetylated Lys14 in Tetrahymena (α-H3K9/14ac). Cells were counterstained with DAPI to visualize both the macronucleus and the micronucleus. Histone H3 Lys9/Lys14 acetylation was detected only in the macronuclei of wild-type cells, but also in the micronuclei of thd2 Δ cells. (D) Total nuclear proteins from purified macronuclei and micronuclei were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblot analysis using antiserum against acetylated Lys16 on histone H4 (α-H4K16ac) or against general histone H4 (α-H4). In both wild-type and thd2 Δ cells, H4 Lys16 was acetylated only in macronuclei.

Techniques Used: Immunofluorescence, Staining, Purification, SDS Page

Cells lacking Thd2 exhibit chromatin phenotypes. (A) Immunofluorescence using antiserum against micronuclear linker histone H1 (α-Mlh1) was performed on wild-type (WT) and thd2 Δ cells. DAPI staining was used to visualize both the macronucleus (M) and the micronucleus (m). A higher incidence of elongated micronuclei in close association with macronuclei was observed in the mutant cells. (B) Total proteins from purified macronuclei and micronuclei were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and hybridized with antiserum against general histone H3 (α-H3) or with antiserum against phosphorylated serine 10 on histone H3 (α-H3S10ph). The full-length form of histone H3 (H3 s ) was detected in both macronuclei and micronuclei of all cells, but only wild-type micronuclei contained the faster-migrating proteolytically cleaved form (H3 f ). Likewise, phosphorylation of Ser10, which is specific for H3 f , occurred only in wild-type micronuclei. (Note: the blot was first hybridized with α-H3S10ph and then stripped and hybridized with α-H3.)
Figure Legend Snippet: Cells lacking Thd2 exhibit chromatin phenotypes. (A) Immunofluorescence using antiserum against micronuclear linker histone H1 (α-Mlh1) was performed on wild-type (WT) and thd2 Δ cells. DAPI staining was used to visualize both the macronucleus (M) and the micronucleus (m). A higher incidence of elongated micronuclei in close association with macronuclei was observed in the mutant cells. (B) Total proteins from purified macronuclei and micronuclei were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and hybridized with antiserum against general histone H3 (α-H3) or with antiserum against phosphorylated serine 10 on histone H3 (α-H3S10ph). The full-length form of histone H3 (H3 s ) was detected in both macronuclei and micronuclei of all cells, but only wild-type micronuclei contained the faster-migrating proteolytically cleaved form (H3 f ). Likewise, phosphorylation of Ser10, which is specific for H3 f , occurred only in wild-type micronuclei. (Note: the blot was first hybridized with α-H3S10ph and then stripped and hybridized with α-H3.)

Techniques Used: Immunofluorescence, Staining, Mutagenesis, Purification, SDS Page

THD2 is a nonessential gene. (A) Diagram of the THD2 deletion construct used to replace THD2 with NEO in the somatic macronucleus. Depicted in the diagram are the flanking regions (thin black lines), the coding sequence (thick dark gray lines), introns (white boxes), the histone H4 promoter (thick black lines), the neomycin resistance gene ( NEO ), and the BTU2 polyadenylation region (light gray line). The arrows represent the primers used to confirm correct integration of the replacement allele. (B) PCR amplification of genomic DNA from wild-type (WT) and thd2 Δ (Δ) cells confirmed that all THD2 alleles were replaced with the NEO cassette. THD2 PCR was performed using Ta(+) and T2-3 primers (WT allele), NEO PCR with NF and NR primers ( NEO cassette), and THD2-NEO PCR with F1 and NS primers (incorporation of the NEO cassette in the THD2 locus). HHP1 PCR was performed as a positive control for the genomic DNA. (C) Total cDNA derived from WT and thd2 Δ cells was used in PCRs to test for the presence of THD2 mRNA in these cells. HHP1 was used as a control for cDNA synthesis and PCR amplification. Genomic DNA (G) was used as a template to control for genomic-DNA contamination in cDNA.
Figure Legend Snippet: THD2 is a nonessential gene. (A) Diagram of the THD2 deletion construct used to replace THD2 with NEO in the somatic macronucleus. Depicted in the diagram are the flanking regions (thin black lines), the coding sequence (thick dark gray lines), introns (white boxes), the histone H4 promoter (thick black lines), the neomycin resistance gene ( NEO ), and the BTU2 polyadenylation region (light gray line). The arrows represent the primers used to confirm correct integration of the replacement allele. (B) PCR amplification of genomic DNA from wild-type (WT) and thd2 Δ (Δ) cells confirmed that all THD2 alleles were replaced with the NEO cassette. THD2 PCR was performed using Ta(+) and T2-3 primers (WT allele), NEO PCR with NF and NR primers ( NEO cassette), and THD2-NEO PCR with F1 and NS primers (incorporation of the NEO cassette in the THD2 locus). HHP1 PCR was performed as a positive control for the genomic DNA. (C) Total cDNA derived from WT and thd2 Δ cells was used in PCRs to test for the presence of THD2 mRNA in these cells. HHP1 was used as a control for cDNA synthesis and PCR amplification. Genomic DNA (G) was used as a template to control for genomic-DNA contamination in cDNA.

Techniques Used: Construct, Sequencing, Polymerase Chain Reaction, Amplification, Positive Control, Derivative Assay

22) Product Images from "Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition"

Article Title: Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.21.7719-7731.2003

The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).
Figure Legend Snippet: The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).

Techniques Used: Cell Culture, Isolation, Chromatin Immunoprecipitation, Amplification, Autoradiography, Luciferase, Activity Assay, Reporter Assay

Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.
Figure Legend Snippet: Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.

Techniques Used: Isolation, Cell Culture, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Autoradiography, Immunoprecipitation

23) Product Images from "Valproate Treatment of Human Cord Blood CD4-positive Effector T Cells Confers on Them the Molecular Profile (MicroRNA Signature and FOXP3 Expression) of Natural Regulatory CD4-positive Cells through Inhibition of Histone Deacetylase *"

Article Title: Valproate Treatment of Human Cord Blood CD4-positive Effector T Cells Confers on Them the Molecular Profile (MicroRNA Signature and FOXP3 Expression) of Natural Regulatory CD4-positive Cells through Inhibition of Histone Deacetylase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.119628

Valproate treatment induces acetylation of H4 lysines and increases transcription factors accessibility to FOXP3 promoter. A , the acetylation status of histone H4 in the nucleosomes associated with the FOXP3 core promoter region was assessed by ChIP assay
Figure Legend Snippet: Valproate treatment induces acetylation of H4 lysines and increases transcription factors accessibility to FOXP3 promoter. A , the acetylation status of histone H4 in the nucleosomes associated with the FOXP3 core promoter region was assessed by ChIP assay

Techniques Used: Chromatin Immunoprecipitation

24) Product Images from "Real-time imaging of histone H4 hyperacetylation in living cells"

Article Title: Real-time imaging of histone H4 hyperacetylation in living cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0902150106

( A ) Pseudocolored images of the 480 nm/535 nm emission ratio obtained from a COS7 cell expressing the acetylation indicator during mitosis. ( B ) Time courses of the 480 nm/535 nm emission ratio of Histac (●) and Histac-4KR (○) during mitosis. ( C ) Acetylation of histone H4 at K5 and K8 of asynchronous (A) and nocodazole-treated (N) COS7 cells was analyzed by immunoblotting using antibodies against histone H4 acetylated at K5 and K8. Phosphorylated histone H3 (pS10), a mitotic marker, was analyzed by immunoblotting using an antibody against phosphorylated histone H3. COS7 cells were arrested in mitosis by treatment with 10 μg/mL nocodazole for 12 h.
Figure Legend Snippet: ( A ) Pseudocolored images of the 480 nm/535 nm emission ratio obtained from a COS7 cell expressing the acetylation indicator during mitosis. ( B ) Time courses of the 480 nm/535 nm emission ratio of Histac (●) and Histac-4KR (○) during mitosis. ( C ) Acetylation of histone H4 at K5 and K8 of asynchronous (A) and nocodazole-treated (N) COS7 cells was analyzed by immunoblotting using antibodies against histone H4 acetylated at K5 and K8. Phosphorylated histone H3 (pS10), a mitotic marker, was analyzed by immunoblotting using an antibody against phosphorylated histone H3. COS7 cells were arrested in mitosis by treatment with 10 μg/mL nocodazole for 12 h.

Techniques Used: Expressing, Marker

( A ) COS7 cells expressing Histac and nontransfected COS7 cells were treated with 1 μM TSA. Immunoblot analyses were performed with antibodies against histone H4 acetylated at Lys-5, 8, 12, and 16. ( B and C ) Pseudocolored images and a time course of the emission ratio in the nucleus of a COS7 cell expressing Histac. TSA at a final concentration of 1 μM or vehicle alone was added to the culture at 0 min. ( D ) After photobleaching of Venus within Histac, the cells were treated with 1 μM TSA for 3 h.
Figure Legend Snippet: ( A ) COS7 cells expressing Histac and nontransfected COS7 cells were treated with 1 μM TSA. Immunoblot analyses were performed with antibodies against histone H4 acetylated at Lys-5, 8, 12, and 16. ( B and C ) Pseudocolored images and a time course of the emission ratio in the nucleus of a COS7 cell expressing Histac. TSA at a final concentration of 1 μM or vehicle alone was added to the culture at 0 min. ( D ) After photobleaching of Venus within Histac, the cells were treated with 1 μM TSA for 3 h.

