Journal: bioRxiv

Article Title: Alternative splicing links histone modifications to stem cell fate decision

doi: 10.1101/181875

Figure Lengend Snippet: (A) Genome browser view shows the AS event and H3K36me3 signals of PBX1 upon hESC differentiation. The green horizontal bars below the ChIP-seq tracks indicate the narrow peaks called by MACS2. (B) The inclusion level for exon 7 of PBX1 is significantly correlated to the H3K36me3 signals over this exon across cell lineages. (C) The sequence difference of three protein isoforms of PBX1 and the main functional domains. (D) The relative expressions of PBX1a and PBX1b in 56 cells/tissues, representing the differential expressions of two isoforms in three groups based on their developmental states. (E) The expression levels of NANOG and OCT4 genes are negatively correlated with the expression of PBX1b. (F) The expression levels of PSIP1 and SRSF1 show significant positive correlations with the expression level of PBX1a. Also, see Figure S9-S10 .

Article Snippet: The SRSF1-bound PSIP1 protein (also known as LEDGF) level was determined with the PSIP1 antibody (Human LEDGF Antibody, R&D systems) using Western blot as described above.

Techniques: ChIP-sequencing, Sequencing, Functional Assay, Expressing

Journal: bioRxiv

Article Title: Alternative splicing links histone modifications to stem cell fate decision

doi: 10.1101/181875

Figure Lengend Snippet: (A) qRT-PCR and western blot show the expression levels of Yamanaka factors in H1, MSC and IMR90 cells. Whiskers denote the standard deviations of three replicates. (B) RT-PCR and western blot show the isoform switches between PBX1a and PBX1b from H1 cells to differentiated cells. (C) i . ChIP-PCR shows the differential binding of PBX1b to NANOG promoter in H1 cells and differentiated cells; ii . ChIP-PCR shows the reduced H3K36me3 signal in differentiated cells; iii. ChIP-PCR shows the differential recruitment of PSIP1 to the exon 7 of PBX1. (D) RIP-PCR show the differential recruitment of SRSF1 around the exon 7 of PBX1. (E) Co-IP shows the overall physical interaction between PSIP1 and SRSF1 in all studied cell types. (F) The mechanism by which H3K36me3 is linked to cell fate decision by regulating the isoform switch of PBX1, which functions upstream of the pluripotency regulatory network. Also, see Figures S9-S10 .

Article Snippet: The SRSF1-bound PSIP1 protein (also known as LEDGF) level was determined with the PSIP1 antibody (Human LEDGF Antibody, R&D systems) using Western blot as described above.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1101/2021.01.06.425580

Figure Lengend Snippet: A , Representative ChIP-seq tracks of H3K36me2, LEDGF, and HDGF2 chromatin co-occupancy in H3-WT (DIPG10 and pcGBM2) and H3K27M (DIPG4, DIPG6, and DIPG13) cells. B , Violin plots showing enrichment of H3K36me2, LEDGF, or HDGF2, respectively, for genes categorized as low, mid, or high based on their mRNA expression. The central thick dash line indicates the mean value of each plot; the upper thin dash line indicates the top 25% percentile and the lower one for the bottom 25% percentile. C , Metaprofile plots of H3K27me3, H3K36me2, LEDGF, and HDGF2 ChIP-seq data in HEK293T cells ectopically expressing wildtype histone H3 (H3-WT) or H3K27M for 24 hrs. Genes exhibiting loss of H3K27me3 by H3K27M were categorized by its loss upstream or downstream from transcription start sites (TSS) and subsequent changes in H3K36me2, LEDGF, and HDGF2 occupancies were presented below. Data were presented within a 500-kb window upstream or downstream from TSS. D , Top, metaprofile plots of LEDGF and HDGF2 occupancy in wildtype or NSD2-KO DIPG13 cells (clone#7 and clone#10). Bottom, representative ChIP-seq tracks for the top panel. Overlayed panels were presented at the bottom to better illustrate the differences. ****p<0.0001 by Student’s t-test.

