The histone variant macroH2A occupies large repressive domains throughout the genome; however, mechanisms underlying its precise deposition remain poorly understood. Here, we characterize de novo chromatin deposition of macroH2A2 using temporal genomic profiling in murine-derived fibroblasts devoid of all macroH2A isoforms. We find that macroH2A2 is first pervasively deposited genome wide at both steady-state domains and adjacent transcribed regions, the latter of which are subsequently pruned, establishing mature macroH2A2 domains. Pruning of macroH2A2 can be counteracted by chemical inhibition of transcription. Further, locus-specific transcriptional manipulation reveals that gene activation depletes pre-existing macroH2A2, while silencing triggers ectopic macroH2A2 accumulation. We demonstrate that the FACT (facilitates chromatin transcription) complex is required for macroH2A2 pruning within transcribed chromatin. Taken together, we have identified active chromatin as a boundary for macroH2A domains through a transcription-associated ‘pruning’ mechanism that establishes and maintains the faithful genomic localization of macroH2A variants.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 06 November 2020
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Weber, C. M. & Henikoff, S. Histone variants: dynamic punctuation in transcription. Genes Dev. 28, 672–682 (2014).
Buschbeck, M. & Hake, S. B. Variants of core histones and their roles in cell fate decisions, development and cancer. Nat. Rev. Mol. Cell Biol. 18, 299–314 (2017).
Doyen, C. et al. Mechanism of polymerase II transcription repression by the histone variant macroH2A. Mol. Cell. Biol. 26, 1156–1164 (2006).
Angelov, D. et al. The histone variant macroH2A interferes with transcription factor binding and SWI/SNF nucleosome remodeling. Mol. Cell 11, 1033–1041 (2003).
Gamble, M. J., Frizzell, K. M., Yang, C., Krishnakumar, R. & Kraus, W. L. The histone variant macroH2A1 marks repressed autosomal chromatin, but protects a subset of its target genes from silencing. Genes Dev. 24, 21–32 (2010).
Chen, H. 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).
Timinszky, G. et al. A macrodomain-containing histone rearranges chromatin upon sensing PARP1 activation. Nat. Struct. Mol. Biol. 16, 923–929 (2009).
Kim, J. et al. Replication stress shapes a protective chromatin environment across fragile genomic regions. Mol. Cell 69, 36–47 (2018).
Pasque, V., Gillich, A., Garrett, N. & Gurdon, J. B. Histone variant macroH2A confers resistance to nuclear reprogramming. EMBO J. 30, 2373–2387 (2011).
Gaspar-Maia, A. et al. MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency. Nat. Commun. 4, 1565 (2013).
Kapoor, A. et al. The histone variant macroH2A suppresses melanoma progression through regulation of CDK8. Nature 468, 1105–1109 (2010).
Buschbeck, M. et al. The histone variant macroH2A is an epigenetic regulator of key developmental genes. Nat. Struct. Mol. Biol. 16, 1074–1079 (2009).
Zhang, R. et al. Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Dev. Cell 8, 19–30 (2005).
Douet, J. et al. MacroH2A histone variants maintain nuclear organization and heterochromatin architecture. J. Cell Sci. 130, 1570–1582 (2017).
Changolkar, L. N. et al. Genome-wide distribution of macroH2A1 histone variants in mouse liver chromatin. Mol. Cell. Biol. 30, 5473–5483 (2010).
Costanzi, C. & Pehrson, J. R. Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393, 599–601 (1998).
Hernández-Muñoz, I. et al. Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase. Proc. Natl Acad. Sci. USA 102, 7635–7640 (2005).
Pehrson, J. R., Changolkar, L. N., Costanzi, C. & Leu, N. A. Mice without macroH2A histone variants. Mol. Cell. Biol. 34, 4523–4533 (2014).
Oksuz, O. et al. Capturing the onset of PRC2-mediated repressive domain formation. Mol. Cell 70, 1149–1162 (2018).
Zhang, K., Mosch, K., Fischle, W. & Grewal, S. I. S. Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin. Nat. Struct. Mol. Biol. 15, 381–388 (2008).
Boyarchuk, E., Filipescu, D., Vassias, I., Cantaloube, S. & Almouzni, G. The histone variant composition of centromeres is controlled by the pericentric heterochromatin state during the cell cycle. J. Cell Sci. 127, 3347–3359 (2014).