Techniques Used: Expressing, Concentration Assay

( A ) Schematic representation of the domain structure of Histac. ( B ) Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4 N-terminal tail peptides (upper panel), or partly acetylated peptides (middle and lower panels). Pull-downs were analyzed by immunoblotting using an antibody against GFP.
Figure Legend Snippet: ( A ) Schematic representation of the domain structure of Histac. ( B ) Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4 N-terminal tail peptides (upper panel), or partly acetylated peptides (middle and lower panels). Pull-downs were analyzed by immunoblotting using an antibody against GFP.

Techniques Used: Pull Down Assay

Immunoblot analysis was performed with antibodies against histone H4 acetylated at Lys-5, 8, 12, and 16. COS7 cells were treated with various concentrations of TSA at 37 °C for 3 h ( A and B ). Emission ratio time courses ( C ) and changes in emission ratios ( D ) of cells expressing Histac treated with TSA. Asterisk indicates P
Figure Legend Snippet: Immunoblot analysis was performed with antibodies against histone H4 acetylated at Lys-5, 8, 12, and 16. COS7 cells were treated with various concentrations of TSA at 37 °C for 3 h ( A and B ). Emission ratio time courses ( C ) and changes in emission ratios ( D ) of cells expressing Histac treated with TSA. Asterisk indicates P

Techniques Used: Expressing

( A ) The schematic representation shows the domain structure of BRDT. YA is a bromodomain mutant, in which Tyr-65 and 308 of BRDT are replaced with Ala. ( B ) Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4 N-terminal tail peptides. Pull-downs were analyzed by immunoblotting using an antibody against GFP. ( C ) Changes in emission ratio of Histac mutants in response to 1 μM TSA for 3 h.
Figure Legend Snippet: ( A ) The schematic representation shows the domain structure of BRDT. YA is a bromodomain mutant, in which Tyr-65 and 308 of BRDT are replaced with Ala. ( B ) Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4 N-terminal tail peptides. Pull-downs were analyzed by immunoblotting using an antibody against GFP. ( C ) Changes in emission ratio of Histac mutants in response to 1 μM TSA for 3 h.

Techniques Used: Mutagenesis, Pull Down Assay

25) Product Images from "Inhibition of Histone Deacetylases 1 and 6 Enhances Cytarabine-Induced Apoptosis in Pediatric Acute Myeloid Leukemia Cells"

Article Title: Inhibition of Histone Deacetylases 1 and 6 Enhances Cytarabine-Induced Apoptosis in Pediatric Acute Myeloid Leukemia Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017138

Effects of equal doses (IC 20 ) of structurally-diverse HDACIs on acetylation of histones H3 and H4 and α-tubulin, and cytarabine-induced apoptosis in pediatric AML cells. Panel A: THP-1 cells were treated with equal doses (IC 20 s, determined by MTT assays) of MS-275, VPA or SAHA for 48 hrs. Acetylation of histones H3 and H4, and α-tubulin were determined by western blots probed by anti-ac-H3, -ac-H4, -ac-tubulin, or –H4 antibodies. Panel B: THP-1 cells were treated with cytarabine (900 nM, IC 20 ) or equal doses of MS-275, VPA, or SAHA, alone or in combination for 48 hrs. Early and late apoptosis events were determined by annexin V/PI staining and flow cytometry analysis. **, p
Figure Legend Snippet: Effects of equal doses (IC 20 ) of structurally-diverse HDACIs on acetylation of histones H3 and H4 and α-tubulin, and cytarabine-induced apoptosis in pediatric AML cells. Panel A: THP-1 cells were treated with equal doses (IC 20 s, determined by MTT assays) of MS-275, VPA or SAHA for 48 hrs. Acetylation of histones H3 and H4, and α-tubulin were determined by western blots probed by anti-ac-H3, -ac-H4, -ac-tubulin, or –H4 antibodies. Panel B: THP-1 cells were treated with cytarabine (900 nM, IC 20 ) or equal doses of MS-275, VPA, or SAHA, alone or in combination for 48 hrs. Early and late apoptosis events were determined by annexin V/PI staining and flow cytometry analysis. **, p

Techniques Used: MTT Assay, Mass Spectrometry, Western Blot, Staining, Flow Cytometry, Cytometry

26) Product Images from "Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling"

Article Title: Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12550

Proteins identified in association with hyperacetylated histone H4 in Bel treated NB4 cells. 2 μM Bel treated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).
Figure Legend Snippet: Proteins identified in association with hyperacetylated histone H4 in Bel treated NB4 cells. 2 μM Bel treated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).

Techniques Used: Chromatin Immunoprecipitation, Mass Spectrometry

Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).
Figure Legend Snippet: Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).

Techniques Used: Chromatin Immunoprecipitation, Mass Spectrometry

Bel effect on acetylated histone H4 association with p27, C/EBPα and C/EBPε promoter regions. ChIP with antibody against hyperacetylated histone H4 was performed with control (C) and NB4 cells treated with 2 μM Bel for 6 hrs (Bel). Specimens were further tested using qPCR analysis. Data are represented as percent input (± SD, n = 2).
Figure Legend Snippet: Bel effect on acetylated histone H4 association with p27, C/EBPα and C/EBPε promoter regions. ChIP with antibody against hyperacetylated histone H4 was performed with control (C) and NB4 cells treated with 2 μM Bel for 6 hrs (Bel). Specimens were further tested using qPCR analysis. Data are represented as percent input (± SD, n = 2).

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

27) Product Images from "Regulation of TCR ? and ? repertoires by local and long-distance control of variable gene segment chromatin structure"

Article Title: Regulation of TCR ? and ? repertoires by local and long-distance control of variable gene segment chromatin structure

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20050680

V gene segment histone modifications. Histone H3 and H4 acetylation was measured in chromatin prepared from DN thymocytes ( Rag2 −/− ), DP thymocytes (Rxβ), Eα −/− DP thymocytes (Eα −/− Rxβ), and non–T cells (T-deficient splenocytes of Tcrb −/− Tcrd −/− mice). Data for all individual gene segments are displayed according to the order in which the gene segments appear in the TcraTcrd locus. In cases where multiple family members are analyzed in a single PCR, the data is displayed according to the locus position of the most distal family member. Arrows identify the dominant adult Vδ gene segments. The data represent the mean ± SEM of triplicate PCR reactions. Analysis of two independent chromatin preparations gave similar results.
Figure Legend Snippet: V gene segment histone modifications. Histone H3 and H4 acetylation was measured in chromatin prepared from DN thymocytes ( Rag2 −/− ), DP thymocytes (Rxβ), Eα −/− DP thymocytes (Eα −/− Rxβ), and non–T cells (T-deficient splenocytes of Tcrb −/− Tcrd −/− mice). Data for all individual gene segments are displayed according to the order in which the gene segments appear in the TcraTcrd locus. In cases where multiple family members are analyzed in a single PCR, the data is displayed according to the locus position of the most distal family member. Arrows identify the dominant adult Vδ gene segments. The data represent the mean ± SEM of triplicate PCR reactions. Analysis of two independent chromatin preparations gave similar results.

Techniques Used: Mouse Assay, Polymerase Chain Reaction

28) Product Images from "Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells"

Article Title: Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.071043198

Changes in histone acetylation at the hTERT promoter and TSA-mediated hyperacetylation of histones during HL60 differentiation. A ChIP assay was performed on logarithmically growing and DMSO-treated HL60 cells as described in Materials and Methods . ( a ) A decrease in acetylation of H3 and H4 histones at the hTERT promoter in differentiated HL60 cells. The absence of acetylated histones at the E22a fragment that contains an E-box but is not transcribed. Log, logarithmically growing cells; DMSO, DMSO-treated (differentiated) cells. ( b ) Abolishment of the differentiation-associated deacetylation of histones H3 and H4 at the hTERT promoter by TSA treatment in HL60 cells. Cells were induced to differentiate by DMSO overnight in the absence or presence of TSA as indicated. ( c ) TSA-mediated dose-dependent accumulation of histone H3 at the hTERT promoter in differentiating HL60 cells. Cells were treated with DMSO overnight in the absence or presence of various concentrations of TSA as indicated.
Figure Legend Snippet: Changes in histone acetylation at the hTERT promoter and TSA-mediated hyperacetylation of histones during HL60 differentiation. A ChIP assay was performed on logarithmically growing and DMSO-treated HL60 cells as described in Materials and Methods . ( a ) A decrease in acetylation of H3 and H4 histones at the hTERT promoter in differentiated HL60 cells. The absence of acetylated histones at the E22a fragment that contains an E-box but is not transcribed. Log, logarithmically growing cells; DMSO, DMSO-treated (differentiated) cells. ( b ) Abolishment of the differentiation-associated deacetylation of histones H3 and H4 at the hTERT promoter by TSA treatment in HL60 cells. Cells were induced to differentiate by DMSO overnight in the absence or presence of TSA as indicated. ( c ) TSA-mediated dose-dependent accumulation of histone H3 at the hTERT promoter in differentiating HL60 cells. Cells were treated with DMSO overnight in the absence or presence of various concentrations of TSA as indicated.

