A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis.

Histone variants play an important role in numerous biological processes through changes in nucleosome structure and stability and possibly through mechanisms influenced by posttranslational modifications unique to a histone variant. The family of histone H2A variants includes members such as H2A.Z, the DNA damage-associated H2A.X, macroH2A (mH2A), and H2ABbd (Barr body-deficient). Here, we have undertaken the challenge to decipher the posttranslational modification-mediated "histone code" of mH2A, a variant generally associated with certain forms of condensed chromatin such as the inactive X chromosome in female mammals. By using female human cells as a source of mH2A, endogenous mH2A was purified and analyzed by mass spectrometry. Although mH2A is in low abundance compared with conventional histones, we identified a phosphorylation site, S137ph, which resides within the "hinge" region of mH2A. This lysine-rich hinge is an approximately 30-aa stretch between the H2A and macro domains, proposed to bind nucleic acids. A specific antibody to S137ph was raised; by using this reagent, S137 phosphorylation was found to be present in both male and female cells and on both splice variants of the mH2A1 gene. Although mH2A is generally enriched on the inactive X chromosome in female cells, mH2AS137ph is excluded from this heterochromatic structure. Thus, a phosphorylated subpopulation of mH2A appears to play a unique role in chromatin regulation beyond X inactivation. We provide evidence that S137ph is enriched in mitosis, suggestive of a role in the regulation of mH2A posttranslational modifications throughout the cell cycle.

Mutant U2AF1-induced alternative splicing of H2afy (macroH2A1) regulates B-lymphopoiesis in mice.

Somatic mutations in spliceosome genes are found in ∼50% of patients with myelodysplastic syndromes (MDS), a myeloid malignancy associated with low blood counts. Expression of the mutant splicing factor U2AF1(S34F) alters hematopoiesis and mRNA splicing in mice. Our understanding of the functionally relevant alternatively spliced target genes that cause hematopoietic phenotypes in vivo remains incomplete. Here, we demonstrate that reduced expression of H2afy1.1, an alternatively spliced isoform of the histone H2A variant gene H2afy, is responsible for reduced B cells in U2AF1(S34F) mice. Deletion of H2afy or expression of U2AF1(S34F) reduces expression of Ebf1 (early B cell factor 1), a key transcription factor for B cell development, and mechanistically, H2AFY is enriched at the EBF1 promoter. Induced expression of H2AFY1.1 in U2AF1(S34F) cells rescues reduced EBF1 expression and B cells numbers in vivo. Collectively, our data implicate alternative splicing of H2AFY as a contributor to lymphopenia induced by U2AF1(S34F) in mice and MDS.

Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA.

In senescent cells, specialized domains of transcriptionally silent senescence-associated heterochromatic foci (SAHF), containing heterochromatin proteins such as HP1, are thought to repress expression of proliferation-promoting genes. We have investigated the composition and mode of assembly of SAHF and its contribution to cell cycle exit. SAHF is enriched in a transcription-silencing histone H2A variant, macroH2A. As cells approach senescence, a known chromatin regulator, HIRA, enters PML nuclear bodies, where it transiently colocalizes with HP1 proteins, prior to incorporation of HP1 proteins into SAHF. A physical complex containing HIRA and another chromatin regulator, ASF1a, is rate limiting for formation of SAHF and onset of senescence, and ASF1a is required for formation of SAHF and efficient senescence-associated cell cycle exit. These data indicate that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.

The Sgp3 locus derived from the 129 strain is responsible for enhanced endogenous retroviral expression in macroH2A1-deficient mice.

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine lupus nephritis is secreted by hepatocytes, and its expression is largely regulated by the Sgp3 (serum gp70 production 3) locus derived from lupus-prone mice. Because of the localization of the macroH2A1 gene encoding macroH2A histone variants within the Sgp3 interval and of an up-regulated transcription of endogenous retroviral sequences in macroH2A1-deficient C57BL/6 (B6) mice, we investigated whether macroH2A1 is a candidate gene for Sgp3. macroH2A1-deficient B6 mice carrying the 129-derived Sgp3 locus, which was co-transferred with the 129 macroH2A1 mutant gene, displayed increased levels of serum gp70 and hepatic retroviral gp70 RNAs comparable to those of B6.NZB-Sgp3 congenic mice bearing the Sgp3 locus of lupus-prone NZB mice. In contrast, the abundance of retroviral gp70 RNAs in macroH2A1-deficient 129 mice was not elevated at all as compared with wild-type 129 mice. Furthermore, Sgp3 subcongenic B6 mice devoid of the NZB-derived macroH2A1 gene displayed an Sgp3 phenotype identical to that of B6.NZB-Sgp3 congenic mice carrying the NZB-derived macroH2A1 gene, thus excluding macroH2A1 as a candidate Sgp3 gene. Collectively, our data indicate that enhanced transcription of endogenous retroviral sequences observed in macroH2A1-deficient B6 mice is not a result of the macroH2A1 mutation, but due to the presence of the 129-derived Sgp3 locus. In contrast, the effect of a macroH2A1 knockout mutation on the expression of several non-retroviral cellular genes was very similar on the B6 and 129 backgrounds, indicating that these effects were due to the macroH2A1 knockout.

