Journal: Oncology Letters

Article Title: Metformin inhibits histone H2B monoubiquitination and downstream gene transcription in human breast cancer cells

doi: 10.3892/ol.2014.2158

Figure Lengend Snippet: Metformin activates AMPK and suppresses histone H2B monoubiquitination and downstream gene transcription. (A) The T47D cells were treated with 4mM metformin for 12 h, and then the cells were lysed and immnoblotted to determine AMPKα1 T172 phosphorylation and H2B K120 monoubiquitination. β-actin was used as a loading control. (B) The T47D cells were treated with 4mM metformin for 12 h, then mRNA was extracted and the transcription level of p21, cyclin D1 and Tulp4 were examined by quantitative (q)PCR. Data are presented as the mean ± standard deviation (n=3). DMSO, dimethyl sulfoxide; AMPK, 5′-adenosine monophosphate-activated protein kinase.

Article Snippet: Monoclonal rabbit anti-human anti-phospho-acetyl-CoA carboxylase (Ser79) (pACC1), monoclonal rabbit anti-human anti-phospho-AMPKα (Thr172)(p-AMPKα1), monoclonal rabbit anti-human anti-ubiquityl-histone H2B (Lys120) (H2B K120ub) and rabbit monoclonal anti-human anti-AMPKα1 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), with the exception of monoclonal mouse anti-human β-actin, was purchased from Sigma-Aldrich.

Techniques: Standard Deviation

Journal: Oncology Letters

Article Title: Metformin inhibits histone H2B monoubiquitination and downstream gene transcription in human breast cancer cells

doi: 10.3892/ol.2014.2158

Figure Lengend Snippet: Metformin effect on T47D cell proliferation depends on AMPK. (A) Western blotting showing the AMPKα1 protein level in the T47D cells following transfection with AMPKα1 siRNA. β-actin was used as a loading control. (B) The T47D cells transfected with control siRNA or AMPKα1 siRNA were treated with 4 mM metformin for 12 h, and the cell proliferation was examined by Cell Titer-Blue cell counting. Data are presented as the mean ± standard deviation (n=3). DMSO, dimethyl sulfoxide; AMPK, 5′-adenosine monophosphate-activated protein kinase.

Article Snippet: Monoclonal rabbit anti-human anti-phospho-acetyl-CoA carboxylase (Ser79) (pACC1), monoclonal rabbit anti-human anti-phospho-AMPKα (Thr172)(p-AMPKα1), monoclonal rabbit anti-human anti-ubiquityl-histone H2B (Lys120) (H2B K120ub) and rabbit monoclonal anti-human anti-AMPKα1 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), with the exception of monoclonal mouse anti-human β-actin, was purchased from Sigma-Aldrich.

Techniques: Western Blot, Transfection, Cell Counting, Standard Deviation

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Synthetic lethal/sick phenotypes of rkr1∆ hta1 and rkr1∆ htb1 mutants were assessed through ten-fold serial dilution assays. Double mutant cells, as well as control RKR1 hta1 and RKR1 htb1 cells, were plated on SC-His medium as a growth control and on SC-His + 5-FOA medium to select for histone mutant plasmids and against the URA3 -marked HTA1-HTB1 plasmid. Library plasmids were transformed into the rkr1∆ strain KY981 and wild-type strain KY943. KY2265 and KY2249 were used as respective negative and positive growth controls on 5-FOA plates. ( B) X-ray crystal structure of the nucleosome, denoting histones H2A, H2B, H3, and H4 in cyan, green, yellow, and white, respectively. As depicted in red, the majority of histone residues identified in the rkr1∆ synthetic lethality screen form a surface-exposed patch on the nucleosome. (C) Electrostatic potential (red is negative, blue is positive) of the nucleosome core particle. This figure was created using Pymol (PDB 1ID3 ).

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques: Serial Dilution, Mutagenesis, Plasmid Preparation, Transformation Assay

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Western analysis of H2B K123ub, as well as total H2B and G6PDH, both of which served as loading controls. KY1599 ( rtf1∆ ) was used as a negative control. The bar graph shows H2B K123ub levels normalized to total H2B levels. These relative H2B K123ub levels were normalized to the wild-type value. Error bars represent SEM of three independent experiments. (B) ChIP analysis of H2B K123ub occupancy at the 5'- and 3'- ends of PYK1 and PMA1 and at a nontranscribed region, TELVI . H2B K123ub ChIP values were normalized to total H2B ChIP values. The error bars represent SEM of three independent experiments.

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques: Western Blot, Negative Control

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Western analysis of H2B, H2A, and H3 levels in the H2A mutant strains. G6PDH levels served as a loading control. (B, C, D) Analysis of H2B (KY2674), H2A (KY2675), and H3 (KY943) occupancy at the 5’- and 3’ ends of PYK1 and at TELVI by ChIP. The error bars denote SEM of three independent experiments. (E) Northern analysis assessing the effects of the H2A substitutions and H2B-K123R (KY2044) on SER3 , SRG1 , FLO8 and FLO8 cryptic transcript levels. Upper band (*) corresponds to the full-length FLO8 transcript and the lower band (**) corresponds to the cryptic internally initiated transcript. The spt6-1004 (KY2678) and spt16-197 (KY2679) temperature-sensitive alleles serve as positive controls for cryptic initiation and SER3 derepression and are isogenic to the wild-type strain KY2677. SCR1 was used as a loading control.