Techniques Used: Chromatin Immunoprecipitation

29) Product Images from "Axin gene methylation status correlates with radiosensitivity of lung cancer cells"

Article Title: Axin gene methylation status correlates with radiosensitivity of lung cancer cells

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-368

The effect of X-ray irradiation on DNMTs, MeCP2, acetylated histones and factors of the Wnt signaling pathway. Western blot analysis shows the effects of X-ray irradiation on the expression of DNMTs, MeCP2, acetylated histones, Axin, β-catenin, Cyclin D1 and MMP-7 at 24h after X-ray treatment. DNMT1, DNMT3B and MeCP2 are significantly down-regulated in H157 cells, and acetylated histone H3 and H4 are up-regulated in H157 cells (A , B) is the histogram of A. DNMT 1 and 3B are also down-regulated significantly in LTE cells. Slightly decreased MeCP2 expression and up-regulation of acetylated H3, H4 are noted in LTE cells (C and D) , with the degree of change less significant than in H157 cells. Increased Axin expression and decreased β-catenin, Cyclin D1 and MMP-7 expression are noted in the H157 cell line after X-ray irradiation (E and F) , but no significant change in these factors are detected in LTE cells (G and H) . Note a dose dependent pattern of the changes in both cell lines, with more prominent changes in the H157 than in the LTE cell line. * P
Figure Legend Snippet: The effect of X-ray irradiation on DNMTs, MeCP2, acetylated histones and factors of the Wnt signaling pathway. Western blot analysis shows the effects of X-ray irradiation on the expression of DNMTs, MeCP2, acetylated histones, Axin, β-catenin, Cyclin D1 and MMP-7 at 24h after X-ray treatment. DNMT1, DNMT3B and MeCP2 are significantly down-regulated in H157 cells, and acetylated histone H3 and H4 are up-regulated in H157 cells (A , B) is the histogram of A. DNMT 1 and 3B are also down-regulated significantly in LTE cells. Slightly decreased MeCP2 expression and up-regulation of acetylated H3, H4 are noted in LTE cells (C and D) , with the degree of change less significant than in H157 cells. Increased Axin expression and decreased β-catenin, Cyclin D1 and MMP-7 expression are noted in the H157 cell line after X-ray irradiation (E and F) , but no significant change in these factors are detected in LTE cells (G and H) . Note a dose dependent pattern of the changes in both cell lines, with more prominent changes in the H157 than in the LTE cell line. * P

Techniques Used: Irradiation, Western Blot, Expressing

30) Product Images from "Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition"

Article Title: Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.21.7719-7731.2003

The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).
Figure Legend Snippet: The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).

Techniques Used: Cell Culture, Isolation, Chromatin Immunoprecipitation, Amplification, Autoradiography, Luciferase, Activity Assay, Reporter Assay

Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.
Figure Legend Snippet: Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.

Techniques Used: Isolation, Cell Culture, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Autoradiography, Immunoprecipitation

31) Product Images from "Histone deacetylase inhibitors stimulate mitochondrial HMG-CoA synthase gene expression via a promoter proximal Sp1 site"

Article Title: Histone deacetylase inhibitors stimulate mitochondrial HMG-CoA synthase gene expression via a promoter proximal Sp1 site

Journal: Nucleic Acids Research

doi:

Mitochondrial HMG-CoA synthase, a target gene for HDAC inhibitors: TSA induces mitochondrial HMG-CoA synthase mRNA expression through mitochondrial HMG-CoA synthase promoter acetylation in the chromatin complex. ( A ) HT-29 and Caco-2 cells were treated with 0.3 µM TSA (+) or DMSO (–) for 24 h. The mitochondrial HMG-CoA synthase mRNA levels were analyzed by northern blot. Levels of 18S rRNA are also shown to correct for gel loading variation. ( B ) ChIPs were performed using chromatin isolated from HT-29 and CaCo-2 cells either treated with TSA (+) for 24 h or untreated (–). The chromatin preparations were immunoprecipitated with antibodies against acetylated histone H4 (α-AcH4) and analyzed by PCR to detect the HMG-CoA synthase promoter associated to acetylated histones. Aliquots of chromatin were also analyzed before immunoprecipitation (Input). Relative histone acetylation levels (Fold of acetylation) were determined by quantitation of PCR product using BIOPROFIL® image analysis software, BIO-1D (Vilber Lourmat) and by correction with the input data and represented as the ratio of relative acetylation in the presence versus the absence of TSA treatment. ( C ) ChIP assay of HDAC1 bound to endogenous mitochondrial HMG-CoA synthase promoter. CaCo-2 chromatin, isolated as described above, was immunoprecipitated with increasing amounts of anti-HDAC1 or preimmune serum (IgG). PCRs shown in (B) and (C) were performed with 5 µl of DNA and 30 cycles of amplification were used.
Figure Legend Snippet: Mitochondrial HMG-CoA synthase, a target gene for HDAC inhibitors: TSA induces mitochondrial HMG-CoA synthase mRNA expression through mitochondrial HMG-CoA synthase promoter acetylation in the chromatin complex. ( A ) HT-29 and Caco-2 cells were treated with 0.3 µM TSA (+) or DMSO (–) for 24 h. The mitochondrial HMG-CoA synthase mRNA levels were analyzed by northern blot. Levels of 18S rRNA are also shown to correct for gel loading variation. ( B ) ChIPs were performed using chromatin isolated from HT-29 and CaCo-2 cells either treated with TSA (+) for 24 h or untreated (–). The chromatin preparations were immunoprecipitated with antibodies against acetylated histone H4 (α-AcH4) and analyzed by PCR to detect the HMG-CoA synthase promoter associated to acetylated histones. Aliquots of chromatin were also analyzed before immunoprecipitation (Input). Relative histone acetylation levels (Fold of acetylation) were determined by quantitation of PCR product using BIOPROFIL® image analysis software, BIO-1D (Vilber Lourmat) and by correction with the input data and represented as the ratio of relative acetylation in the presence versus the absence of TSA treatment. ( C ) ChIP assay of HDAC1 bound to endogenous mitochondrial HMG-CoA synthase promoter. CaCo-2 chromatin, isolated as described above, was immunoprecipitated with increasing amounts of anti-HDAC1 or preimmune serum (IgG). PCRs shown in (B) and (C) were performed with 5 µl of DNA and 30 cycles of amplification were used.

Techniques Used: Expressing, Northern Blot, Isolation, Immunoprecipitation, Polymerase Chain Reaction, Quantitation Assay, Software, Chromatin Immunoprecipitation, Amplification

Recruitment of HDAC1 over transfected mitochondrial HMG-CoA synthase promoter: deacetylation of promoter associated histones and down-regulation of mitochondrial HMG-CoA synthase transcriptional activity. ( A ) Caco-2 cells were transiently co-transfected with 4 µg of the reporter plasmid –116pGl3 in combination with increasing amounts of an expression vector for the HDAC1 (pCINeoHDAC1). Luciferase activities are normalized by micrograms of protein assayed. Data are means and standard deviations from four independent experiments with two plates each. ( B ) ChIP assay to demonstrate histone deacetylation as a result of expression and recruitment of HDAC1 over mitochondrial HMG-CoA synthase proximal promoter. Caco-2 cells were transiently transfected with 10 µg of the reporter plasmid –116pGl3 (–) or in combination with 5 µg of an expression vector for the Flag-epitope-tagged HDAC1 (pCDNA3FlagHDAC1) (+). Thirty-six hours after transfection chromatin preparations were obtained and immunoprecipitated with antibodies against acetylated histone H4 (α-AcH4) and Flag epitope (M2). The immunoprecipitates were then analyzed by PCR using specific primers for mitochondrial HMG-CoA synthase promoter and luciferase gene. Aliquots of chromatin were also analyzed before immunoprecipitation (Input). Thirty cycles of PCR were used. Western blot analysis of Flag-HDAC1 protein in whole cell extracts from control (–) or pCDNA3FlagHDAC1 transfected (+) CaCo-2 cells is shown on the right.
Figure Legend Snippet: Recruitment of HDAC1 over transfected mitochondrial HMG-CoA synthase promoter: deacetylation of promoter associated histones and down-regulation of mitochondrial HMG-CoA synthase transcriptional activity. ( A ) Caco-2 cells were transiently co-transfected with 4 µg of the reporter plasmid –116pGl3 in combination with increasing amounts of an expression vector for the HDAC1 (pCINeoHDAC1). Luciferase activities are normalized by micrograms of protein assayed. Data are means and standard deviations from four independent experiments with two plates each. ( B ) ChIP assay to demonstrate histone deacetylation as a result of expression and recruitment of HDAC1 over mitochondrial HMG-CoA synthase proximal promoter. Caco-2 cells were transiently transfected with 10 µg of the reporter plasmid –116pGl3 (–) or in combination with 5 µg of an expression vector for the Flag-epitope-tagged HDAC1 (pCDNA3FlagHDAC1) (+). Thirty-six hours after transfection chromatin preparations were obtained and immunoprecipitated with antibodies against acetylated histone H4 (α-AcH4) and Flag epitope (M2). The immunoprecipitates were then analyzed by PCR using specific primers for mitochondrial HMG-CoA synthase promoter and luciferase gene. Aliquots of chromatin were also analyzed before immunoprecipitation (Input). Thirty cycles of PCR were used. Western blot analysis of Flag-HDAC1 protein in whole cell extracts from control (–) or pCDNA3FlagHDAC1 transfected (+) CaCo-2 cells is shown on the right.