Ahmad, K. & Henikoff, S. The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 9, 1191–1200 (2002).
Fontanals-Cirera, B. et al. Harnessing BET inhibitor sensitivity reveals AMIGO2 as a melanoma survival gene. Mol. Cell 68, 731–744 (2017).
Vardabasso, C. et al. Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma. Mol. Cell 59, 75–88 (2015).
Bulut-Karslioglu, A. et al. Suv39h-dependent H3K9me3 marks intact retrotransposons and silences LINE elements in mouse embryonic stem cells. Mol. Cell 55, 277–290 (2014).
Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).
Yildirim, E., Sadreyev, R. I., Pinter, S. F. & Lee, J. T. X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription. Nat. Struct. Mol. Biol. 19, 56–62 (2012).
Posavec Marjanović, M. et al. MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption. Nat. Struct. Mol. Biol. 24, 902–910 (2017).
Yue, F. et al. A comparative encyclopedia of DNA elements in the mouse genome. Nature 515, 355–364 (2014).
Mousavi, K., Zare, H., Wang, A. H. & Sartorelli, V. Polycomb protein Ezh1 promotes RNA polymerase II elongation. Mol. Cell 45, 255–262 (2012).
Jonkers, I., Kwak, H. & Lis, J. T. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. eLife 3, e02407 (2014).
Chao, S. H. & Price, D. H. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J. Biol. Chem. 276, 31793–31799 (2001).
Ratnakumar, K. et al. ATRX-mediated chromatin association of histone variant macroH2A1 regulates α-globin expression. Genes Dev. 26, 433–438 (2012).
Lu, H. et al. Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism. eLife 4, e06535 (2015).
McCabe, M. T. et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 492, 108–112 (2012).
Hammond, C. M., Strømme, C. B., Huang, H., Patel, D. J. & Groth, A. Histone chaperone networks shaping chromatin function. Nat. Rev. Mol. Cell Biol. 18, 141–158 (2017).
Ouararhni, K. et al. The histone variant mH2A1.1 interferes with transcription by down-regulating PARP-1 enzymatic activity. Genes Dev. 20, 3324–3336 (2006).
Mehrotra, P. V. et al. DNA repair factor APLF is a histone chaperone. Mol. Cell 41, 46–55 (2011).
Belotserkovskaya, R. et al. FACT facilitates transcription-dependent nucleosome alteration. Science 301, 1090–1093 (2003).
Jeronimo, C., Watanabe, S., Kaplan, C. D., Peterson, C. L. & Robert, F. The histone chaperones FACT and Spt6 restrict H2A.Z from intragenic locations. Mol. Cell 58, 1113–1123 (2015).
Choi, E. S. et al. Factors that promote H3 chromatin integrity during transcription prevent promiscuous deposition of CENP-ACnp1 in fission yeast. PLoS Genet. 8, e1002985 (2012).
Safina, A. et al. Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells. Cell Cycle 12, 2423–2434 (2013).
Deal, R. B., Henikoff, J. G. & Henikoff, S. Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328, 1161–1164 (2010).
Ray-Gallet, D. et al. Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity. Mol. Cell 44, 928–941 (2011).
Kraushaar, D. C. et al. Genome-wide incorporation dynamics reveal distinct categories of turnover for the histone variant H3.3. Genome Biol. 14, R121 (2013).
Costanzi, C., Stein, P., Worrad, D. M., Schultz, R. M. & Pehrson, J. R. Histone macroH2A1 is concentrated in the inactive X chromosome of female preimplantation mouse embryos. Development 127, 2283–2289 (2000).
Chang, C.-C. et al. A maternal store of macroH2A is removed from pronuclei prior to onset of somatic macroH2A expression in preimplantation embryos. Dev. Biol. 278, 367–380 (2005).
Sansoni, V. et al. The histone variant H2A.Bbd is enriched at sites of DNA synthesis. Nucleic Acids Res. 42, 6405–6420 (2014).
Okuwaki, M., Kato, K. & Nagata, K. Functional characterization of human nucleosome assembly protein 1-like proteins as histone chaperones. Genes Cells 15, 13–27 (2010).
Chakravarthy, S. & Luger, K. The histone variant macro-H2A preferentially forms ‘hybrid nucleosomes’. J. Biol. Chem. 281, 25522–25531 (2006).
