Histone variant macroH2A contains two distinct macrochromatin domains capable of directing macroH2A to the inactive X chromosome

Chromatin on the inactive X chromosome (Xi) of female mammals is enriched for the histone variant macroH2A that can be detected at interphase as a distinct nuclear structure referred to as a macro chromatin body (MCB). Green fluorescent protein-tagged and Myc epitope-tagged macroH2A readily form an MCB in the nuclei of transfected female, but not male, cells. Using targeted disruptions, we have identified two macrochromatin domains within macroH2A that are independently capable of MCB formation and association with the Xi. Complete removal of the non-histone C-terminal tail does not reduce the efficiency of association of the variant histone domain of macroH2A with the Xi, indicating that the histone portion alone can target the Xi. The non-histone domain by itself is incapable of MCB formation. However, when directed to the nucleosome by fusion to core histone H2A or H2B, the non-histone tail forms an MCB that appears identical to that of the endogenous protein. Mutagenesis of the non-histone portion of macroH2A localized the region required for MCB formation and targeting to the Xi to an ~190 amino acid region.     

Centrosomal association of histone macroH2A1.2 in embryonic stem cells and somatic cells

The histone 2A variant macroH2A1.2 is expressed in female and male mammals and is implicated in X-chromosome inactivation and autosomal gene silencing. In undifferentiated and early differentiating murine embryonic stem (ES) cells a cytosolic pool of macroH2A1.2 has recently been reported and found to be associated with the centrosome. Here, we show that the centrosomal association of macroH2A1.2 is a widespread phenomenon and is not restricted to undifferentiated and early differentiating ES cells. By indirect immunofluorescence we detect macroH2A1.2 protein in a juxtanuclear structure that duplicates once per cell cycle and colocalizes with centrosomal gamma-tubulin in both XX and XY ES cells prior to and throughout their differentiation. MacroH2A1.2 localization to the centrosome is also observed in female and male somatic cells, both in interphase and in mitosis. Biochemical analysis demonstrates that the association between macroH2A1.2 and the centrosome in somatic cells is stable, as macroH2A1.2 copurifies with centrosomes isolated from human lymphoblasts. Therefore, in addition to a nuclear pool of macroH2A1.2 a fraction of the histone is associated with the centrosome in various cell types and throughout ES cell differentiation.     

Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A)

Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.     

Evolutionary conservation of histone macroH2A subtypes and domains.

Histone macroH2A is an unusual core histone that contains a large non-histone region, and a region that resembles a full length H2A. We examined theconservation of this novel structural arrangement by cloning chicken macroH2A cDNAs and comparing them to their rat counterparts. The amino acid sequences of the two known macroH2A subtypes are >95% identical between these species despite evolutionary separation of approximately 300 million years. The H2A region of macroH2A is completely conserved, and thus is even more conserved than conventional H2A in these species. The origin of the non-histone domain was examined by comparing its sequence to proteins found in bacteria and RNA viruses. These comparisons indicate that this domain is derived from a gene that originated prior to the appearance of eukaryotes, and suggest that the non-histone region has retained the basic function of its ancestral gene.     

Cell cycle-dependent localization of macroH2A in chromatin of the inactive X chromosome

One of several features acquired by chromatin of the inactive X chromosome (Xi) is enrichment for the core histone H2A variant macroH2A within a distinct nuclear structure referred to as a macrochromatin body (MCB). In addition to localizing to the MCB, macroH2A accumulates at a perinuclear structure centered at the centrosome. To better understand the association of macroH2A1 with the centrosome and the formation of an MCB, we investigated the distribution of macroH2A1 throughout the somatic cell cycle. Unlike Xi-specific RNA, which associates with the Xi throughout interphase, the appearance of an MCB is predominantly a feature of S phase. Although the MCB dissipates during late S phase and G2 before reforming in late G1, macroH2A1 remains associated during mitosis with specific regions of the Xi, including at the X inactivation center. This association yields a distinct macroH2A banding pattern that overlaps with the site of histone H3 lysine-4 methylation centered at the DXZ4 locus in Xq24. The centrosomal pool of macroH2A1 accumulates in the presence of an inhibitor of the 20S proteasome. Therefore, targeting of macroH2A1 to the centrosome is likely part of a degradation pathway, a mechanism common to a variety of other chromatin proteins.     