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques: Western Blot, Mutagenesis, Northern Blot

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Western analysis of H2B K123ub and total H2B in the indicated wild-type and H2A mutant strains. (B) The relative levels of H2B K123ub between ubp8∆ (KY2086) and UBP8 (KY943) backgrounds are shown. The ratio of H2B K123ub in ubp8∆ to H2B K123ub in UBP8 was calculated after normalizing to total H2B levels. To determine the fold change of H2B K123ub levels between H2A mutants, these ratios were normalized to the wild-type H2A background. Error bars represent SEMs of three biological replicates. (C) Western analysis of H3 K4me 3 and H3 K79me 2/3 levels in H2A mutants in the presence or absence of UBP8 . Total H3 served as a loading control.

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques: Western Blot, Mutagenesis

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Diagram of experimental procedure. Cells were grown in medium containing 2% galactose (zero time point) and then 2% glucose was added to shut off transcription. Samples were taken at zero, two, four, and eight-minute time points for cross-linking. ChIP of the Rpb3 subunit of Pol II across YLR454W was performed in (B) wild type (WT), (C) H2A-E65A, (D) H2A-L66A, (E) H2A-E93A, and (F) H2B-K123R strains, which were transformants of KY2676. Values were normalized to the zero time point for each locus. Error bars represent SEM of three biological replicates.

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques:

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Western analysis of H2B K123ub and total H2B in the indicated wild-type and H2A mutant strains. (B) The relative levels of H2B K123ub between ubp8∆ (KY2086) and UBP8 (KY943) backgrounds are shown. The ratio of H2B K123ub in ubp8∆ to H2B K123ub in UBP8 was calculated after normalizing to total H2B levels. To determine the fold change of H2B K123ub levels between H2A mutants, these ratios were normalized to the wild-type H2A background. Error bars represent SEMs of three biological replicates. (C) Western analysis of H3 K4me 3 and H3 K79me 2/3 levels in H2A mutants in the presence or absence of UBP8 . Total H3 served as a loading control.

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques: Western Blot, Mutagenesis

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Western blots were probed with antibodies to detect di- and tri-methylation of H3 K4, K36, and K79 as indicated. Total H3 and G6PDH levels were used as loading controls. Strains lacking SET1 (KY1715), DOT1 (KY1717), and SET2 (KY1716) show the specificity of the antibodies used. (B, C) ChIP analysis of methylated H3 K79 and K4 at PYK1 , PMA1 and TELVI . The H3 K79 antibody used in these experiments can detect both the di- and tri-methylated states (Abcam). The error bars represent SEM of three independent experiments.

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques: Western Blot, Methylation

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Western analysis of H2B K123ub and total H2B in the indicated wild-type and H2A mutant strains. (B) The relative levels of H2B K123ub between ubp8∆ (KY2086) and UBP8 (KY943) backgrounds are shown. The ratio of H2B K123ub in ubp8∆ to H2B K123ub in UBP8 was calculated after normalizing to total H2B levels. To determine the fold change of H2B K123ub levels between H2A mutants, these ratios were normalized to the wild-type H2A background. Error bars represent SEMs of three biological replicates. (C) Western analysis of H3 K4me 3 and H3 K79me 2/3 levels in H2A mutants in the presence or absence of UBP8 . Total H3 served as a loading control.

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques: Western Blot, Mutagenesis

Journal: PLoS Genetics

Article Title: The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005420

Figure Lengend Snippet: (A) Western analysis of H2B K123ub and total H2B in the indicated wild-type and H2A mutant strains. (B) The relative levels of H2B K123ub between ubp8∆ (KY2086) and UBP8 (KY943) backgrounds are shown. The ratio of H2B K123ub in ubp8∆ to H2B K123ub in UBP8 was calculated after normalizing to total H2B levels. To determine the fold change of H2B K123ub levels between H2A mutants, these ratios were normalized to the wild-type H2A background. Error bars represent SEMs of three biological replicates. (C) Western analysis of H3 K4me 3 and H3 K79me 2/3 levels in H2A mutants in the presence or absence of UBP8 . Total H3 served as a loading control.

Article Snippet: For histone ChIPs, sheared chromatin was incubated overnight at 4°C with antibodies specific to H2B, (0.5 μl, Active Motif, 39237), human H2B K120ub (2.5 μl, Cell Signaling 5546), H3 K4me 3 (2.5 μl, Abcam ab8580), H3 K79me 2/3 (2.5 μl, Abcam ab2621), or total H3 (5 μl) [ ].

Techniques: Western Blot, Mutagenesis