Developmental changes in histone macroH2A1-mediated gene regulation.

macroH2A histone variants have been implicated to function in gene silencing by several studies, including ones showing a preferential association of macroH2A on the inactive X chromosome. To examine macroH2A function in vivo, we knocked out macroH2A1. macroH2A1 knockout mice are viable and fertile. A broad screen of liver gene expression showed no evidence of defects in X inactivation but did identify genes that have increased expression levels in macroH2A1 knockouts. macroH2A1-containing nucleosomes are enriched on the coding and/or upstream regions of these genes, suggesting that their increased expression levels are a direct effect of the absence of macroH2A1. The concentrations of macroH2A1 nucleosomes on these genes are low in the livers of newborn mice, and the macroH2A1 knockout had little effect on the expression levels of these genes in newborn liver. Our results indicate that an increase in liver macroH2A1 during the transition from newborn to young-adult status contributes to a decrease in the expression levels of these genes. These genes cluster in the area of lipid metabolism, and we observed metabolic effects in macroH2A1 knockouts. Our results indicate that the function of macroH2A1 histones is not restricted to gene silencing but also involves fine tuning the expression of specific genes.

macroH2A1 histone variants are depleted on active genes but concentrated on the inactive X chromosome.

Using a novel thiol affinity chromatography approach to purify macroH2A1-containing chromatin fragments, we examined the distribution of macroH2A1 histone variants in mouse liver chromatin. We found that macroH2A1 was depleted on the transcribed regions of active genes. This depletion was observed on all of the 20 active genes that we probed, with only one site showing a small amount of enrichment. In contrast, macroH2A1 was concentrated on the inactive X chromosome, consistent with our previous immunofluorescence studies. This preferential localization was seen on genes that are active in liver, genes that are inactive in liver, and intergenic regions but was absent from four regions that escape X inactivation. These results support the hypothesis that macroH2As function as transcriptional repressors. Also consistent with this hypothesis is our finding that the heterochromatin protein HP1beta copurifies with the macroH2A1-containing chromatin fragments. This study presents the first detailed examination of the distribution of macroH2A1 variants on specific sequences. Our results indicate that macroH2As have complex distribution patterns that are influenced by both local factors and long-range mechanisms.

Structural characterization of the histone variant macroH2A.

macroH2A is an H2A variant with a highly unusual structural organization. It has a C-terminal domain connected to the N-terminal histone domain by a linker. Crystallographic and biochemical studies show that changes in the L1 loop in the histone fold region of macroH2A impact the structure and potentially the function of nucleosomes. The 1.6-A X-ray structure of the nonhistone region reveals an alpha/beta fold which has previously been found in a functionally diverse group of proteins. This region associates with histone deacetylases and affects the acetylation status of nucleosomes containing macroH2A. Thus, the unusual domain structure of macroH2A integrates independent functions that are instrumental in establishing a structurally and functionally unique chromatin domain.

The histone variant macroH2A confers functional robustness to the intestinal stem cell compartment.

A stem cell's epigenome directs cell fate during development, homeostasis, and regeneration. Epigenetic dysregulation can lead to inappropriate cell fate decisions, aberrant cell function, and even cancer. The histone variant macroH2A has been shown to influence gene expression, guide cell fate, and safeguard against genotoxic stress. Interestingly, mice lacking functional macroH2A histones (hereafter referred to as macroH2A DKO) are viable and fertile; yet suffer from increased perinatal death and reduced weight and size compared to wildtype (WT). Here, we ask whether the ostensible reduced vigor of macroH2A DKO mice extends to intestinal stem cell (ISC) function during homeostasis, regeneration, and oncogenesis. Lgr5-eGFP-IRES-CreERT2 or Hopx-CreERT2::Rosa26-LSL-tdTomato ISC reporter mice or the C57BL/6J-Apcmin/J murine intestinal adenoma model were bred into a macroH2A DKO or strain-matched WT background and assessed for ISC functionality, regeneration and tumorigenesis. High-dose (12Gy) whole-body γ-irradiation was used as an injury model. We show that macroH2A is dispensable for intestinal homeostasis and macroH2A DKO mice have similar numbers of active crypt-base columnar ISCs (CBCs). MacroH2A DKO intestine exhibits impaired regeneration following injury, despite having significantly more putative reserve ISCs. DKO reserve ISCs disproportionately undergo apoptosis compared to WT after DNA damage infliction. Interestingly, a macroH2A DKO background does not significantly increase tumorigenesis in the Apcmin model of intestinal adenoma. We conclude that macroH2A influences reserve ISC number and function during homeostasis and regeneration. These data suggest macroH2A enhances reserve ISC survival after DNA damage and thus confers functional robustness to the intestinal epithelium.