Techniques Used: Transfection, Activity Assay, Plasmid Preparation, Expressing, Luciferase, Chromatin Immunoprecipitation, FLAG-tag, Immunoprecipitation, Polymerase Chain Reaction, Western Blot

32) Product Images from "Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition"

Article Title: Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.21.7719-7731.2003

The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).
Figure Legend Snippet: The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).

Techniques Used: Cell Culture, Isolation, Chromatin Immunoprecipitation, Amplification, Autoradiography, Luciferase, Activity Assay, Reporter Assay

Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.
Figure Legend Snippet: Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.

Techniques Used: Isolation, Cell Culture, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Autoradiography, Immunoprecipitation

33) Product Images from "Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition"

Article Title: Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.21.7719-7731.2003

The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).
Figure Legend Snippet: The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).

Techniques Used: Cell Culture, Isolation, Chromatin Immunoprecipitation, Amplification, Autoradiography, Luciferase, Activity Assay, Reporter Assay

Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.
Figure Legend Snippet: Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α- 32 P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.

Techniques Used: Isolation, Cell Culture, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Autoradiography, Immunoprecipitation

34) Product Images from "Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling"

Article Title: Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12550

Proteins identified in association with hyperacetylated histone H4 in Bel treated NB4 cells. 2 μM Bel treated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).
Figure Legend Snippet: Proteins identified in association with hyperacetylated histone H4 in Bel treated NB4 cells. 2 μM Bel treated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).

Techniques Used: Chromatin Immunoprecipitation, Mass Spectrometry

Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).
Figure Legend Snippet: Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP - MS analysis. Association network of identified proteins was studied and represented using STRING database ( http://string.embl.de ).

Techniques Used: Chromatin Immunoprecipitation, Mass Spectrometry

Bel effect on acetylated histone H4 association with p27, C/EBPα and C/EBPε promoter regions. ChIP with antibody against hyperacetylated histone H4 was performed with control (C) and NB4 cells treated with 2 μM Bel for 6 hrs (Bel). Specimens were further tested using qPCR analysis. Data are represented as percent input (± SD, n = 2).
Figure Legend Snippet: Bel effect on acetylated histone H4 association with p27, C/EBPα and C/EBPε promoter regions. ChIP with antibody against hyperacetylated histone H4 was performed with control (C) and NB4 cells treated with 2 μM Bel for 6 hrs (Bel). Specimens were further tested using qPCR analysis. Data are represented as percent input (± SD, n = 2).

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

35) Product Images from "An insulator embedded in the chicken ?-globin locus regulates chromatin domain configuration and differential gene expression"

Article Title: An insulator embedded in the chicken ?-globin locus regulates chromatin domain configuration and differential gene expression

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq740

Distribution of histone post-translational modifications over the 6.6-kb genomic region containing the αEHS-1.4 insulator. ( A ) Histone covalent modifications were evaluated semi-quantitatively using duplex-PCR with primers P1–P5 (primers locations are shown in the bottom scheme) in DT40 (lymphoid), 6C2 (pre-erythroblasts) and HD3 (erythroblasts) cells. ( B ) ChIP assays as in ( A ) using 5d and 10dRBCs. ( C ) Representative gel of the duplex-PCR amplification using primers P2 located over the CTCF-binding site with its corresponding control (Ctrl). The ChIP was performed on 10-day-old embryonic erythrocytes (10dRBC). All the histone modification enrichments are statistically significant (ANOVA with P ≤ 0.001) with respect to the control IgG except for the acH3 enrichment. Results are shown as averages (±standard error).
Figure Legend Snippet: Distribution of histone post-translational modifications over the 6.6-kb genomic region containing the αEHS-1.4 insulator. ( A ) Histone covalent modifications were evaluated semi-quantitatively using duplex-PCR with primers P1–P5 (primers locations are shown in the bottom scheme) in DT40 (lymphoid), 6C2 (pre-erythroblasts) and HD3 (erythroblasts) cells. ( B ) ChIP assays as in ( A ) using 5d and 10dRBCs. ( C ) Representative gel of the duplex-PCR amplification using primers P2 located over the CTCF-binding site with its corresponding control (Ctrl). The ChIP was performed on 10-day-old embryonic erythrocytes (10dRBC). All the histone modification enrichments are statistically significant (ANOVA with P ≤ 0.001) with respect to the control IgG except for the acH3 enrichment. Results are shown as averages (±standard error).

Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification, Binding Assay, Modification

Reduction of CTCF alters the profile of histone covalent modifications at the αEHS-1.4 insulator region and α-globin genes expression. ( A ) ChIP analysis of histone modifications in HD3 cells (HD3) and HD3 cells knocked down for CTCF (HD3+CTCF-kd). Each ChIP assay was performed in triplicate. Representative radioactive duplex PCR gels of the HD3-scramble (HD3-scr) and HD3 cells knocked down for CTCF (HD3+CTCF-kd) are shown. ( B ) Evaluation of α-globin gene expression in HD3 cells (HD3) and HD3 cells knocked down for CTCF in normal conditions (undifferentiated, HD3+CTCF-kd) and differentiated HD3 cells (dif-HD3). The results are representative of nine independent PCR reactions from two independent total RNA purifications and reverse transcriptase reactions. There is a premature chromatin opening of the insulator region in the CTCF knock down cells, and the adult genes are unable to be expressed in the HD3 differentiated cells. * P ≤ 0.05 (MANOVA) and *** P ≤ 0.001 (MANOVA) with respect to the corresponding histone covalent modification or expressed gene in HD3 or dif-HD3 scramble controls, respectively.
Figure Legend Snippet: Reduction of CTCF alters the profile of histone covalent modifications at the αEHS-1.4 insulator region and α-globin genes expression. ( A ) ChIP analysis of histone modifications in HD3 cells (HD3) and HD3 cells knocked down for CTCF (HD3+CTCF-kd). Each ChIP assay was performed in triplicate. Representative radioactive duplex PCR gels of the HD3-scramble (HD3-scr) and HD3 cells knocked down for CTCF (HD3+CTCF-kd) are shown. ( B ) Evaluation of α-globin gene expression in HD3 cells (HD3) and HD3 cells knocked down for CTCF in normal conditions (undifferentiated, HD3+CTCF-kd) and differentiated HD3 cells (dif-HD3). The results are representative of nine independent PCR reactions from two independent total RNA purifications and reverse transcriptase reactions. There is a premature chromatin opening of the insulator region in the CTCF knock down cells, and the adult genes are unable to be expressed in the HD3 differentiated cells. * P ≤ 0.05 (MANOVA) and *** P ≤ 0.001 (MANOVA) with respect to the corresponding histone covalent modification or expressed gene in HD3 or dif-HD3 scramble controls, respectively.

Techniques Used: Expressing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Modification

Summary and model for the αEHS-1.4 insulator role at the chicken α-globin locus. ( A ) Summary of histone post-translational modifications and CTCF in vivo enrichment over the 6.6-kb genomic region containing the αEHS-1.4 insulator. As adult erythroid differentiation proceeds there is an increment in histone modifications corresponding to a permissive chromatin environment over the αEHS-1.4 insulator and the CTCF site (see P2 primer location) that expands and further increases over the entire region in 10dRBC when the adult genes α D and α A are switched on. CTCF is present during differentiation with a peak at the erythroblast stage (HD3 cells) and then released at the mature 10dRBC when the adult α D and α A genes are being expressed (see right side green graph bar). P1–P5 represent the primers covering the 6.6-kb genomic region used for ChIP assays and P2 are the primers located over the CTCF-binding site. ( B ) Model suggesting a mechanism of action for the αEHS-1.4 insulator at the level of the chromatin structure at the domain scale and adult α-globin gene expression. Vertical arrows represent DNase I hypersensitive sites. We propose that at the erythroblast stage the αEHS-1.4 insulator blocks the effects coming from the α-MRE concomitant with the fact that the adult α D and α A globin genes remain silenced. At the late erythrocyte stage CTCF dissociation from the insulator facilitates α-MRE activity allowing trans -activation of α-globin adult genes.
Figure Legend Snippet: Summary and model for the αEHS-1.4 insulator role at the chicken α-globin locus. ( A ) Summary of histone post-translational modifications and CTCF in vivo enrichment over the 6.6-kb genomic region containing the αEHS-1.4 insulator. As adult erythroid differentiation proceeds there is an increment in histone modifications corresponding to a permissive chromatin environment over the αEHS-1.4 insulator and the CTCF site (see P2 primer location) that expands and further increases over the entire region in 10dRBC when the adult genes α D and α A are switched on. CTCF is present during differentiation with a peak at the erythroblast stage (HD3 cells) and then released at the mature 10dRBC when the adult α D and α A genes are being expressed (see right side green graph bar). P1–P5 represent the primers covering the 6.6-kb genomic region used for ChIP assays and P2 are the primers located over the CTCF-binding site. ( B ) Model suggesting a mechanism of action for the αEHS-1.4 insulator at the level of the chromatin structure at the domain scale and adult α-globin gene expression. Vertical arrows represent DNase I hypersensitive sites. We propose that at the erythroblast stage the αEHS-1.4 insulator blocks the effects coming from the α-MRE concomitant with the fact that the adult α D and α A globin genes remain silenced. At the late erythrocyte stage CTCF dissociation from the insulator facilitates α-MRE activity allowing trans -activation of α-globin adult genes.