Nakayama, T., Nishioka, K., Dong, Y. X., Shimojima, T. & Hirose, S. Drosophila GAGA factor directs histone H3.3 replacement that prevents the heterochromatin spreading. Genes Dev. 21, 552–561 (2007).
Bernstein, E. et al. A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis. Proc. Natl Acad. Sci. USA 105, 1533–1538 (2008).
Boyer, La et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).
Pavri, R. et al. Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II. Cell 125, 703–717 (2006).
Schmitges, F. W. et al. Histone methylation by PRC2 is inhibited by active chromatin marks. Mol. Cell 42, 330–341 (2011).
Wang, X. et al. Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA. Nat. Struct. Mol. Biol. 24, 1028–1038 (2017).
Mendenhall, E. M. et al. GC-rich sequence elements recruit PRC2 in mammalian ES cells. PLoS Genet. 6, e1001244 (2010).
Hosogane, M., Funayama, R., Shirota, M. & Nakayama, K. Lack of transcription triggers H3K27me3 accumulation in the gene body. Cell Rep. 16, 696–706 (2016).
Riising, E. M. et al. Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide. Mol. Cell 55, 347–360 (2014).
Højfeldt, J. W. et al. Accurate H3K27 methylation can be established de novo by SUZ12-directed PRC2. Nat. Struct. Mol. Biol. 25, 225–232 (2018).
Cas9 Activator Tool, http://sam.genome-engineering.org/database
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).
Chung, C.-Y. et al. Cbx8 acts non-canonically with Wdr5 to promote mammary tumorigenesis. Cell Rep. 16, 472–486 (2016).
Buenrostro, J. D., Wu, B., Chang, H. Y. & Greenleaf, W. J. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 109, 21.29.1–21.29.9 (2015).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Liang, K. & Keleş, S. Normalization of ChIP-seq data with control. BMC Bioinformatics 13, 199 (2012).
Zang, C. et al. A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 25, 1952–1958 (2009).
Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).
Sandve, G. K. et al. The Genomic HyperBrowser: inferential genomics at the sequence level. Genome Biol. 11, R121 (2010).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
Peled, M. et al. Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor. Proc. Natl Acad. Sci. USA 115, E468–E477 (2018).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Schwanhüusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).
Dignam, J. D., Lebovitz, R. M. & Roeder, R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).
The authors thank J. Pehrson from the University of Pennsylvania for sharing macroH2A dKO mice; R. Fisher, J. Jin, R. Parsons, Y. Hoshida and S. Aaronson from Icahn School of Medicine at Mount Sinai for sharing advice, equipment or reagents; A. Cook and D. Hasson from the Bernstein laboratory for critically reading the manuscript; the Tisch Cancer Institute Genomics Core Facility at ISMMS, particularly G. Panda, for sequencing assistance; the Microscopy CoRE at ISMMS; and the Flow Cytometry Core at ISMMS for help with cell sorting. This work was supported by Scientific Computing at ISMMS, the Office of Research Infrastructure of the NIH to ISMMS (grant no. S10OD018522), ISMMS Cancer Center Support Grant P30CA196521 and NIH/NCI R01CA154683 (E.B.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Chromatin deposition of macroH2A variants is replication independent (related to Fig. 1).
(a) Western blot analysis of whole cell lysate showing the expression of SNAP-tagged histones in NIH 3T3 cells. Arrowheads indicate SNAP-tagged proteins and asterisks indicate endogenous proteins. Amido black staining of histones used as loading control. (b) Profiles of cell cycle distribution for SNAP-H3.1 or macroH2A2-SNAP in NIH 3T3 cells based on DNA content. Numbers represent percentage of nuclei in the indicated cell cycle phase. (c) FACS analysis showing quantification of median SNAPc in NIH 3T3 cells stably expressing SNAP-H3.1 or macroH2A2-SNAP with indicated labeling strategies as well as parental cells. (d) Same as in (a) in WT iDFs. (e) qPCR analysis of WT iDFs in distinct cell cycle stages sorted based on DNA content, showing the mRNA expression level of endogenous macroH2A1.1, macroH2A1.2 and macroH2A2. E2f1 (G1), Pcna (G1), H4 (S) and Cenpa (G2) are used as controls. Rpl7 is used as internal reference for normalization. Error bars represent s.d. from n = 2 independent experiments. P values are calculated from two-tailed t test comparing cell cycle enriched fractions with the asynchronous population (*P < 0.05, ** P < 0.01, *** P < 0.001).