Article Snippet: The antibodies used in this study are listed below: LEDGF (Proteintech) Rabbit Polyclonal, Cat # 25504-1-AP HDGF2 (Proteintech) Rabbit Polyclonal, Cat # 15134-1-AP H3K27me3 (Cell Signaling) Rabbit Monoclonal C36B11, Cat # 9733 H3K36me2 (Cell Signaling) Rabbit Monoclonal C75H12, Cat # 2901 H3K36me3 (Cell Signaling) Rabbit Monoclonal D5A7, Cat # 4909 NSD1 (UC Davis/NIH NeuroMab Facility) Mouse Monoclonal,N312/10 NSD2 (Millipore) Mouse Monoclonal 29D1, Cat# MABE191 NSD3 (Cell Signaling) Rabbit Monoclonal D4N9N, Cat # 92056 ASH1L (Bethyl Laboratories) Rabbit Polyclonal, Cat # A301-748A SETD2 (Bio-Rad) Mouse Monoclonal OTI1E1, Cat # VMA00449 GAPDH (Cell Signaling) Rabbit Monoclonal D16H11, Cat # 5174 Histone H3 (Abcam) Rabbit Monoclonal EPR16987, Cat # ab176842 Anti-Flag (Sigma) Mouse Monoclonal M2, Cat # F1804 H2Av (Active Motif) Rabbit Polyclonal, Cat # 39715

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1101/2021.01.06.425580

Figure Lengend Snippet: A , Top, a schematic illustration of the design of Cell Penetrating Peptide (CPP). A HIV-based cell entry peptide was linked to a H3K36me2 peptide (histone H3 21-43 a.a., Cargo Peptide) by a disulfide bond. Bottom, Amino acid (a.a.) sequence of H3K36me2 linked CPP. B . A CellTiter-Glo® cell survival assay for H3-WT (DIPG10 and pcGBM2) and H3K27M (DIPG6 and DIPG13) cells treated with control (vehicle only) or H3K36me2-CPP. Cells were assayed at 72 hours after dosing with a titration of control or H3K36me2-CPP and data were presented by ratios of CellTiter-Glo signals in control versus H3K36me2-CPP treated cells. C , A metaprofile of ChIP-seq analysis for changes in LEDGF and HDGF2 occupancy of genes decorated by H3K36me2 and H3K36me3 in DIPG13 cells treated with a control vehicle (Red) or a H3K36me2-CPP (Blue).

Article Snippet: The antibodies used in this study are listed below: LEDGF (Proteintech) Rabbit Polyclonal, Cat # 25504-1-AP HDGF2 (Proteintech) Rabbit Polyclonal, Cat # 15134-1-AP H3K27me3 (Cell Signaling) Rabbit Monoclonal C36B11, Cat # 9733 H3K36me2 (Cell Signaling) Rabbit Monoclonal C75H12, Cat # 2901 H3K36me3 (Cell Signaling) Rabbit Monoclonal D5A7, Cat # 4909 NSD1 (UC Davis/NIH NeuroMab Facility) Mouse Monoclonal,N312/10 NSD2 (Millipore) Mouse Monoclonal 29D1, Cat# MABE191 NSD3 (Cell Signaling) Rabbit Monoclonal D4N9N, Cat # 92056 ASH1L (Bethyl Laboratories) Rabbit Polyclonal, Cat # A301-748A SETD2 (Bio-Rad) Mouse Monoclonal OTI1E1, Cat # VMA00449 GAPDH (Cell Signaling) Rabbit Monoclonal D16H11, Cat # 5174 Histone H3 (Abcam) Rabbit Monoclonal EPR16987, Cat # ab176842 Anti-Flag (Sigma) Mouse Monoclonal M2, Cat # F1804 H2Av (Active Motif) Rabbit Polyclonal, Cat # 39715

Techniques: Sequencing, Clonogenic Cell Survival Assay, Control, Titration, ChIP-sequencing

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: INS and INS-derived peptides bind LEDGF/p75 and promote partial dissociation of the IN-LEDGF/p75 complex . (A) LEDGF/p75 was incubated in ELISA plates coated with the indicated peptide or with BSA as a negative control, and binding was determined as described in Methods. Wells containing the buffer carbonate (BC) in the absence of peptide were used as a background control. (B) The IN protein was first bound to the ELISA plates which were then incubated with LEDGF/p75 to obtain LEDGF/p75-IN complex. The complex was then incubated with either the indicated peptide or BSA at the designated IN:peptide (or BSA) ratios. The amount of bound LEDGF/p75 was then determined. (C) Same as (B) but the peptides (or BSA) were added at the same time as LEDGF/p75 to determine competition. (D) Formation of Rev-IN, IN-LEDGF/p75 and Rev-LEDGF/p75 complexes and their dissociation by the INS and INS-derived peptides. Co-IP was performed in lysates obtained from virus-infected cells. All other experimental conditions are described in Methods.