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Binding Assay, Expressing, Activity Assay, Activation Assay

36) Product Images from "Obatoclax Potentiates the Cytotoxic Effect of Cytarabine on Acute Myeloid Leukemia Cells by Enhancing DNA Damage"

Article Title: Obatoclax Potentiates the Cytotoxic Effect of Cytarabine on Acute Myeloid Leukemia Cells by Enhancing DNA Damage

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2014.09.008

Ectopic overexpression of Mcl‐1 or Bcl‐xL in THP‐1 cells blocks apoptosis induced by cytarabine or cytarabine plus obatoclax. THP‐1 cells were infected with Precision LentiORF Mcl‐1, Bcl‐xL, (THP‐1/Mcl‐1 or THP‐1/Bcl‐xL respectively) or red fluorescent protein control (THP‐1/RFP) lentivirus overnight, washed and then incubated for 48 h before selection drug (Blasticidin) was added to the culture medium. The cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti‐Bcl‐xL, ‐Mcl‐1, ‐PARP, ‐cleaved caspase‐3, or ‐β‐actin antibody (Panels A  E). Apoptotic events in the cells post drug treatment were determined by annexin V/7‐AAD staining and flow cytometry analyses (Panels B  F). THP‐1/RFP and THP‐1/Bcl‐xL cells were treated with cytarabine and/or obatoclax for 48 h. Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. Due to the extent of overexpression of Bcl‐xL 1/10th of the total protein loaded for the THP‐1/RFP was loaded for the THP‐1/Bcl‐xL samples in order to visualize nuclear Bcl‐xL level changes on the same blot. The membranes were probed with anti‐Bcl‐xL, ‐MEK1/2, or ‐Histone H4 antibody (Panels C  D). Experiments were repeated at least 3 independent times with one representative shown. The data are presented as mean ± standard error. *** indicates p 
Figure Legend Snippet: Ectopic overexpression of Mcl‐1 or Bcl‐xL in THP‐1 cells blocks apoptosis induced by cytarabine or cytarabine plus obatoclax. THP‐1 cells were infected with Precision LentiORF Mcl‐1, Bcl‐xL, (THP‐1/Mcl‐1 or THP‐1/Bcl‐xL respectively) or red fluorescent protein control (THP‐1/RFP) lentivirus overnight, washed and then incubated for 48 h before selection drug (Blasticidin) was added to the culture medium. The cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti‐Bcl‐xL, ‐Mcl‐1, ‐PARP, ‐cleaved caspase‐3, or ‐β‐actin antibody (Panels A E). Apoptotic events in the cells post drug treatment were determined by annexin V/7‐AAD staining and flow cytometry analyses (Panels B F). THP‐1/RFP and THP‐1/Bcl‐xL cells were treated with cytarabine and/or obatoclax for 48 h. Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. Due to the extent of overexpression of Bcl‐xL 1/10th of the total protein loaded for the THP‐1/RFP was loaded for the THP‐1/Bcl‐xL samples in order to visualize nuclear Bcl‐xL level changes on the same blot. The membranes were probed with anti‐Bcl‐xL, ‐MEK1/2, or ‐Histone H4 antibody (Panels C D). Experiments were repeated at least 3 independent times with one representative shown. The data are presented as mean ± standard error. *** indicates p 

Techniques Used: Over Expression, Infection, Incubation, Selection, Western Blot, Staining, Flow Cytometry, Cytometry

The effects of cytarabine and obatoclax treatments on Bcl‐2, Bcl‐xL, and Mcl‐1 expression and subcellular localization in OCI‐AML3 cells. OCI‐AML3 cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting, and then probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, or –β‐actin antibody (Panel A, upper panel). Densitometry for Mcl‐1 expression was measured and graphed as fold change compared to the no drug control (Panel A, lower panel). Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. The membranes were probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, or ‐Histone H4 antibody (Panel B). OCI‐AML3 cells were treated with vehicle control or 2 μM cytarabine plus 175 nM obatoclax for 48 h. Cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panel C). OCI‐AML3 cells were treated with 2 μM cytarabine with or without 175 nM obatoclax for up to 48 h. Nuclear proteins were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1 or ‐Histone H4 antibody (Panel D). The data are presented as mean ± standard error from at least 3 independent experiments. * indicates p 
Figure Legend Snippet: The effects of cytarabine and obatoclax treatments on Bcl‐2, Bcl‐xL, and Mcl‐1 expression and subcellular localization in OCI‐AML3 cells. OCI‐AML3 cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting, and then probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, or –β‐actin antibody (Panel A, upper panel). Densitometry for Mcl‐1 expression was measured and graphed as fold change compared to the no drug control (Panel A, lower panel). Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. The membranes were probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, or ‐Histone H4 antibody (Panel B). OCI‐AML3 cells were treated with vehicle control or 2 μM cytarabine plus 175 nM obatoclax for 48 h. Cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panel C). OCI‐AML3 cells were treated with 2 μM cytarabine with or without 175 nM obatoclax for up to 48 h. Nuclear proteins were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1 or ‐Histone H4 antibody (Panel D). The data are presented as mean ± standard error from at least 3 independent experiments. * indicates p 

Techniques Used: Expressing, Western Blot, Staining, Confocal Microscopy

37) Product Images from "Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation"

Article Title: Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0407933101

HDAC inhibitors overcome the growth defect of an IE1-deficient mutant HCMV. ( A ) Comparison of viral protein accumulation in MRC-5 cells infected with WT (Towne) and mutant (CR208) viruses in the absence or continuous presence (beginning 24 h before infection) of 300 nM TSA. MRC-5 cells were infected at a multiplicity of 0.1 TCID 50 per cell, and expression of IE1 and IE2 (IE1/2), as well as the late viral protein pp28, was monitored at 24–96 h postinfection (hpi) by Western blot assay. ( B ) Multistep growth analysis of WT (Towne, circles) and mutant (CR208, triangles) viruses in the absence (filled symbols) or continuous presence (open symbols) of 300 nM TSA. MRC-5 cells were infected at a multiplicity of 0.1 TCID 50 per cell, and virus yields were monitored for 144 h. Symbols identify mean values from two experiments. ( C ) Titers of WT (Towne) and mutant (CR208) viruses in the absence or continuous presence of the HDAC inhibitors HC toxin (HCT, 100 nM), sodium butyrate (NaB, 1 mM), or TSA (300 nM). MRC-5 cells were infected at a multiplicity of 0.1 TCID 50 per cell, and yields were determined 120 h later. Bars represent mean values and SEs from three separate infections. ( D ) HDAC inhibitors induce histone hyperacetylation. MRC-5 cells were treated with 100 nM HCT, 300 nM TSA, or 1 mM NaB for 24 h, and Western blot analyses were performed by using Abs against acetylated histone H3 (Ac-H3) or the unmodified protein (H3).
Figure Legend Snippet: HDAC inhibitors overcome the growth defect of an IE1-deficient mutant HCMV. ( A ) Comparison of viral protein accumulation in MRC-5 cells infected with WT (Towne) and mutant (CR208) viruses in the absence or continuous presence (beginning 24 h before infection) of 300 nM TSA. MRC-5 cells were infected at a multiplicity of 0.1 TCID 50 per cell, and expression of IE1 and IE2 (IE1/2), as well as the late viral protein pp28, was monitored at 24–96 h postinfection (hpi) by Western blot assay. ( B ) Multistep growth analysis of WT (Towne, circles) and mutant (CR208, triangles) viruses in the absence (filled symbols) or continuous presence (open symbols) of 300 nM TSA. MRC-5 cells were infected at a multiplicity of 0.1 TCID 50 per cell, and virus yields were monitored for 144 h. Symbols identify mean values from two experiments. ( C ) Titers of WT (Towne) and mutant (CR208) viruses in the absence or continuous presence of the HDAC inhibitors HC toxin (HCT, 100 nM), sodium butyrate (NaB, 1 mM), or TSA (300 nM). MRC-5 cells were infected at a multiplicity of 0.1 TCID 50 per cell, and yields were determined 120 h later. Bars represent mean values and SEs from three separate infections. ( D ) HDAC inhibitors induce histone hyperacetylation. MRC-5 cells were treated with 100 nM HCT, 300 nM TSA, or 1 mM NaB for 24 h, and Western blot analyses were performed by using Abs against acetylated histone H3 (Ac-H3) or the unmodified protein (H3).