Supplementary Figure 2 Characterization of the clonal macroH2A-GFP inducible dKO iDF cell lines (related to Fig. 2).
(a) Western blots for indicated histone variants and histone modifications with isolated chromatin. Amido black staining of histones is used as loading control. (b) Immunofluorescence analysis of WT and dKO iDFs stained with antibodies against macroH2A1 or macroH2A2. (c) Proliferation curve of WT and dKO iDFs. Error bars represent s.d. from n = 3 independent experiments. (d) FACS analysis with live cells for the expression of macroH2A2-GFP and macroH2A1.1-GFP at different time points after dox induction. (e) Reverse transcription coupled with qPCR (RT-qPCR) analysis for macroH2A2 (H2afy2) gene expression level at indicated time points after dox induction. Error bars represent s.d. from n = 2 independent experiments. (f-h) Western blots for inducibly expressed macroH2A2-GFP and macroH2A1.1-GFP using isolated chromatin at indicated time points after dox addition with respective clonal cell lines. Chromatin extract from WT iDFs was used to show level of endogenous macroH2As. Amido black staining of histones and histone H3 are used as loading controls. Numbers below are quantifications of endogenous or inducibly expressed macroH2As. (i) Relative amount of endogenous macroH2A variants and inducibly expressed ones at 72 h post induction calculated based on quantifications from f to h. The amount of total endogenous macroH2A (sum of macroH2A1 and macroH2A2) in WT iDFs was set to 1. (j) Relative amount of macroH2A2-GFP expression level at indicated time points post induction in dKO iDFs compared to total macroH2A in WT iDFs. Calculation was based on f and i.
Supplementary Figure 3 Delayed macroH2A2 deposition at a subset of steady-state peaks is associated with heterochromatic features (related to Fig. 2).
(a) Spearman correlation heatmap of nChIP-seq profiles genome-wide in DFs10 and iDFs. Black box highlights macroH2A nChIPs. (b) Heatmap of macroH2A relative enrichment at all macroH2A peaks in WT DFs. (c) Separation of steady-state peaks into three categories based on the time when they are first detected after dox induction by nChIP-seq. (d) Box plots of H3K27me3 (DF)10 and H3K9me3 (MEF)25 ChIP-seq occupancy level at early (E), intermediate (I) and late (L) steady-state peak regions of macroH2A2. Boxes span the lower to upper quartile; median is indicated with a black line; whiskers show locations of the minimum and maximum; P values are calculated using a two-tailed t test, **** P < 2.2 × 10−16.
Supplementary Figure 4 MacroH2A acts as a redundant layer of epigenetic repression (related to Fig. 3).
(a) MA plot showing log2 fold change (y axis) of gene expression during macroH2A2-GFP induction in dKO iDFs, 6 h versus 0 h (left panel) and 24 h versus 0 h (right panel), against the ranked expression level at 0 h (x axis). Red and blue dots represent genes significantly up or downregulated, respectively (FDR < 0.05, log2[fold change] > 1 or < −1, n = 3 biological replicates). (b) RT-qPCR analysis of indicated genes at different time points after dox induction. Error bars represent s.d. from n = 2 independent experiments. (c) Experimental scheme showing treatment of WT and dKO iDFs with a combination of GSK126 (EZH2 inhibitor), 5AzaC (DNA methyltransferase inhibitor), TSA (histone deacetylase inhibitor). (d) Genome browser tracks showing occupancy of indicated histone variant/modifications. Expression levels in FPKM in dKO iDFs are showing in the boxes below. (e) RT-qPCR anaylsis showing activation of repressed genes in WT and dKO cells after simultaneous alleviation of multiple layers of epigenetic repression. Runx1 used as a negative control. Dotted line highlighted value 1 representing no change between drug treated and untreated. Error bars represent s.d. (n = 2). P values are calculated from two-tailed t test (*P < 0.05). (f) Percentage of inactive or active genes overlapping with transient macroH2A2 peaks. (g) Metagene plot showing the occupancy of elongating RNA polymerase II (Pol II S2P) in MEFs27 at active and inactive genes in iDFs. (h) Expression levels (FPKM) in inducible macroH2A2-GFP dKO iDFs (0 hour) and their promoter association with CpG islands (CGI) for the panel of genes selected for qPCR validations. (i) nChIP-qPCR analysis of macroH2A2-GFP occupancy 6 h and 24 h after induction. Same data was used as in Fig. 3e, but macroH2A-GFP ChIP was normalized to input. Three negative control regions (chr2, chr6 and chr14) were used for normalization to calculate relative enrichment. Error bars represent s.d. from n = 3 independent experiments. (j) nChIP-qPCR analysis of H2A occupancy 6 hour and 24 hour after macroH2A2-GFP induction. H2A ChIP was normalized to H2B. Error bars represent s.d. from n = 2 independent experiments.