Dynamic relocalization of histone MacroH2A1 from centrosomes to inactive X chromosomes during X inactivation

Histone variant macroH2A1 (macroH2A1) contains an NH(2)-terminal domain that is highly similar to core histone H2A and a larger COOH-terminal domain of unknown function. MacroH2A1 is expressed at similar levels in male and female embryonic stem (ES) cells and adult tissues, but a portion of total macroH2A1 protein localizes to the inactive X chromosomes (Xi) of differentiated female cells in concentrations called macrochromatin bodies. Here, we show that centrosomes of undifferentiated male and female ES cells harbor a substantial store of macroH2A1 as a nonchromatin-associated pool. Greater than 95% of centrosomes from undifferentiated ES cells contain macroH2A1. Cell fractionation experiments confirmed that macroH2A1 resides at a pericentrosomal location in close proximity to the known centrosomal proteins gamma-tubulin and Skp1. Retention of macroH2A1 at centrosomes was partially labile in the presence of nocodazole suggesting that intact microtubules are necessary for accumulation of macroH2A1 at centrosomes. Upon differentiation of female ES cells, Xist RNA expression became upregulated and monoallelic as judged by fluorescent in situ hybridization, but early Xist signals lacked associated macroH2A1. Xi acquired macroH2A1 soon thereafter as indicated by the colocalization of Xist RNA and macroH2A1. Accumulation of macroH2A1 on X chromosomes occurred with a corresponding loss of centrosomal macroH2A1. Our results define a sequence for the loading of macroH2A1 on the Xi and place this event in the context of differentiation and Xist expression. Furthermore, these results suggest a role for the centrosome in the X inactivation process.     

Poly(ADP-ribose) polymerase 1 is inhibited by a histone H2A variant, MacroH2A, and contributes to silencing of the inactive X chromosome

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in modulating chromatin structure, regulation of gene expression, and sensing DNA damage. Here, we report that PARP-1 enzymatic activity is inhibited by macroH2A, a vertebrate histone H2A variant that is enriched on facultative heterochromatin. MacroH2A family members have a large C-terminal non-histone domain (NHD) and H2A-like histone domain. MacroH2A1.2 and PARP-1 interact in vivo and in vitro via the NHD. The NHD of each macroH2A family member was sufficient to inhibit PARP-1 enzymatic activity in vitro. The NHD of macroH2A1.2 was a mixed inhibitor of PARP-1 catalytic activity, with affects on both catalytic activity and the substrate binding affinity of PARP-1. Depletion of PARP-1 by RNA interference caused reactivation of a reporter gene on the inactive X chromosome, demonstrating that PARP-1 participates in the maintenance of silencing. These results suggest that one function of macroH2A in gene silencing is to inhibit PARP-1 enzymatic activity, and this may affect PARP-1 association with chromatin.     

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 linker region of macroH2A promotes self-association of nucleosomal arrays

MacroH2A is a histone variant found in higher eukaryotes localized at the inactive X chromosome and is known to maintain heterochromatic regions in the genome. MacroH2A consists of a conserved histone domain and a macro domain connected by a linker region. To understand the contributions of the three domains to chromatin condensation, we incorporated various constructs of macroH2A into defined nucleosomal arrays and analyzed their impact on in vitro chromatin compaction. The folding and oligomerization properties of arrays containing full-length macroH2A (macroH2A(FL)), macroH2A(1-161) (encompassing the histone domain and linker region), and macroH2A(1-122) (histone domain only) were compared with major-type H2A arrays. Analytical ultracentrifugation and atomic force microscope imaging indicate that macroH2A(1-161)-containing arrays favor condensation under conditions where major-type arrays are nearly fully extended. In contrast, arrays with macroH2A(FL) exhibit behavior similar to that of major-type arrays. This suggests that the linker region of macroH2A facilitates array condensation and that this behavior is inhibited by the macro domain. Furthermore, chimeric major-type H2A arrays containing the macroH2A linker domain (H2A(ML)) exhibited the same condensation properties as macroH2A(1-161) arrays, thus emphasizing the intriguing behavior of the macroH2A linker region.     