Techniques Used: Mutagenesis, Infection, Expressing, Western Blot

The HCMV major IE and UL44 promoters are regulated by histone acetylation and IE1. ( A ) Quantitative real-time RT-PCR was performed with total RNA isolated from MRC-5 cells at 6–72 h postinfection (hpi) with WT (Towne) or mutant (CR208) virus at a multiplicity of 0.1 TCID 50 per cell by using UL44- or UL122 (IE2)-specific primers. ( B ) Luciferase assays were performed in MRC-5 cells, which were transfected with reporter plasmids pGL3-ICP36 (UL44) or pGL3-MIEP (major IE) and treated with 100 nM HC toxin at 24–40 h after transfection, as indicated. At 24 h after transfection, cells were infected with WT (Towne) or mutant (CR208) viruses at a multiplicity of 1 TCID 50 per cell for 16 h. ( C ) Luciferase assays were performed in which MRC-5 cells were cotransfected with reporter plasmids pGL3-ICP36 (UL44 promoter) or pGL3-MIEP (major IE promoter) and empty vector (w/o) or expression plasmids encoding HDAC3, IE1, or both. As indicated, cells were treated with 100 nM HC toxin (HCT), 2 mM sodium butyrate (NaB), or 500 nM TSA at 24–40 h after transfection. Transfections were performed in triplicate, and mean relative light units with SEs (× 10 3 for the UL44 promoter and × 10 6 for the major IE promoter) are presented.
Figure Legend Snippet: The HCMV major IE and UL44 promoters are regulated by histone acetylation and IE1. ( A ) Quantitative real-time RT-PCR was performed with total RNA isolated from MRC-5 cells at 6–72 h postinfection (hpi) with WT (Towne) or mutant (CR208) virus at a multiplicity of 0.1 TCID 50 per cell by using UL44- or UL122 (IE2)-specific primers. ( B ) Luciferase assays were performed in MRC-5 cells, which were transfected with reporter plasmids pGL3-ICP36 (UL44) or pGL3-MIEP (major IE) and treated with 100 nM HC toxin at 24–40 h after transfection, as indicated. At 24 h after transfection, cells were infected with WT (Towne) or mutant (CR208) viruses at a multiplicity of 1 TCID 50 per cell for 16 h. ( C ) Luciferase assays were performed in which MRC-5 cells were cotransfected with reporter plasmids pGL3-ICP36 (UL44 promoter) or pGL3-MIEP (major IE promoter) and empty vector (w/o) or expression plasmids encoding HDAC3, IE1, or both. As indicated, cells were treated with 100 nM HC toxin (HCT), 2 mM sodium butyrate (NaB), or 500 nM TSA at 24–40 h after transfection. Transfections were performed in triplicate, and mean relative light units with SEs (× 10 3 for the UL44 promoter and × 10 6 for the major IE promoter) are presented.

Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Luciferase, Transfection, Infection, Plasmid Preparation, Expressing

Decreased histone H4 acetylation at the HCMV major IE and UL44 promoters in the absence of IE1. Chromatin immunoprecipitation assays were performed on chromatin from MRC-5 cells infected with WT (Towne) or mutant (CR208) viruses at a multiplicity of 1 TCID 50 per cell by using an Ab specific to amino-terminally acetylated histone H4- and UL44-specific, major IE-specific, or c-fos-specific primers for quantitative real-time PCR of coprecipitated DNA. PCRs were performed in triplicate, and mean values normalized to input DNA levels are presented, representing changes in acetylation at 6 and 12 h postinfection (hpi) relative to 1 h postinfection (set at 1).
Figure Legend Snippet: Decreased histone H4 acetylation at the HCMV major IE and UL44 promoters in the absence of IE1. Chromatin immunoprecipitation assays were performed on chromatin from MRC-5 cells infected with WT (Towne) or mutant (CR208) viruses at a multiplicity of 1 TCID 50 per cell by using an Ab specific to amino-terminally acetylated histone H4- and UL44-specific, major IE-specific, or c-fos-specific primers for quantitative real-time PCR of coprecipitated DNA. PCRs were performed in triplicate, and mean values normalized to input DNA levels are presented, representing changes in acetylation at 6 and 12 h postinfection (hpi) relative to 1 h postinfection (set at 1).

Techniques Used: Chromatin Immunoprecipitation, Infection, Mutagenesis, Real-time Polymerase Chain Reaction

38) Product Images from "PIASx acts as an Elk-1 coactivator by facilitating derepression"

Article Title: PIASx acts as an Elk-1 coactivator by facilitating derepression

Journal:

doi: 10.1038/sj.emboj.7600690

PIASxα affects HDAC-2 binding and histone acetylation levels at Elk-1-regulated promoters. (A–C) Reporter gene analysis of the activities of GAL-Elk-1 constructs on a GAL-driven E1B promoter-reporter construct in 293 cells. ( A ) The activity
Figure Legend Snippet: PIASxα affects HDAC-2 binding and histone acetylation levels at Elk-1-regulated promoters. (A–C) Reporter gene analysis of the activities of GAL-Elk-1 constructs on a GAL-driven E1B promoter-reporter construct in 293 cells. ( A ) The activity

Techniques Used: Binding Assay, Construct, Activity Assay

39) Product Images from "Corepressors selectively control the transcriptional activity of PPAR? in adipocytes"

Article Title: Corepressors selectively control the transcriptional activity of PPAR? in adipocytes

Journal: Genes & Development

doi: 10.1101/gad.1263305

Exogenous ligand is differentially required for coactivator recruitment to PPARγ target genes in adipocytes. ChIP analysis of factor binding and histone acetylation on adipocyte aP2 and GyK genes in preadipocytes and adipocytes ± rosiglitazone
Figure Legend Snippet: Exogenous ligand is differentially required for coactivator recruitment to PPARγ target genes in adipocytes. ChIP analysis of factor binding and histone acetylation on adipocyte aP2 and GyK genes in preadipocytes and adipocytes ± rosiglitazone

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

40) Product Images from "Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus"

Article Title: Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Cross-modulation between chromatin structure, NM organization, and the acinar phenotype. Confocal fluorescence images (0.2-μm optical sections) of NuMA ( a , e , and i ), collagen IV ( b , f , and j ), β-catenin ( c , g , and k ), and acetylated histone H4 ( d , h , and l ) in control, trichostatin A (TSA)-treated, and NuMA mAb-incubated acini (day 10 of 3D rBM culture). ( a – d ) Nuclear organization and acinar phenotype in controls. Acini exhibit NuMA foci ( a ), an organized endogenous collagen IV-rich BM ( b ), cell–cell localized β-catenin ( c ), and dispersed acetylated H4 histone ( d ). ( e – h ) Effects of TSA on nuclear architecture and acinar phenotype. After 24 hr of TSA treatment (40 nM), > 55% of the cells entered the cell cycle, as indicated by an increase in Ki-67 labeling index ( m ) and the appearance of mitotic cells (arrow in e ). NuMA was uniformly distributed in the nuclei ( e ), collagen IV disappeared ( f ), β-catenin was released from the cell–cell interface ( g ), and the pattern of histone H4 acetylation was altered ( h ). ( i – l ) Effects of mAb-induced NuMA foci disruption on nuclear organization and acinar phenotype. Introduction of a NuMA mAb into the nuclei of the acini by using reversible digitonin permeabilization led to the disruption of NuMA foci ( i ), degradation of the collagen IV-rich BM (arrows in j ), and the nuclear marginalization of acetylated H4 histone ( l ). There was no consistent alteration observed for β-catenin other than increased basal labeling ( k ). These effects were not observed with mock IgGs or mAbs to lamins A/C or B. ( n ) BM degradation after mAb-induced NuMA disruption in acini. Analysis of the percentage of acini with intact collagen IV-rich BMs in relation to control/digitonin-permeabilized (DP) acini ( a ), mock-IgG mAb-treated/DP acini ( b ), NuMA mAb-treated/nonpermeabilized acini ( c ), NuMA mAb-treated/DP acini ( d ), NuMA mAb-treated/DP acini + the metalloproteinase inhibitor GM6001 ( e ), NuMA mAb-treated/DP acini + the inactive metalloproteinase inhibitor GM1210 ( f ), NuMA mAb-treated/DP acini + the uPA inhibitor, aprotinin ( g ), and Lamin B mAb-treated/DP acini ( h ). Acini ( > 35%) degraded their endogenous BMs in response to disruption of NuMA ( d ). The BM loss could be rescued by treatment with the metalloproteinase inhibitor GM6001 ( e ), but not its inactive analogue ( f ) or a uPA protease inhibitor ( g ). (Bar = 10 μm.)
Figure Legend Snippet: Cross-modulation between chromatin structure, NM organization, and the acinar phenotype. Confocal fluorescence images (0.2-μm optical sections) of NuMA ( a , e , and i ), collagen IV ( b , f , and j ), β-catenin ( c , g , and k ), and acetylated histone H4 ( d , h , and l ) in control, trichostatin A (TSA)-treated, and NuMA mAb-incubated acini (day 10 of 3D rBM culture). ( a – d ) Nuclear organization and acinar phenotype in controls. Acini exhibit NuMA foci ( a ), an organized endogenous collagen IV-rich BM ( b ), cell–cell localized β-catenin ( c ), and dispersed acetylated H4 histone ( d ). ( e – h ) Effects of TSA on nuclear architecture and acinar phenotype. After 24 hr of TSA treatment (40 nM), > 55% of the cells entered the cell cycle, as indicated by an increase in Ki-67 labeling index ( m ) and the appearance of mitotic cells (arrow in e ). NuMA was uniformly distributed in the nuclei ( e ), collagen IV disappeared ( f ), β-catenin was released from the cell–cell interface ( g ), and the pattern of histone H4 acetylation was altered ( h ). ( i – l ) Effects of mAb-induced NuMA foci disruption on nuclear organization and acinar phenotype. Introduction of a NuMA mAb into the nuclei of the acini by using reversible digitonin permeabilization led to the disruption of NuMA foci ( i ), degradation of the collagen IV-rich BM (arrows in j ), and the nuclear marginalization of acetylated H4 histone ( l ). There was no consistent alteration observed for β-catenin other than increased basal labeling ( k ). These effects were not observed with mock IgGs or mAbs to lamins A/C or B. ( n ) BM degradation after mAb-induced NuMA disruption in acini. Analysis of the percentage of acini with intact collagen IV-rich BMs in relation to control/digitonin-permeabilized (DP) acini ( a ), mock-IgG mAb-treated/DP acini ( b ), NuMA mAb-treated/nonpermeabilized acini ( c ), NuMA mAb-treated/DP acini ( d ), NuMA mAb-treated/DP acini + the metalloproteinase inhibitor GM6001 ( e ), NuMA mAb-treated/DP acini + the inactive metalloproteinase inhibitor GM1210 ( f ), NuMA mAb-treated/DP acini + the uPA inhibitor, aprotinin ( g ), and Lamin B mAb-treated/DP acini ( h ). Acini ( > 35%) degraded their endogenous BMs in response to disruption of NuMA ( d ). The BM loss could be rescued by treatment with the metalloproteinase inhibitor GM6001 ( e ), but not its inactive analogue ( f ) or a uPA protease inhibitor ( g ). (Bar = 10 μm.)