Mice without macroH2A histone variants.

MacroH2A core histone variants have a unique structure that includes a C-terminal nonhistone domain. They are highly conserved in vertebrates and are thought to regulate gene expression. However, the nature of genes regulated by macroH2As and their biological significance remain unclear. Here, we examine macroH2A function in vivo by knocking out both macroH2A1 and macroH2A2 in the mouse. While macroH2As are not required for early development, the absence of macroH2As impairs prenatal and postnatal growth and can significantly reduce reproductive efficiency. The distributions of macroH2A.1- and macroH2A.2-containing nucleosomes show substantial overlap, as do their effects on gene expression. Our studies in fetal and adult liver indicate that macroH2As can exert large positive or negative effects on gene expression, with macroH2A.1 and macroH2A.2 acting synergistically on the expression of some genes and apparently having opposing effects on others. These effects are very specific and in the adult liver preferentially involve genes related to lipid metabolism, including the leptin receptor. MacroH2A-dependent gene regulation changes substantially in postnatal development and can be strongly affected by fasting. We propose that macroH2As produce adaptive changes to gene expression, which in the liver focus on metabolism.

MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency.

The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Using fibroblasts derived from macroH2A double knockout (dKO) mice, here we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together with H3K27me3, co-occupy pluripotency genes in wild-type (wt) fibroblasts. In particular, we find macroH2A isoforms to be highly enriched at target genes of the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. Finally, while macroH2A dKO-induced pluripotent cells are able to differentiate properly in vitro and in vivo, such differentiated cells retain the ability to return to a stem-like state. Therefore, we propose that macroH2A isoforms provide a redundant silencing layer or terminal differentiation 'lock' at critical pluripotency genes that presents as an epigenetic barrier when differentiated cells are challenged to reprogram.

A unified phylogeny-based nomenclature for histone variants.

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

Genome-wide distribution of macroH2A1 histone variants in mouse liver chromatin.

Studies of macroH2A histone variants indicate that they have a role in regulating gene expression. To identify direct targets of the macroH2A1 variants, we produced a genome-wide map of the distribution of macroH2A1 nucleosomes in mouse liver chromatin using high-throughput DNA sequencing. Although macroH2A1 nucleosomes are widely distributed across the genome, their local concentration varies over a range of 100-fold or more. The transcribed regions of most active genes are depleted of macroH2A1, often in sharply localized domains that show depletion of 4-fold or more relative to bulk mouse liver chromatin. We used macroH2A1 enrichment to help identify genes that appear to be directly regulated by macroH2A1 in mouse liver. These genes functionally cluster in the area of lipid metabolism. All but one of these genes has increased expression in macroH2A1 knockout mice, indicating that macroH2A1 functions primarily as a repressor in adult liver. This repressor activity is further supported by the substantial and relatively uniform macroH2A1 enrichment along the inactive X chromosome, which averages 4-fold. Genes that escape X inactivation stand out as domains of macroH2A1 depletion. The rarity of such genes indicates that few genes escape X inactivation in mouse liver, in contrast to what has been observed in human cells.

macroH2A1-dependent silencing of endogenous murine leukemia viruses.

We show that macroH2A1 histone variants are important for repressing the expression of endogenous murine leukemia viruses (MLVs) in mouse liver. Intact MLV proviruses and proviruses with deletions in env were nearly silent in normal mouse liver and showed substantial derepression in macroH2A1 knockout liver. In contrast, MLV proviruses with a deletion in the 5' end of pro-pol were expressed in normal liver and showed relatively low levels of derepression in knockout liver. macroH2A1 nucleosomes were enriched on endogenous MLVs, with the highest enrichment occurring on the 5' end of pro-pol. The absence of macroH2A1 also led to a localized loss of DNA methylation on the 5' ends of MLV proviruses. These results demonstrate that macroH2A1 histones have a significant role in silencing endogenous MLVs in vivo and suggest that specific internal MLV sequences are targeted by a macroH2A1-dependent silencing mechanism.

Reconstitution of nucleosomes with histone macroH2A1.2.

