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MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation.

Chen H, Ruiz PD, Novikov L, Casill AD, Park JW, Gamble MJ - Nat. Struct. Mol. Biol. (2014)

Bottom Line: The histone variant macroH2A1 regulates gene expression important for differentiation, stem-cell reprogramming and tumor suppression.Here, we demonstrate that in primary human cells, macroH2A1 participates in two physically and functionally distinct types of chromatin marked by either H3K27me3 or nine histone acetylations.Through the recruitment of PARP-1, macroH2A1.1 promotes the CBP-mediated acetylation of H2B K12 and K120, which either positively or negatively regulates the expression of macroH2A1-target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.

ABSTRACT
The histone variant macroH2A1 regulates gene expression important for differentiation, stem-cell reprogramming and tumor suppression. Here, we demonstrate that in primary human cells, macroH2A1 participates in two physically and functionally distinct types of chromatin marked by either H3K27me3 or nine histone acetylations. Using RNA sequencing, we found that macroH2A1-regulated genes, which have roles in cancer progression, are specifically found in macroH2A1-containing acetylated chromatin. Of the two macroH2A1 variants, macroH2A1.1 and macroH2A1.2, the former is suppressed in cancer and can interact with PARP-generated poly(ADP-ribose). Through the recruitment of PARP-1, macroH2A1.1 promotes the CBP-mediated acetylation of H2B K12 and K120, which either positively or negatively regulates the expression of macroH2A1-target genes. Although macroH2A1-regulated H2B acetylation is a common feature of primary cells, this regulation is typically lost in cancer cells. Consequently, our results provide insight into macroH2A1.1's role in cancer suppression.

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MacroH2A1 and PARP-1 recruit CBP to mediate acetylation of H2B K12 and K120 and regulate macroH2A1-target gene expression.(a) Immunoblots for H2BK12ac, H2BK120ac, H3K4me3 or H3 of acid-extracted lysates from IMR90 cells expressing shRNA against luciferase (L) as a control or macroH2A1 (M), either untreated, treated with 25 nM of TSA or 75 μM of Sirtinol for 24hrs.(b) ChIP for H2BK12ac, H2BK120ac, macroH2A1 and H3 from IMR90 cells expressing shRNA targeted against luciferase (L) as a control or macroH2A1 (M). Cells were treated with 25 nM of TSA where indicated. The horizontal dotted line, upper limit of the 95% confidence interval of the signal from no-antibody control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(c) Reverse transcription coupled to qPCR (RT-qPCR) of IMR90 cells described in (b) for the indicated genes. Downregulated and upregulated genes are plotted on the left or right y-axis, respectively. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(d) ChIP for CBP at indicated genes from IMR90 cells expressing shRNA against either Luciferase (L) or macroH2A1 (M). Horizontal dotted line, upper limit of a 95% confidence interval of signal from no-antibody control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(e) As for (a) except IMR90 cells treated with or without 10 μM PJ-34 for three days.(f) RT-qPCR for the indicated genes in IMR90 cells treated with or without 20 μM of CBP inhibitor C646 for 3 days. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests. Downregulated and upregulated genes are plotted on the left or right y-axis, respectively.(g) RT-qPCR as described in (f) except IMR90 cells expressing shRNA against luciferase (L) as a control or CBP.
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Figure 6: MacroH2A1 and PARP-1 recruit CBP to mediate acetylation of H2B K12 and K120 and regulate macroH2A1-target gene expression.(a) Immunoblots for H2BK12ac, H2BK120ac, H3K4me3 or H3 of acid-extracted lysates from IMR90 cells expressing shRNA against luciferase (L) as a control or macroH2A1 (M), either untreated, treated with 25 nM of TSA or 75 μM of Sirtinol for 24hrs.(b) ChIP for H2BK12ac, H2BK120ac, macroH2A1 and H3 from IMR90 cells expressing shRNA targeted against luciferase (L) as a control or macroH2A1 (M). Cells were treated with 25 nM of TSA where indicated. The horizontal dotted line, upper limit of the 95% confidence interval of the signal from no-antibody control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(c) Reverse transcription coupled to qPCR (RT-qPCR) of IMR90 cells described in (b) for the indicated genes. Downregulated and upregulated genes are plotted on the left or right y-axis, respectively. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(d) ChIP for CBP at indicated genes from IMR90 cells expressing shRNA against either Luciferase (L) or macroH2A1 (M). Horizontal dotted line, upper limit of a 95% confidence interval of signal from no-antibody control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(e) As for (a) except IMR90 cells treated with or without 10 μM PJ-34 for three days.(f) RT-qPCR for the indicated genes in IMR90 cells treated with or without 20 μM of CBP inhibitor C646 for 3 days. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests. Downregulated and upregulated genes are plotted on the left or right y-axis, respectively.(g) RT-qPCR as described in (f) except IMR90 cells expressing shRNA against luciferase (L) as a control or CBP.