LSH catalyzes ATP-driven exchange of histone variants macroH2A1 and macroH2A2

LSH, a homologue of the ISWI/SNF2 family of chromatin remodelers, is required in vivo for deposition of the histone variants macroH2A1 and macroH2A2 at specific genomic locations. However, it remains unknown whether LSH is directly involved in this process or promotes other factors. Here we show that recombinant LSH interacts in vitro with macroH2A1–H2B and macroH2A2–H2B dimers, but not with H2A.Z–H2B dimers. Moreover, LSH catalyzes the transfer of macroH2A into mono-nucleosomes reconstituted with canonical core histones in an ATP dependent manner. LSH requires the ATP binding site and the replacement process is unidirectional leading to heterotypic and homotypic nucleosomes. Both variants macroH2A1 and macroH2A2 are equally well incorporated into the nucleosome. The histone exchange reaction is specific for histone variant macroH2A, since LSH is not capable to incorporate H2A.Z. These findings define a previously unknown role for LSH in chromatin remodeling and identify a novel molecular mechanism for deposition of the histone variant macroH2A.     

QKI-mediated alternative splicing of the histone variant MacroH2A1 regulates cancer cell proliferation

The histone variant macroH2A1 contains a carboxyl-terminal ～30-kDa domain called a macro domain. MacroH2A1 is produced as one of two alternatively spliced forms, macroH2A1.1 and macroH2A1.2. While the macro domain of macroH2A1.1 can interact with NAD(+)-derived small molecules, such as poly(ADP-ribose), macroH2A1.2's macro domain cannot. Here, we show that changes in the alternative splicing of macroH2A1 pre-mRNA, which lead to a decrease in macroH2A1.1 expression, occur in a variety of cancers, including testicular, lung, bladder, cervical, breast, colon, ovarian, and endometrial. Furthermore, reintroduction of macroH2A1.1 suppresses the proliferation of lung and cervical cancer cells in a manner that requires the ability of macroH2A1.1 to bind NAD(+)-derived metabolites. MacroH2A1.1-mediated suppression of proliferation occurs, at least in part, through the reduction of poly(ADP-ribose) polymerase 1 (PARP-1) protein levels. By analyzing publically available expression and splicing microarray data, we identified splicing factors that correlate with alterations in macroH2A1 splicing. Using RNA interference, we demonstrate that one of these factors, QKI, regulates the alternative splicing of macroH2A1 pre-mRNA, resulting in increased levels of macroH2A1.1. Finally, we demonstrate that QKI expression is significantly reduced in many of the same cancer types that demonstrate a reduction in macroH2A1.1 splicing.     

MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP‐ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1‐dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform‐specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.     

Mapping post-translational modifications of the histone variant MacroH2A1 using tandem mass spectrometry

Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although post-translational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys(115) is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the epsilon amino group of lysine residues Lys(17), Lys(122), and Lys(238) and phosphorylated on Thr(128). Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.     

The histone domain of macroH2A1 contains several dispersed elements that are each sufficient to direct enrichment on the inactive X chromosome

Histone variants replace the core histones in a substantial fraction of nucleosomes, affecting chromatin structure and impacting chromatin-templated processes. In many instances incorporation of histone variants results in formation of specialized regions of chromatin. Proper localization of histone variants to distinct regions of the genome is critical for their function, yet how this specific localization is achieved remains unclear. macroH2A1 is enriched on the inactive X chromosome in female mammalian cells, where it functions to maintain gene silencing. macroH2A1 consists of a histone H2A-like histone domain and a large, globular C-terminal macro domain that is not present in other histone proteins. The histone domain of macroH2A1 is alone sufficient to direct enrichment on the inactive X chromosome when expressed in female cells, indicating that sequences important for correct localization lie in this domain. Here we investigate whether divergent sequences of the H2A variant macroH2A1 contribute to its correct localization. We mapped the regions of the macroH2A1 histone domain that are sufficient for localization to the inactive X chromosome using chimeras between H2A and the histone domain of macroH2A1. Multiple short sequences dispersed along the macroH2A1 histone domain individually supported enrichment on the inactive X chromosome when introduced into H2A. These sequences map to the surface of the macroH2A1/H2B dimer, but are buried in the crystal structure of the macroH2A1 containing nucleosome, suggesting that they may contribute to recognition by macroH2A1/H2B deposition factors.