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

Nature Structural & Molecular Biology volume 21, pages 981–989 (2014)Cite this article

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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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Figure 1: MacroH2A1 is enriched in two distinct types of chromatin.
Figure 2: MacroH2A1-regulated genes are marked by specific histone acetylations.
Figure 3: MacroH2A1 directly regulates the acetylation of H2B at K12 and K120 in primary cell types but not in cancer cells.
Figure 4: MacroH2A1.1 is specifically required to regulate H2B K12 and K120 acetylation.
Figure 5: MacroH2A1.1 and PARP-1 enzymatic activity are required to regulate H2BK12ac and H2BK120ac and macroH2A1-target-gene expression.
Figure 6: MacroH2A1 and PARP-1 recruit CBP to mediate acetylation of H2B K12 and K120 and regulate macroH2A1-target-gene expression.
Figure 7: CBP is recruited to macroH2A1-target genes in a macroH2A1- and PARP-1–dependent fashion.

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Acknowledgements

We thank S.B. Horwitz, C.S. Rubin, J.M. Backer and C.W. Chow from Albert Einstein College of Medicine for sharing equipment and reagents. We thank T. Zhang (Regeneron Pharmaceuticals) for critical reading of the manuscript. The high-throughput sequencing was performed by the Einstein Epigenomics Core Facility. We thank specimen donors and The Cancer Genome Atlas (TCGA) genome characterization center at the University of North Carolina for their contributions to the lung squamous-cell carcinoma data set from TCGA. This work was supported by the US National Institutes of Health training grant T32GM007491-38 (P.D.R.) and grant R01CA155232 (M.J.G.).

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  1. Jong Woo Park

    Present address: Present address: Department of Biochemistry and Molecular Biology, College of Pharmacy, Sungkyunkwan University, Suwon, Korea.,

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

    Hongshan Chen, Penelope D Ruiz, Leonid Novikov, Alyssa D Casill, Jong Woo Park & Matthew J Gamble

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Contributions

H.C. designed and performed experiments, analyzed the data and wrote the manuscript. L.N., P.D.R. and J.W.P. performed experiments. A.D.C. performed the analysis of TCGA data. M.J.G. designed experiments, analyzed data and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 ISOR-analyzed macroH2A1 ChIP-seq data are both sensitive and reproducible.

(a) Three-way Venn diagram depicting the overlap of significantly (p < 0.01) enriched regions from macroH2A1 ChIP-seq replicate 1 (yellow), ChIP-seq replicate 2 (purple), and previously published ChIP-chip data1 (blue). The total regions of the genome considered was limited to autosomal DNA sufficiently covered by sequencing depth (regions with at least one read/kb) or, in the case of the ChIP-chip data probe tiling (at least 6 probes/kb). Data shown in the figures is from macroH2A1 ChIP-seq replicate 1 unless indicated. However, all results were independently confirmed with data from replicate 2 (data not shown). (b) Confirmation analysis of ISOR identified regions. MacroH2A1 ChIP-qPCR for 9 loci in macroH2A1 depleted regions and 54 macroH2A1 enriched regions. No antibody (NA) ChIP reactions were included as a negative control. The ISOR identified depleted regions show macroH2A1 binding in ChIP-qPCR assays similar to no antibody controls. All ISOR identified bound regions were confirmed by ChIP-qPCR for macroH2A1. Overall, we estimate an FDR less than 0.02. (c) Boxplot summaries of the sizes of enriched domains for the indicated histone marks. (d) Percent of the autosomal genome covered by the enriched domains for each of the indicated histone marks. Sequencing gaps greater than 1 kb have been subtracted from the results.

Supplementary Figure 2 MacroH2A1 depletion leads to cancer-relevant changes in gene expression and cellular phenotype.

(a) Mean percent macroH2A1.1 expressed in normal lung tissue (n=50) or primary tumor (n=489) samples in the Lung Squamous Cell Cancer dataset from TCGA. Error bars represent +/− s.e.m. of all samples in the group, **** p value < 1x10-75 from a two tailed Student’s t-test. (b) Genes regulated by macroH2A1-depletion in IMR90 cells are significantly enriched for genes altered in squamous cell lung cancer. Analysis of the squamous cell lung cancer data set identified 7392 differentially regulated genes between normal and primary tumors. We calculated the enrichment, expressed as the odds ratio, for genes altered in squamous cell lung cancer in genes regulated by macroH2A1 depletion in IMR90 cells. Error bars represent the 95% confidence interval of the odds ratio. (c) Percent macroH2A1.1 plotted for each sample in the dataset, showing that many squamous cell tumors have reduced levels of macroH2A1.1 splicing. (d) Correlation analysis between macroH2A1.1 splicing levels and gene expression across the 549 samples in the squamous cell lung cancer samples yielded 3431 genes that significantly correlated with macroH2A1.1 splicing. Depicted are the odds ratios for the enrichment of gene regulated by macroH2A1 depletion in IMR90 cells and genes whose expression significantly correlates with macroH2A1.1 splicing changes. Error bars indicate the 95% confidence interval of the odds ratio. (b,d) * p value < 0.001, ** p value < 1x10-4, *** p value < 1x10-12 from a Fisher’s exact test. (e) Growth curves of IMR90 cell expressing shRNA targeting luciferase (Luc KD) or macroH2A1 (mH2A1 KD) showing that macroH2A1 suppresses cell proliferation. (f) Histograms of doubling times calculated from the growth curves shown in (e). (g) Representative fields of soft agar colony formation assays for the cells described in (e). (h) Summary of soft agar colony formation assays indicating the number of colonies per 5000 cells plated. The results demonstrate that macroH2A1 suppresses anchorage independent growth of IMR90 cells. Error bars represent the s.e.m. of three cell cultures. *, p value < 0.001 from a two-tailed Student’s t-test.

