Limits...
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: 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.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

MacroH2A1.1 and PARP-1 enzymatic activity are required to regulate H2BK12ac and H2BK120ac and macroH2A1-target gene expression.(a) Immunoblots for macroH2A1, H3 and indicated PTMs from the indicated primary and cancer treated with or without 10 μM of PJ-34 for three days.(b) Immunoblots for macroH2A1, H3 and indicated PTMs in IMR90 primary lung fibroblasts and A549 lung cancer cells treated with or without 10 μM BYK204165 for three days.(c) Immunoblots for macroH2A1, H3 and indicated PTMs from IMR90 primary lung fibroblasts and A549 lung cancer cells expressing shRNA against Luciferase (L, as a control) or PARP-1 (P).(d) (left) Heat map depicting the fold change of macroH2A1-target genes upon macroH2A1 depletion (mH2A1 KD), treatment with 10 μM PJ-34 for two days, treatment with 10 μM BYK204165 for two days, or ectopic expression of macroH2A1.2 (mH2A1.2). (right) * p value < 0.05 from two-tailed Student’s t-tests for the indicated gene and condition (n = 3 independent cell passages).(e) ChIP for PARP-1 at the indicated genes from IMR90 cells expressing shRNA against either Luciferase (L) or macroH2A1 (M). Dotted line indicates upper 95% confidence interval of signal from no-antibody control ChIPs. Error bars, +/− the s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(f) As for (e) except with IMR90 cells expressing ectopic GFP (as a control) or macroH2A1.2 (mH2A1.2).(g) As for (e) except with IMR90 cells treated with or without 10 μM PJ-34 for three days.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4221384&req=5

Figure 5: MacroH2A1.1 and PARP-1 enzymatic activity are required to regulate H2BK12ac and H2BK120ac and macroH2A1-target gene expression.(a) Immunoblots for macroH2A1, H3 and indicated PTMs from the indicated primary and cancer treated with or without 10 μM of PJ-34 for three days.(b) Immunoblots for macroH2A1, H3 and indicated PTMs in IMR90 primary lung fibroblasts and A549 lung cancer cells treated with or without 10 μM BYK204165 for three days.(c) Immunoblots for macroH2A1, H3 and indicated PTMs from IMR90 primary lung fibroblasts and A549 lung cancer cells expressing shRNA against Luciferase (L, as a control) or PARP-1 (P).(d) (left) Heat map depicting the fold change of macroH2A1-target genes upon macroH2A1 depletion (mH2A1 KD), treatment with 10 μM PJ-34 for two days, treatment with 10 μM BYK204165 for two days, or ectopic expression of macroH2A1.2 (mH2A1.2). (right) * p value < 0.05 from two-tailed Student’s t-tests for the indicated gene and condition (n = 3 independent cell passages).(e) ChIP for PARP-1 at the indicated genes from IMR90 cells expressing shRNA against either Luciferase (L) or macroH2A1 (M). Dotted line indicates upper 95% confidence interval of signal from no-antibody control ChIPs. Error bars, +/− the s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(f) As for (e) except with IMR90 cells expressing ectopic GFP (as a control) or macroH2A1.2 (mH2A1.2).(g) As for (e) except with IMR90 cells treated with or without 10 μM PJ-34 for three days.

Mentions: The data described above implicates macroH2A1.1’s ligand binding function in regulating H2B K12 and K120 acetylation. Two of macroH2A1.1’s ligands, PAR and monoADP-ribose, are covalent PTMs catalyzed by PARPs30. Through binding the ends of PAR chains, the macrodomain of macroH2A1.1 has been implicated in repression of PARP activity in vitro20. To determine if the activity of a PARP family member is required for macroH2A1-mediated regulation of H2BK12ac and H2BK120ac, we inhibited PARP enzymatic activity with PJ-34, a pan-specific PARP inhibitor31. Reminiscent of macroH2A1 depletion, PJ-34 treatment decreased H2B K12 and K120 acetylation in IMR90 primary lung fibroblasts, skin fibroblasts, prostate and mammary epithelium (Fig. 5a). PJ-34 does not affect H2B K12 and K120 acetylation in cancer cells. Other histone PTMs, like H3K4me3, were unaffected either in primary or cancer cells (Fig. 5a).