Supplementary Figure 5 MCDs are flanked by actively transcribed chromatin across differentiated cell types (related to Fig. 4).
(a) Size distribution of all MCDs in iDFs. (b) Observed over expected random distribution of active and inactive genes falling within MCDs. The dashed line represents the expected value of a random distribution. Error bars represent s.d. P values are calculated using Monte Carlo testing, *** P < 0.001. (c) Metagene plots of Pol II S2P (MEF)27, total Pol II (MEF)29, H3K27me3 (DF)10, H3K27ac (DF)10, and H2A.Z (DF) occupancy at iDF MCDs. (d-e) Metagene plots of fetal liver and C2C12 myoblast MCDs18,28, occupancy of Pol II29,30 and indicated histone modifications29 of respective cell types at corresponding MCDs.
Supplementary Figure 6 Pruning of pervasively deposited macroH2A requires active transcription (related to Fig. 5).
(a) Metagene plot showing the occupancy of Pol II S2P (MEF)27 at indicated classes of genes in iDFs. (b) Box plots showing Pol II S2P (MEF)27 density at gene body. Boxes span the lower to upper quartile; median is indicated with a black line; whiskers show locations of the minimum and maximum; P values are calculated using a two-tailed t test, **** P < 2.2 × 10−16. (c) RT-qPCR for indicated genes 18 h after FP treatment. 18 S rRNA used as internal reference. Error bars represent s.d. from n = 2 independent experiments. P values are calculated from two-tailed t test comparing FP treated with untreated (* P < 0.05, ** P < 0.01, *** P < 0.001). (d) Genome browser snapshot showing retention of transient macroH2A2 peaks. (e) Metagene profiles of macroH2A2-GFP relative enrichment at transient or steady-state macroH2A2 peak regions during de novo deposition ±FP. (f) Spearman correlation heatmap and clustering analysis of nChIP-seq profiles genome-wide. Black box highlights clustering of macroH2A2 ChIP profile at 24 h (+FP) with that at 6 h (untreated). (g) Heatmap of macroH2A2-GFP occupancy at inactive gene loci.
Supplementary Figure 7 Transcriptional inhibition by FP leads to accumulation of macroH2A2 at steady state (related to Fig. 6).
(a) Genotyping genomic PCR analysis of iDF clones with WT (+/+), heterozygous KO (±) or homozygous KO (-/-) TSS of Tks4. (b) Western blots of whole cell extract from WT iDFs after 4-hour FP treatment; NT, no treatment. S2P, serine-2 phosphorylation. S5P, serine-5 phosphorylation. β-tubulin used as loading control. (c-d and g-h) RT-qPCR analysis of WT iDFs (c and g) and iDFs constitutively expressing macroH2A2-GFP (d and h) treated with FP for 18 h. 18 S rRNA used as internal reference. Error bars represent s.d. from n = 2 independent experiments. P values are calculated from a two-tailed t test comparing FP treated with untreated (*P < 0.05, ** P < 0.01, *** P < 0.001). (e-f and i-j) nChIP-qPCR analysis of WT iDFs (e and i) and iDFs constitutively expressing macroH2A2-GFP (f and j) showing the occupancy of endogenous macroH2A2 or macroH2A2-GFP after indicated time of FP treatment. MacroH2A2 ChIP was normalized to H2B. Primers target non-CGI gene body regions. Error bars represent s.d. from n = 2 independent experiments. Insets in (e and f) shows macroH2A2 (H2afy2) gene expression by RT-qPCR. Error bars represent s.d. from n = 2 independent experiments.