Techniques Used: Fluorescence, Incubation, Labeling, Protease Inhibitor

Related Articles

Immunoprecipitation:

Article Title: Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis
Article Snippet: .. Antibodies used for immunoprecipitation were diacetylated histone H3 (Upstate Biotechnology, 06-599), tetraacetylated histone H4 (Upstate Biotechnology, 06-866), CBP (Santa Cruz Biotechnology Inc., A-22), histone H3 trimethylated lysine 27 (Abcam, ab6002), histone H3 trimethylated lysine 9 (Abcam, ab8893), histone H3 dimethylated lysine 4 (Abcam, ab7766), P300 (Santa Cruz Biotechnology Inc., C-20, SC-585X), USF1 (Santa Cruz Biotechnology Inc., H-86, sc-8983), USF2 (Santa Cruz Biotechnology Inc., C-20, sc-862), and Brg1/SMARCA4 (Santa Cruz Biotechnology Inc., H-88, SC-10768X). .. After elution and extraction, immunoprecipitated DNA was analyzed by quantitative real-time PCR (iCycler, Bio-Rad) using primers shown in Supplemental Table 1.

Article Title: Epigenetic Regulation of Tumor Necrosis Factor Alpha ▿
Article Snippet: .. Chromatin was immunoprecipitated overnight with antibodies for total histone H3 (05-928), acetylated histone H3 (06-599), or acetylated histone H4 (06-866) (Upstate Biotechnology, Waltham, MA) or dimethylated histone H3K4 (ab7766), trimethylated histone H3K9 (ab8898), or trimethylated histone H3K4 (ab8580) (Abcam, Cambridge, MA). .. A negative control antibody (anti-glutathione S -transferase [GST]; Abcam) was always included but never gave a significant signal and is omitted from some of the figures for simplicity.

Incubation:

Article Title: Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia
Article Snippet: .. The slides were then incubated with antibodies that specifically recognize the acetylated forms of histone H3 and histone H4 (Upstate Biotechnology Inc., Lake Placid, New York, USA) for 1 hour. .. Staining for the PML protein was carried out as described previously ( , ).

Article Title: FoxA Proteins Regulate H19 Endoderm Enhancer E1 and Exhibit Developmental Changes in Enhancer Binding In Vivo
Article Snippet: .. After preclearance with salmon sperm DNA-protein A agarose, samples were incubated overnight at 4°C with rabbit polyclonal antibodies to FoxA1 and FoxA2 (gifts from Robert Costa, University of Illinois at Chicago), C/EBPα, C/EBPδ proteins (obtained from Santa Cruz Biotechnology), or acetylated histone H4 (Upstate Biotechnology). ..

Derivative Assay:

Article Title: A PHD FINGER DOMAIN IN RAG-2 THAT BINDS HYPERMETHYLATED LYSINE 4 OF HISTONE H3 IS NECESSARY FOR EFFICIENT V(D)J REARRANGEMENT
Article Snippet: .. Biotinylated peptides derived from histone H3 were purchased from Upstate Biotechnology. .. Whole cell lysates were prepared from 293T cells by lysis in a buffer containing 50 mM Tris (pH 7.5), 300 mM NaCl, 1 mM PMSF, 1% NP-40, 1% deoxycholic acid, 0.1% SDS, and a cocktail of protease inhibitors (Roche).

Purification:

Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor
Article Snippet: .. Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein. .. Purified KSHV v-cyclin protein was included as a control.

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    Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular <t>histone</t> H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.
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    SHP interacts with mSin3A-Swi/Snf-Brm through its C-terminal repression domain in vitro. (A) Schematic diagrams of GST, full-length (FL) GST-SHP, and deletion mutants. RID and RD, receptor interacting and intrinsic repression domains, respectively; aa, amino acids. (B) 35 S-labeled Brm and 35 S-labeled mSin3A were synthesized in vitro, and GST pull-down assays were performed as described in Materials and Methods. Input, 20% of the total 35 S-labeled proteins. (C) Swi/Snf complexes were purified using anti-Flag M2 agarose from nuclear extracts of HeLa cells stably expressing Flag-Ini 1 (F:Ini) (BAF47). GST or GST-SHP was incubated with purified Swi/Snf complex, and association of the complex with SHP was detected by Western blotting. Input, 20% of the amounts used in the binding reaction. (D and E) GST-SHP or the GST-SHP mutants were incubated with purified Swi/Snf complex (D) or HepG2 nuclear extracts (E), and association of the complex with SHP was analyzed by Western blotting using anti-Flag M2 antibody (D) or antisera against Brm, BAF155, mSin3A, and <t>HDAC-1</t> (E).
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    Monomethylated H4K20 is strongly reduced in PR-Set7 brains. (a) Wild-type (WT) and PR-Set7/PR-Set7 third-instar larval brains were stained with Hoechst. The two strongly staining rings (dense nuclei) observed in wild type are the optic lobes. (b) Western blots of extracts from wild-type and PR-Set7/PR-Set7 third-instar larval brains probed with anti–mono-, anti–di-, and anti–trimethylated H4K20 (mono-, di-, and tri-me), anti–histone H4, and anti-lamin antibodies. The intensity of the bands was quantified by ImageJ, and the value of mono-, di-, or trimethylated H4K20 was normalized to the values of both <t>histone</t> H4 and lamin (see Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200607178/DC1 ). The graph shows the ratio of PR-Set7/PR-Set7 mutant to wild-type values. Error bars show two SDs ( n = 3). (c) Neuroblasts were costained with anti–monomethylated H4K20 (mono-me; red) and anti-PH3 antibodies (green). DNA was stained with Hoechst (blue). (d) Monomethylated H4K20 (red) is distributed all along the chromosomes. Bars: (a) 100 μm; (c and d) 5 μm.
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    Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Over Expression, HAT Assay, Transfection, Mutagenesis

    Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Immunofluorescence, In Vivo, Staining, Microscopy

    Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Inhibition, HAT Assay, Activity Assay, Recombinant, Purification, Radioactivity, Labeling, Incubation

    Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: In Vivo, Expressing

    SHP interacts with mSin3A-Swi/Snf-Brm through its C-terminal repression domain in vitro. (A) Schematic diagrams of GST, full-length (FL) GST-SHP, and deletion mutants. RID and RD, receptor interacting and intrinsic repression domains, respectively; aa, amino acids. (B) 35 S-labeled Brm and 35 S-labeled mSin3A were synthesized in vitro, and GST pull-down assays were performed as described in Materials and Methods. Input, 20% of the total 35 S-labeled proteins. (C) Swi/Snf complexes were purified using anti-Flag M2 agarose from nuclear extracts of HeLa cells stably expressing Flag-Ini 1 (F:Ini) (BAF47). GST or GST-SHP was incubated with purified Swi/Snf complex, and association of the complex with SHP was detected by Western blotting. Input, 20% of the amounts used in the binding reaction. (D and E) GST-SHP or the GST-SHP mutants were incubated with purified Swi/Snf complex (D) or HepG2 nuclear extracts (E), and association of the complex with SHP was analyzed by Western blotting using anti-Flag M2 antibody (D) or antisera against Brm, BAF155, mSin3A, and HDAC-1 (E).