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)

MacroH2A1.1 and PARP-1 enzymatic activity are required to regulate H2BK12ac and H2BK120ac and macroH2A1-target gene expression.(a) Immunoblots for macroH2A1, H3 and indicated PTMs from the indicated primary and cancer treated with or without 10 μM of PJ-34 for three days.(b) Immunoblots for macroH2A1, H3 and indicated PTMs in IMR90 primary lung fibroblasts and A549 lung cancer cells treated with or without 10 μM BYK204165 for three days.(c) Immunoblots for macroH2A1, H3 and indicated PTMs from IMR90 primary lung fibroblasts and A549 lung cancer cells expressing shRNA against Luciferase (L, as a control) or PARP-1 (P).(d) (left) Heat map depicting the fold change of macroH2A1-target genes upon macroH2A1 depletion (mH2A1 KD), treatment with 10 μM PJ-34 for two days, treatment with 10 μM BYK204165 for two days, or ectopic expression of macroH2A1.2 (mH2A1.2). (right) * p value < 0.05 from two-tailed Student’s t-tests for the indicated gene and condition (n = 3 independent cell passages).(e) ChIP for PARP-1 at the indicated genes from IMR90 cells expressing shRNA against either Luciferase (L) or macroH2A1 (M). Dotted line indicates upper 95% confidence interval of signal from no-antibody control ChIPs. Error bars, +/− the s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(f) As for (e) except with IMR90 cells expressing ectopic GFP (as a control) or macroH2A1.2 (mH2A1.2).(g) As for (e) except with IMR90 cells treated with or without 10 μM PJ-34 for three days.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4221384&req=5

Figure 5: MacroH2A1.1 and PARP-1 enzymatic activity are required to regulate H2BK12ac and H2BK120ac and macroH2A1-target gene expression.(a) Immunoblots for macroH2A1, H3 and indicated PTMs from the indicated primary and cancer treated with or without 10 μM of PJ-34 for three days.(b) Immunoblots for macroH2A1, H3 and indicated PTMs in IMR90 primary lung fibroblasts and A549 lung cancer cells treated with or without 10 μM BYK204165 for three days.(c) Immunoblots for macroH2A1, H3 and indicated PTMs from IMR90 primary lung fibroblasts and A549 lung cancer cells expressing shRNA against Luciferase (L, as a control) or PARP-1 (P).(d) (left) Heat map depicting the fold change of macroH2A1-target genes upon macroH2A1 depletion (mH2A1 KD), treatment with 10 μM PJ-34 for two days, treatment with 10 μM BYK204165 for two days, or ectopic expression of macroH2A1.2 (mH2A1.2). (right) * p value < 0.05 from two-tailed Student’s t-tests for the indicated gene and condition (n = 3 independent cell passages).(e) ChIP for PARP-1 at the indicated genes from IMR90 cells expressing shRNA against either Luciferase (L) or macroH2A1 (M). Dotted line indicates upper 95% confidence interval of signal from no-antibody control ChIPs. Error bars, +/− the s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests.(f) As for (e) except with IMR90 cells expressing ectopic GFP (as a control) or macroH2A1.2 (mH2A1.2).(g) As for (e) except with IMR90 cells treated with or without 10 μM PJ-34 for three days.
Mentions: The data described above implicates macroH2A1.1’s ligand binding function in regulating H2B K12 and K120 acetylation. Two of macroH2A1.1’s ligands, PAR and monoADP-ribose, are covalent PTMs catalyzed by PARPs30. Through binding the ends of PAR chains, the macrodomain of macroH2A1.1 has been implicated in repression of PARP activity in vitro20. To determine if the activity of a PARP family member is required for macroH2A1-mediated regulation of H2BK12ac and H2BK120ac, we inhibited PARP enzymatic activity with PJ-34, a pan-specific PARP inhibitor31. Reminiscent of macroH2A1 depletion, PJ-34 treatment decreased H2B K12 and K120 acetylation in IMR90 primary lung fibroblasts, skin fibroblasts, prostate and mammary epithelium (Fig. 5a). PJ-34 does not affect H2B K12 and K120 acetylation in cancer cells. Other histone PTMs, like H3K4me3, were unaffected either in primary or cancer cells (Fig. 5a).

Bottom Line: 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.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