Supplementary Figure 8 H3K27me3 is dispensable for de novo macroH2A2 deposition (related to Fig. 6).
(a) nChIP-qPCR analysis showing the occupancy of H3K27me3 in WT iDFs after FP treatment. Error bars represent s.d. from n = 2 independent experiments. (b) nChIP-qPCR analysis showing H3K27me3 occupancy during de novo macroH2A2-GFP deposition in inducible iDFs. Error bars represent s.d. from n = 2 independent experiments. (c) nChIP-qPCR analysis showing the occupancy of H3K27me3 in inducible iDFs after GSK126 treatment. Error bars represent s.d. from n = 2 independent experiment. (d) Spearman correlation heatmap and clustering analysis of nChIP-seq profiles genome-wide. Black boxes highlight clustering of the 6-h profiles (-/+GSK126) and 24-h profiles (-/+GSK126). (e) Metagene profiles of macroH2A2-GFP occupancy at steady-state or transient macroH2A2 peak regions during de novo deposition after GSK126 pretreatment. (f) Venn diagram showing the overlap of macroH2A2-GFP nChIP-seq peaks after 24 h of dox induction ± pretreatment of GSK126. (g) Example tracks of de novo deposited macroH2A2-GFP after 24 h of dox induction ± GSK126 pretreatment. (h) nChIP-qPCR analysis showing macroH2A2-GFP occupancy after 24 h of dox induction ± pretreatment of GSK126 at regions shown in c. MacroH2A2 ChIP was normalized to H2B. Error bars represent s.d. from n = 2 independent experiment.
Supplementary Figure 9 The FACT complex facilitates transcription-associated pruning of macroH2A2 (related to Fig. 7).
(a) Western blot of isolated chromatin showing the expression level of GFP-tagged macroH2A isoforms in dKO iDFs using antibodies targeting respective macroH2A isoforms. Chromatin of WT iDFs is used to show the level of endogenous macroH2A isoforms. Histone H3 and amido black staining of histones used as loading control. (b) Western blot of whole cell extracts using anti-GFP antibody showing a similar expression level of GFP-tagged histones. GAPDH used as loading control. (c) Agarose gel electrophoresis analysis showing the distribution of DNA isolated from nucleosomes used as input for MNase IP. Bands corresponding to DNA of mono-/di-/tri-nucleosomes are labeled. (d) Western blot validation of MNase IP—qMS showing the interaction between macroH2A1.1 and PARP1. An empty lane between input and IP was removed for clarity. (e) Western blot analysis showing that endogenous macroH2A2 interacts with FACT subunits SPT16 and SSRP1 in iDF soluble nuclear extracts. MacroH2A2 IP with macroH2A dKO iDFs was used as control. (f) Diagram showing domain architecture of macroH2A2 and design of studied macroH2A2 fragments. (g) Western blot analysis showing interactions between FACT subunit SPT16 and different regions of macroH2A2 in iDF soluble nuclear extracts. For SPT16, a shorter (upper) and a longer (lower) exposure are shown. Lanes between the left and right panels were removed for clarity. (h) RT-qPCR analysis of iDFs with SPT16 knockdown. 18 S rRNA is used as internal reference. Error bars represent s.d. from n = 3 independent experiments. P values are calculated from two-tailed t test comparing shSPT16 with shScr (*P < 0.05, ** P < 0.01). (i) Model of the dynamics of de novo macroH2A2 deposition at distinct chromatin regions. The diagram depicts the absolute level (left) and background-normalized relative level (right) of chromatin incorporated macroH2A2 at indicated regions during de novo deposition. Green-shaded areas highlight the time window where transient macroH2A2 peaks are present. (j) Western blot of IP with chromatin-free cell extract in iDFs showing the interaction of macroH2A2 and NAP-1. Different salt concentrations were applied during immunoprecipitation as indicated.
About this article
Cite this article
Sun, Z., Filipescu, D., Andrade, J. et al. Transcription-associated histone pruning demarcates macroH2A chromatin domains. Nat Struct Mol Biol 25, 958–970 (2018). https://doi.org/10.1038/s41594-018-0134-5
This article is cited by
Nature Reviews Cancer (2021)
Nature Communications (2020)
Nature Reviews Molecular Cell Biology (2020)