    Journal: Molecular and Cellular Biology

    Article Title: Role of an mSin3A-Swi/Snf Chromatin Remodeling Complex in the Feedback Repression of Bile Acid Biosynthesis by SHP

    doi: 10.1128/MCB.24.17.7707-7719.2004

    Figure Lengend Snippet: SHP interacts with mSin3A-Swi/Snf-Brm through its C-terminal repression domain in vitro. (A) Schematic diagrams of GST, full-length (FL) GST-SHP, and deletion mutants. RID and RD, receptor interacting and intrinsic repression domains, respectively; aa, amino acids. (B) 35 S-labeled Brm and 35 S-labeled mSin3A were synthesized in vitro, and GST pull-down assays were performed as described in Materials and Methods. Input, 20% of the total 35 S-labeled proteins. (C) Swi/Snf complexes were purified using anti-Flag M2 agarose from nuclear extracts of HeLa cells stably expressing Flag-Ini 1 (F:Ini) (BAF47). GST or GST-SHP was incubated with purified Swi/Snf complex, and association of the complex with SHP was detected by Western blotting. Input, 20% of the amounts used in the binding reaction. (D and E) GST-SHP or the GST-SHP mutants were incubated with purified Swi/Snf complex (D) or HepG2 nuclear extracts (E), and association of the complex with SHP was analyzed by Western blotting using anti-Flag M2 antibody (D) or antisera against Brm, BAF155, mSin3A, and HDAC-1 (E).

    Article Snippet: We also detected BAF155 and BAF57 in mSin3A precipitates and HDAC-1 in the anti-Brm immunoprecipitates (data not shown).

    Techniques: In Vitro, Labeling, Synthesized, Purification, Stable Transfection, Expressing, Incubation, Western Blot, Binding Assay

    Action of SHP on negative feedback regulation of CYP7A1 in bile acid signaling. Bile acid-induced SHP interacts with the mSin3A/Swi/Snf-Brm complex in cells and recruits the complex to the endogenous human CYP7A1 promoter (A), which results in chromatin remodeling at the promoter chromatin (B). (A) Bile acid treatment also causes dissociation of the p300/CBP/SRC-1 coactivator HAT complex from the promoter. (B) Recruitment of mSin3A/HDAC-1 and dissociation of HATs lead to histone deacetylation at the promoter. Bile acid-induced chromatin remodeling and histone deacetylation both contribute to transcriptional repression after bile acid treatment. (A) The bile acid-activated JNK or ligand-activated PXR (SXR) pathway may contribute to bile acid-mediated CYP7A1 repression independent of the SHP pathway, but the details of the mechanisms are not known, so they are indicated with dashed lines.

    Journal: Molecular and Cellular Biology

    Article Title: Role of an mSin3A-Swi/Snf Chromatin Remodeling Complex in the Feedback Repression of Bile Acid Biosynthesis by SHP

    doi: 10.1128/MCB.24.17.7707-7719.2004

    Figure Lengend Snippet: Action of SHP on negative feedback regulation of CYP7A1 in bile acid signaling. Bile acid-induced SHP interacts with the mSin3A/Swi/Snf-Brm complex in cells and recruits the complex to the endogenous human CYP7A1 promoter (A), which results in chromatin remodeling at the promoter chromatin (B). (A) Bile acid treatment also causes dissociation of the p300/CBP/SRC-1 coactivator HAT complex from the promoter. (B) Recruitment of mSin3A/HDAC-1 and dissociation of HATs lead to histone deacetylation at the promoter. Bile acid-induced chromatin remodeling and histone deacetylation both contribute to transcriptional repression after bile acid treatment. (A) The bile acid-activated JNK or ligand-activated PXR (SXR) pathway may contribute to bile acid-mediated CYP7A1 repression independent of the SHP pathway, but the details of the mechanisms are not known, so they are indicated with dashed lines.

    Article Snippet: We also detected BAF155 and BAF57 in mSin3A precipitates and HDAC-1 in the anti-Brm immunoprecipitates (data not shown).

    Techniques: HAT Assay

    Genome-wide location analysis of histone H3-K79, H3-K36, and H3-K4 methylation in human coding regions. (A) Venn diagram comparing candidate genes from H3-K4, H3-K36, and H3-K79 dimethylation data sets. The data from three experiments were averaged, and

    Journal:

    Article Title: Mapping Global Histone Methylation Patterns in the Coding Regions of Human Genes †

    doi: 10.1128/MCB.25.11.4650-4661.2005

    Figure Lengend Snippet: Genome-wide location analysis of histone H3-K79, H3-K36, and H3-K4 methylation in human coding regions. (A) Venn diagram comparing candidate genes from H3-K4, H3-K36, and H3-K79 dimethylation data sets. The data from three experiments were averaged, and

    Article Snippet: Anti-dimethylated-histone H3-K4, K9, K27, K36, K79, H4-K20, anti-trimethylated-histone H3-K4, K9 and anti-acetyl-histone H3-K9/K14 were purchased from Upstate Biotechnology (Lake Placid, NY).

    Techniques: Genome Wide, Methylation

    Genome-wide location analysis of histone H3-K4 and H3-K9 dimethylation (Me2) in human coding regions. (A) Virtual gene array images of histone H3-K4Me2 and H3-K9Me2 patterns. The human 1.7k cDNA arrays used contain 1,781 double-spotted well-characterized

    Journal:

    Article Title: Mapping Global Histone Methylation Patterns in the Coding Regions of Human Genes †

    doi: 10.1128/MCB.25.11.4650-4661.2005

    Figure Lengend Snippet: Genome-wide location analysis of histone H3-K4 and H3-K9 dimethylation (Me2) in human coding regions. (A) Virtual gene array images of histone H3-K4Me2 and H3-K9Me2 patterns. The human 1.7k cDNA arrays used contain 1,781 double-spotted well-characterized

    Article Snippet: Anti-dimethylated-histone H3-K4, K9, K27, K36, K79, H4-K20, anti-trimethylated-histone H3-K4, K9 and anti-acetyl-histone H3-K9/K14 were purchased from Upstate Biotechnology (Lake Placid, NY).

    Techniques: Genome Wide

    Monomethylated H4K20 is strongly reduced in PR-Set7 brains. (a) Wild-type (WT) and PR-Set7/PR-Set7 third-instar larval brains were stained with Hoechst. The two strongly staining rings (dense nuclei) observed in wild type are the optic lobes. (b) Western blots of extracts from wild-type and PR-Set7/PR-Set7 third-instar larval brains probed with anti–mono-, anti–di-, and anti–trimethylated H4K20 (mono-, di-, and tri-me), anti–histone H4, and anti-lamin antibodies. The intensity of the bands was quantified by ImageJ, and the value of mono-, di-, or trimethylated H4K20 was normalized to the values of both histone H4 and lamin (see Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200607178/DC1 ). The graph shows the ratio of PR-Set7/PR-Set7 mutant to wild-type values. Error bars show two SDs ( n = 3). (c) Neuroblasts were costained with anti–monomethylated H4K20 (mono-me; red) and anti-PH3 antibodies (green). DNA was stained with Hoechst (blue). (d) Monomethylated H4K20 (red) is distributed all along the chromosomes. Bars: (a) 100 μm; (c and d) 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: Aberrant monomethylation of histone H4 lysine 20 activates the DNA damage checkpoint in Drosophila melanogaster

    doi: 10.1083/jcb.200607178

    Figure Lengend Snippet: Monomethylated H4K20 is strongly reduced in PR-Set7 brains. (a) Wild-type (WT) and PR-Set7/PR-Set7 third-instar larval brains were stained with Hoechst. The two strongly staining rings (dense nuclei) observed in wild type are the optic lobes. (b) Western blots of extracts from wild-type and PR-Set7/PR-Set7 third-instar larval brains probed with anti–mono-, anti–di-, and anti–trimethylated H4K20 (mono-, di-, and tri-me), anti–histone H4, and anti-lamin antibodies. The intensity of the bands was quantified by ImageJ, and the value of mono-, di-, or trimethylated H4K20 was normalized to the values of both histone H4 and lamin (see Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200607178/DC1 ). The graph shows the ratio of PR-Set7/PR-Set7 mutant to wild-type values. Error bars show two SDs ( n = 3). (c) Neuroblasts were costained with anti–monomethylated H4K20 (mono-me; red) and anti-PH3 antibodies (green). DNA was stained with Hoechst (blue). (d) Monomethylated H4K20 (red) is distributed all along the chromosomes. Bars: (a) 100 μm; (c and d) 5 μm.

    Article Snippet: Rabbit polyclonal anti-monomethylated, anti-dimethylated, anti-trimethylated, and histone H4 antibodies (Upstate Biotechnology) were used at 1:1,000 dilution ( ).

    Techniques: Staining, Western Blot, Mutagenesis