Supplementary Figure 3 Metagene analysis of macroH2A1 and various histone PTMs.

(a) Correlation of macroH2A1 and various histone PTMs with transcription. Metagene analysis of ChIP-seq data of the indicated histone marks from ChIP-seq data from IMR90 cells. Genes were separated into expression pentiles based on GRO-seq data from IMR90 cells28. The data in each group represent ten 1 kb windows upstream of the TSS, 30 windows spanning the gene body, and ten 1 kb windows downstream of the end of the gene. Error bars (too small to be seen distinctly) represent −/+ s.e.m. (b) As for (a) except genes were separated into upregulated (n = 384) or downregulated (n = 212) groups based on expression changes in the macroH2A1 depleted IMR90 cells compared to controls.

Supplementary Figure 4 MacroH2A1-regulated genes are enriched for genes marked by H2B acetylation.

(a) Area-proportional diagram depicting the relative distribution across eight categories defined by the presence or absence of a significantly enriched regions of macroH2A1 (mH2A1), H2BK12ac and H3K27me3 across all Ensemble annotated protein coding autosomal genes (all), genes regulated, upregulated and downregulated by macroH2A1-depeltion, respectively. (b) The enrichment of macroH2A1-regulated genes in each of the eight classes is expressed as the log2 odds ratio. * p < 0.01 and ** p < 1x10-9 from a Fisher’s exact test. Error bars represent the 95% confidence interval on the odds ratio.

Supplementary Figure 5 Controls for shRNA and ChIP specificity.

(a) Effect of macroH2A1 depletion by an independent shRNA on H2BK12ac and H2BK120ac. Immunoblots for PTMs or factors from IMR90 cells expressing an shRNA against luciferase (Luc KD) or an independent shRNA from that used in the main figures. (b,c,d) Control ChIP data corresponding to experiments shown in Figure 5e-g, respectively. (e) Negative control for PARP-1 ChIP for Figure 5e-g. ChIP for PARP-1 (top) and H3 (bottom) from IMR90 cells expressing shRNA against Luciferase or PARP-1. (f) Negative control for CBP ChIP for experiments shown in Figure 7a,b. As for (e) except CBP ChIP was used instead of PARP-1 ChIP. (g) Depletion of mH2A1 in IMR90 cells does not alter the binding of GCN5 or PCAF. ChIP for GCN5, PCAF, macroH2A1 and histone H3 at the indicated genes from IMR90 cells expressing shRNA against either Luciferase or macroH2A1. Error bars represent +/− the s.e.m. of 3 independent cell cultures. * p value < 0.05 from a two-tailed Student’s t-test

Supplementary Figure 6 Reexpression of macroH2A1.1 promotes loci-specific upregulation of H2B K12 and K120 acetylation in a manner that requires chromatin incorporation.

(a) Schematic depicting GFP-macro1.1 in which the histone like region has been replaced with GFP. (b) Immunoblots for PTMs or proteins from IMR90 cells expressing GFP or GFP fused to macroH2A1.1’s macrodomain, showing the macrodomain alone is insufficient to regulate H2BK12ac and H2BK120ac. (c) Immunoblots for factors in A549 cells expressing the macroH2A1 proteins and mutants as described in Figure 4b. (d) ChIP-qPCR for H2BK12ac, H2BK120ac, macroH2A1 and H3 from A549 cells expressing macroH2A1.1 or GFP at regions positive for macroH2A1 in A549 cells (left) regions lacking significant macroH2A1 in A549 cells but where macroH2A1 increases upon macroH2A1.1 expression (center) and regions where macroH2A1.1 expression does not affect occupancy. The horizontal dotted line indicates upper limit of the 95% confidence interval of the signal from no-antibody control ChIPs. Error bars represent +/− the s.e.m. of three independent cell cultures. * p < 0.05 from a two-tailed Student’s t-test (b–g).

Supplementary Figure 7 Chromatin environments of eight macroH2A1-regulated genes.

Individual gene tracks depicting macroH2A1, H3K27me3, H2BK12ac, H2BK120ac, H2BK15ac and H2BK20ac from -50 kb to +50 kb centered at the transcription start site for macroH2A1 down or upregulated loci in IMR90 cells.

Supplementary Figure 8 CBP activity regulates H2BK12ac and H2BK120ac.

(a) Immunoblots for CBP, PARP-1 and GAPDH from whole cell lysates or for macroH2A1 and H3 from acid extracted lysates of IMR90 cells expressing shRNA targeted against luciferase (L) as a control or macroH2A1 (M). (b) Immunoblots for H2BK12ac, H2BK120ac and H3 from acid extracted lysates of IMR90 cells treated with or without 20 µM of CBP inhibitor C646 for 3 days. (c) Immunoblots for CBP and GAPDH from whole cell lysates of IMR90 cells expressing shRNA against luciferase (L) as a control or CBP.

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Chen, H., Ruiz, P., Novikov, L. et al. MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation. Nat Struct Mol Biol 21, 981–989 (2014). https://doi.org/10.1038/nsmb.2903

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