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Negative Control, Binding Assay, Control, Co-Immunoprecipitation Assay, Virus, Infection

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: Binding to LEDGF/p75.

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Binding Assay, Sequencing

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: Effect of INS concentrations on integration and total viral-DNA in infected wt and LEDGF/p75-knockdown HeLa-P4 cells . HeLa P4 and HeLaP4/shp75Cl15 (LEDGF/p75-knockdown) cells were incubated with the indicated concentration of INS and infected with wt or ΔRev HIV-1 at a MOI of 1.0. The average number of integration events per cell (A) and of total viral DNA copies per cell (B) was estimated as described in Methods. Black shading and dark grey shading are LEDGF/p75-knockdown cells infected with WT or ΔRev HIV-1, respectively; white grey shading and white shading are HeLa P4 cells infected with WT or ΔRev HIV-1, respectively. AZT was used at 2 μM concentration.

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Infection, Knockdown, Incubation, Concentration Assay

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: Summary of the results described in Figure 2.

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Virus

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: Effect of increasing HIV-1 MOIs on integration levels in infected wt and LEDGF/p75-knockdown HeLa P4 cells . HeLa P4 and HeLaP4/shp75Cl15 (LEDGF/p75-knockdown) cells were incubated with or without 100 μM INS and infected at the indicated MOIs with (A) WT or (B) ΔRev HIV-1. The average number of integration events per cell was estimated as described in Methods. Black shading and dark grey shading are infected LEDGF/p75-knockdown cells without or with INS treatment, respectively; light grey shading and white shading are infected HeLa P4 cells without or with INS treatment, respectively. AZT was used at 2 μM concentration.

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Infection, Knockdown, Incubation, Concentration Assay

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: Summary of the results described in Figure 3.

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Virus

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: INr peptides stimulate viral cDNA integration in LEDGF/p75-knockdown cells . HeLa P4 (□) and HeLaP4/shp75Cl15 (LEDGF/p75-knockdown) (■) cells were incubated with or without 100 μM INS or INr and infected with wt HIV-1 at a MOI of 1.0. AZT was used at 2 μM concentration. The average number of integration events per cell was estimated as described in Methods.

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Knockdown, Incubation, Infection, Concentration Assay

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: Stimulation of p24 and virus production in LEDGF/p75-knockdown cells by the INS and INr peptides . Sup-T1 and Sup-T1/TL3 (LEDGF/p75-knockdown) cells were incubated with or without 100 μM INS or INr and then infected with wt HIV-1 at a MOI of 0.01. The amount of viral p24 (A) and of infectious virus (B) was estimated every 2 days post-infection (PI) as described in Methods. ■, ● and ▲ represent Sup-T1 cells treated with INS, INr or not treated, respectively; □, ○ and Δ represent LEDGF/p75-knockdown cells treated with INS, INr or not treated, respectively; ◊ are non-infected cells.

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Virus, Knockdown, Incubation, Infection

Journal: Virology Journal

Article Title: Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

doi: 10.1186/1743-422X-7-177

Figure Lengend Snippet: Schematic model of the process of viral cDNA integration in wt and LEDGF/p75-knockdown cells. (A) In LEDGF/p75-knockdown cells, an early expressed Rev (green triangle), from unintegrated viral DNA (brown double line), binds and inactivate all viral IN molecules (red circle) (in both the cytoplasm (light blue) and nucleus (yellow)) before integration occurs (due to the absence of LEDGF/p75 which supports rapid integration). (B) Addition of INS or INr peptides promotes dissociation of the inhibitory Rev-IN complexes, allowing the IN, which bound to viral DNA, to mediate integration into the host genome (black double line).

Article Snippet: Half of the lysate was subjected to SDS-PAGE (an E-PAGETM 48 8 % High-Throughput Pre-Cast Gel System (Invitrogen)) and immunoblotted with either monoclonal anti-Rev antibody (α-Rev) [ ], antiserum raised against IN amino acids 276-288 (α-IN) (NIH AIDS Research & Reference Reagent Program catalog number 758), anti-LEDGF/p75 (α-LEDGF/p75) (R&D Systems, Minneapolis, MN, USA) or anti-actin (α-Actin) antibody (Santa Cruz), and complementary HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) as second antibodies.

Techniques: Knockdown