Mentions: To better understand the role of these acetylations in macroH2A1 regulated transcription, we treated macroH2A1-depleted cells with TSA, a class I and II HDAC inhibitor, or sirtinol, a class III HDAC inhibitor. Only TSA could restore global levels of H2B K12 and K120 acetylation back to that of controls, indicating that macroH2A1 opposes the activity of a class I or II HDAC (Fig. 6a). Using ChIP-qPCR analysis, we confirmed that TSA can reverse the loss of H2BK12ac and H2BK120ac in macroH2A1-depleted cells at macroH2A1-regulated genes (Fig. 6b). Functionally, TSA suppresses the effect of macroH2A1 depletion on expression of macroH2A1 target genes (Fig. 6c). Acetylation is usually studied in relation to its positive role in transcriptional regulation, however, our data indicate that acetylation plays a critical role at genes either positively or negatively regulated by macroH2A1. These results indicate that macroH2A1-mediated regulation of H2B K12 and K120 acetylation is a critical feature of macroH2A1-mediated transcriptional regulation.


MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation.

Chen H, Ruiz PD, Novikov L, Casill AD, Park JW, Gamble MJ - Nat. Struct. Mol. Biol. (2014)

MacroH2A1 and PARP-1 recruit CBP to mediate acetylation of H2B K12 and K120 and regulate macroH2A1-target gene expression.(a) Immunoblots for H2BK12ac, H2BK120ac, H3K4me3 or H3 of acid-extracted lysates from IMR90 cells expressing shRNA against luciferase (L) as a control or macroH2A1 (M), either untreated, treated with 25 nM of TSA or 75 μM of Sirtinol for 24hrs.(b) ChIP for H2BK12ac, H2BK120ac, macroH2A1 and H3 from IMR90 cells expressing shRNA targeted against luciferase (L) as a control or macroH2A1 (M). Cells were treated with 25 nM of TSA where indicated. The horizontal dotted line, upper limit of the 95% confidence interval of the signal from no-antibody control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(c) Reverse transcription coupled to qPCR (RT-qPCR) of IMR90 cells described in (b) for the indicated genes. Downregulated and upregulated genes are plotted on the left or right y-axis, respectively. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(d) ChIP for CBP at indicated genes from IMR90 cells expressing shRNA against either Luciferase (L) or macroH2A1 (M). Horizontal dotted line, upper limit of a 95% confidence interval of signal from no-antibody control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(e) As for (a) except IMR90 cells treated with or without 10 μM PJ-34 for three days.(f) RT-qPCR for the indicated genes in IMR90 cells treated with or without 20 μM of CBP inhibitor C646 for 3 days. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests. Downregulated and upregulated genes are plotted on the left or right y-axis, respectively.(g) RT-qPCR as described in (f) except IMR90 cells expressing shRNA against luciferase (L) as a control or CBP.
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Figure 6: MacroH2A1 and PARP-1 recruit CBP to mediate acetylation of H2B K12 and K120 and regulate macroH2A1-target gene expression.(a) Immunoblots for H2BK12ac, H2BK120ac, H3K4me3 or H3 of acid-extracted lysates from IMR90 cells expressing shRNA against luciferase (L) as a control or macroH2A1 (M), either untreated, treated with 25 nM of TSA or 75 μM of Sirtinol for 24hrs.(b) ChIP for H2BK12ac, H2BK120ac, macroH2A1 and H3 from IMR90 cells expressing shRNA targeted against luciferase (L) as a control or macroH2A1 (M). Cells were treated with 25 nM of TSA where indicated. The horizontal dotted line, upper limit of the 95% confidence interval of the signal from no-antibody control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(c) Reverse transcription coupled to qPCR (RT-qPCR) of IMR90 cells described in (b) for the indicated genes. Downregulated and upregulated genes are plotted on the left or right y-axis, respectively. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(d) ChIP for CBP at indicated genes from IMR90 cells expressing shRNA against either Luciferase (L) or macroH2A1 (M). Horizontal dotted line, upper limit of a 95% confidence interval of signal from no-antibody control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(e) As for (a) except IMR90 cells treated with or without 10 μM PJ-34 for three days.(f) RT-qPCR for the indicated genes in IMR90 cells treated with or without 20 μM of CBP inhibitor C646 for 3 days. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests. Downregulated and upregulated genes are plotted on the left or right y-axis, respectively.(g) RT-qPCR as described in (f) except IMR90 cells expressing shRNA against luciferase (L) as a control or CBP.
Mentions: To better understand the role of these acetylations in macroH2A1 regulated transcription, we treated macroH2A1-depleted cells with TSA, a class I and II HDAC inhibitor, or sirtinol, a class III HDAC inhibitor. Only TSA could restore global levels of H2B K12 and K120 acetylation back to that of controls, indicating that macroH2A1 opposes the activity of a class I or II HDAC (Fig. 6a). Using ChIP-qPCR analysis, we confirmed that TSA can reverse the loss of H2BK12ac and H2BK120ac in macroH2A1-depleted cells at macroH2A1-regulated genes (Fig. 6b). Functionally, TSA suppresses the effect of macroH2A1 depletion on expression of macroH2A1 target genes (Fig. 6c). Acetylation is usually studied in relation to its positive role in transcriptional regulation, however, our data indicate that acetylation plays a critical role at genes either positively or negatively regulated by macroH2A1. These results indicate that macroH2A1-mediated regulation of H2B K12 and K120 acetylation is a critical feature of macroH2A1-mediated transcriptional regulation.

Bottom Line: The histone variant macroH2A1 regulates gene expression important for differentiation, stem-cell reprogramming and tumor suppression.Here, we demonstrate that in primary human cells, macroH2A1 participates in two physically and functionally distinct types of chromatin marked by either H3K27me3 or nine histone acetylations.Through the recruitment of PARP-1, macroH2A1.1 promotes the CBP-mediated acetylation of H2B K12 and K120, which either positively or negatively regulates the expression of macroH2A1-target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.

ABSTRACT
The histone variant macroH2A1 regulates gene expression important for differentiation, stem-cell reprogramming and tumor suppression. Here, we demonstrate that in primary human cells, macroH2A1 participates in two physically and functionally distinct types of chromatin marked by either H3K27me3 or nine histone acetylations. Using RNA sequencing, we found that macroH2A1-regulated genes, which have roles in cancer progression, are specifically found in macroH2A1-containing acetylated chromatin. Of the two macroH2A1 variants, macroH2A1.1 and macroH2A1.2, the former is suppressed in cancer and can interact with PARP-generated poly(ADP-ribose). Through the recruitment of PARP-1, macroH2A1.1 promotes the CBP-mediated acetylation of H2B K12 and K120, which either positively or negatively regulates the expression of macroH2A1-target genes. Although macroH2A1-regulated H2B acetylation is a common feature of primary cells, this regulation is typically lost in cancer cells. Consequently, our results provide insight into macroH2A1.1's role in cancer suppression.

Show MeSH
Related in: MedlinePlus