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.     

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.     

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.     

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.     

Distinct roles for histone chaperones in the deposition of Htz1 in chromatin

Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1–H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1–H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3–H4)₂ tetramers, H2A–H2B dimers and Htz1–H2B dimers. Nap1 can bind H2A–H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone–DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin.     

Distinct Distribution of Ectopically Expressed Histone Variants H2A.Bbd and MacroH2A in Open and Closed Chromatin Domains

Background

It becomes increasingly evident that nuclesomes are far from being identical to each other. This nucleosome diversity is due partially to the existence of histone variants encoded by separate genes. Among the known histone variants the less characterized are H2A.Bbd and different forms of macroH2A. This is especially true in the case of H2A.Bbd as there are still no commercially available antibodies specific to H2A.Bbd that can be used for chromatin immunoprecipitation (ChIP).

Methods

We have generated HeLa S3 cell lines stably expressing epitope-tagged versions of macroH2A1.1, H2A.Bbd or canonical H2A and analyzed genomic distribution of the tagged histones using ChIP-on-chip technique.

Results

The presence of histone H2A variants macroH2A1.1 and H2A.Bbd has been analyzed in the chromatin of several segments of human chromosomes 11, 16 and X that have been chosen for their different gene densities and chromatin status. Chromatin immunoprecipitation (ChIP) followed by hybridization with custom NimbleGene genomic microarrays demonstrated that in open chromatin domains containing tissue-specific along with housekeeping genes, the H2A.Bbd variant was preferentially associated with the body of a subset of transcribed genes. The macroH2A1.1 variant was virtually absent from some genes and underrepresented in others. In contrast, in closed chromatin domains which contain only tissue-specific genes inactive in HeLa S3 cells, both macroH2A1.1 and H2A.Bbd histone variants were present and often colocalized.

Conclusions

Genomic distribution of macro H2A and H2A.Bbd does not follow any simple rule and is drastically different in open and closed genomic domains.     

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.     

Changes in the nuclear deposition of histone H2A variants during pre-implantation development in mice

Histone H2A has several variants, and changes in chromatin composition associated with their replacement might involve chromatin structure remodeling. We examined the dynamics of the canonical histone H2A and its three variants, H2A.X, H2A.Z and macroH2A, in the mouse during oogenesis and pre-implantation development when genome remodeling occurs. Immunocytochemistry with specific antibodies revealed that, although H2A and all variants were deposited in the nuclei of full-grown oocytes, only histone H2A.X was abundant in the pronuclei of one-cell embryos after fertilization, in contrast with the low abundance of histone H2A and the absence of H2A.Z. The decline in H2A and the depletion of H2A.Z and macroH2A after fertilization were confirmed using Flag epitope-tagged H2A, H2A.Z and macroH2A transgenic mouse lines. Microinjection experiments with mRNA encoding the Flag-tagged proteins revealed a similar pattern of nuclear incorporation of the H2A variants. Fusion protein experiments using H2A, H2A.Z and macroH2A fused with the C-terminal 23 amino acids of H2A.X showed that the C-terminal amino acids of H2A.X function specifically to target this variant histone into chromatin in embryos after fertilization and that the absence of H2A.Z and macroH2A from the chromatin is required for normal development. These results suggest that global changes in the composition of histone H2A variants in chromatin play a role in genome remodeling after fertilization.     

The characterization of macroH2A beyond vertebrates supports an ancestral origin and conserved role for histone variants in chromatin

Histone variants play a critical role in chromatin structure and epigenetic regulation. These “deviant” proteins have been historically considered as the evolutionary descendants of ancestral canonical histones, helping specialize the nucleosome structure during eukaryotic evolution. Such view is now challenged by 2 major observations: first, canonical histones present extremely unique features not shared with any other genes; second, histone variants are widespread across many eukaryotic groups. The present work further supports the ancestral nature of histone variants by providing the first in vivo characterization of a functional macroH2A histone (a variant long defined as a specific refinement of vertebrate chromatin) in a non-vertebrate organism (the mussel Mytilus) revealing its recruitment into heterochromatic fractions of actively proliferating tissues. Combined with in silico analyses of genomic data, these results provide evidence for the widespread presence of macroH2A in metazoan animals, as well as in the holozoan Capsaspora, supporting an evolutionary origin for this histone variant lineage before the radiation of Filozoans (including Filasterea, Choanoflagellata and Metazoa). Overall, the results presented in this work help configure a new evolutionary scenario in which histone variants, rather than modern “deviants” of canonical histones, would constitute ancient components of eukaryotic chromatin.     

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.     

The histone variant macroH2A1.1 is recruited to DSBs through a mechanism involving PARP1

The repair of DNA double-strand breaks (DSBs) requires remodeling of the local chromatin architecture to allow the repair machinery to access sites of damage. Here, we report that the histone variant macroH2A1.1 is recruited to DSBs. Cells lacking macroH2A1 have defective recruitment of 53BP1, defective activation of chk2 kinase and increased radiosensitivity. Importantly, macroH2A1.1 is not incorporated into nucleosomes at DSBs, but instead associates with the chromatin through a mechanism which requires PARP1 activity. These results reveal an unusual mechanism involving a direct association of macroH2A1.1 with PARylated chromatin which is critical for retaining 53BP1 at sites of damage.