MacroH2A histones have an unusual hybrid structure, consisting of an N-terminal domain that is approximately 65% identical to a full-length histone H2A and a large C-terminal nonhistone domain. To develop an in vitro approach for investigating the effects of macroH2A proteins on chromatin structure and function, we reconstituted nucleosomes with recombinant macroH2A1.2, substituting for conventional H2A. Recombinant macroH2A1.2 was able to efficiently replace both of the conventional H2As in reconstituted nucleosomes. The substitution of macroH2A1.2 for H2A did not appear to grossly perturb the basic structure of the nucleosome core, as assessed by sedimentation and by digestion with micrococcal nuclease or DNase I. However, two differences were observed. First, the region around the midpoint of the nucleosomal core DNA was more resistant to digestion by DNase I in nucleosome core particles reconstituted with macroH2A1.2. Second, preparations of core particles reconstituted with macroH2A1.2 had a greater amount of material that sedimented more rapidly than mononucleosomes, suggesting that macroH2A1.2 may promote interactions between nucleosomes. Recombinant macroH2A proteins should be valuable tools for examining the effects of macroH2A on nucleosome and chromatin structure.

Dynamic relocation of epigenetic chromatin markers reveals an active role of constitutive heterochromatin in the transition from proliferation to quiescence.

Quiescent lymphocytes have small nuclei, filled with masses of facultative heterochromatin. Upon receiving mitogenic signals, these cells undergo nuclear enlargement, chromatin decondensation, the reactivation of cell proliferation, and changes in the intranuclear positioning of key genes. We examined the levels and intranuclear localization of major histone modifications and non-histone heterochromatin proteins in quiescent and reactivated mouse spleen lymphocytes. Dramatic and selective changes in localization of two heterochromatin-associated proteins, the histone variant macroH2A and HP1alpha occurred during lymphocyte reactivation. Reciprocal changes in the locations of these two proteins were observed in activated lymphocytes and cultured mouse fibroblasts induced into quiescence. We also describe a new apocentric nuclear compartment with a unique set of histone modifications that occurs as a zone of chromatin surrounding centromeric heterochromatin in differentiated lymphocytes. It is within this zone that the most significant changes occur in the transition from proliferation to quiescence. Our results suggest that constitutive centromeric heterochromatin plays an active role in cell differentiation and reactivation.

Localisation of histone macroH2A1.2 to the XY-body is not a response to the presence of asynapsed chromosome axes.

Histone macroH2A1.2 and the murine heterochromatin protein 1, HP1 beta, have both been implicated in meiotic sex chromosome inactivation (MSCI) and the formation of the XY-body in male meiosis. In order to get a closer insight into the function of histone macroH2A1.2 we have investigated the localisation of macroH2A1.2 in surface spread spermatocytes from normal male mice and in oocytes of XX and XYTdym1 mice. Oocytes of XYTdym1 mice have no XY-body or MSCI despite having an XY chromosome constitution, so the presence or absence of 'XY-body' proteins in association with the X and/or Y chromosome of these oocytes enables some discrimination between potential functions of XY-body located proteins. We demonstrate here that macroH2A1.2 localises to the X and Y chromatin of spermatocytes as they condense to form the XY-body but is not associated with the X and Y chromatin of XYTdym1 early pachytene oocytes. MacroH2A1.2 and HP1 beta co-localise to autosomal pericentromeric heterochromatin in spermatocytes. However, the two proteins show temporally and spatially distinct patterns of association to X and Y chromatin.

Meiotic sex chromosome inactivation in male mice with targeted disruptions of Xist.

X chromosome inactivation occurs twice during the life cycle of placental mammals. In normal females, one X chromosome in each cell is inactivated early in embryogenesis, while in the male, the X chromosome is inactivated together with the Y chromosome in spermatogenic cells shortly before or during early meiotic prophase. Inactivation of one X chromosome in somatic cells of females serves to equalise X-linked gene dosage between males and females, but the role of male meiotic sex chromosome inactivation (MSCI) is unknown. The inactive X-chromosome of somatic cells and male meiotic cells share similar properties such as late replication and enrichment for histone macroH2A1.2, suggesting a common mechanism of inactivation. This possibility is supported by the fact that Xist RNA that mediates somatic X-inactivation is expressed in the testis of male mice and humans. In the present study we show that both Xist RNA and Tsix RNA, an antisense RNA that controls Xist function in the soma, are expressed in the testis in a germ-cell-dependent manner. However, our finding that MSCI and sex-body formation are unaltered in mice with targeted mutations of Xist that prevent somatic X inactivation suggests that somatic X-inactivation and MSCI occur by fundamentally different mechanisms.