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.     

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.     

Weak but uniform enrichment of the histone variant macroH2A1 along the inactive X chromosome

We studied the enrichment and distribution of the histone variant mH2A1 in the condensed inactive X (Xi) chromosome. By using highly specific antibodies against mH2A1 and stable HEK 293 cell lines expressing either green fluorescent protein (GFP)-mH2A1 or GFP-H2A, we found that the Xi chromosome contains ~1.5-fold more mH2A1 than the autosomes. To determine the in vivo distribution of mH2A1 along the X chromosome, we used a native chromatin immunoprecipitation-on-chip technique. DNA isolated from mH2A1-immunoprecipitated nucleosomes from either male or female mouse liver were hybridized to tiling microarrays covering 5 kb around most promoters or the entire X chromosome. The data show that mH2A1 is uniformly distributed across the entire Xi chromosome. Interestingly, a stronger mH2A1 enrichment along the pseudoautosomal X chromosome region was observed in both sexes. Our results indicate a potential role for macroH2A in large-scale chromosome structure and genome stability.     

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.     

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.     

Developmental and tissue expression patterns of histone macroH2A1 subtypes

MacroH2A is a novel nucleosomal core histone that contains a large nonhistone region and a region that closely resembles a full length histone H2A. We have cloned a cDNA that contains the entire coding region of macroH2A1.2, one of the two identified subtypes of macroH2A1. MacroH2A1.2 was found to differ from the other known subtype, macroH2A1.1, in a single segment of the nonhistone region. MacroH2A1 specific antibodies revealed relatively high levels of both subtypes in adult liver and kidney. MacroH2A1.1 was much lower in fetal liver and kidney in comparison to their adult counterparts, and was not detected in adult thymus and testis, tissues with active cell division and differentiation. Both subtypes were present at very low levels or absent from mouse embryonic stem cells maintained in an undifferentiated state by growth in the presence of leukemia inhibitory factor. MacroH2A1.2 increased when the embryonic stem cells were induced to differentiate in vitro, while macroH2A1.1 remained undetectable. These results support the idea that macroH2A1.1 and macroH2A1.2 are functionally distinct, and suggest that changes in their expression may play a role in developmentally regulated changes in chromatin structure and function. J. Cell. Biochem. 65:107–113. © 1997 Wiley-Liss, Inc.     

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.     

Seasonal environmental changes regulate the expression of the histone variant macroH2A in an eurythermal fish

Adaptation to cold and warm conditions requires dramatic change in gene expression. The acclimatization process of the common carp Cyprinus carpio L. in its natural habitat has been used to study how organisms respond to natural environmental changes. At the cellular level, adaptation to cold condition is accompanied by a dramatic alteration in nucleolar structure and a down regulation of the expression of ribosomal genes. We show that the enrichment of condensed chromatin in winter adapted cells is not correlated with an increase of the heterochromatin marker trimethyl and monomethyl K20H4. However, the expression of the tri methyl K4 H3 and of the variant histone macroH2A is significantly increased during the winter season together with a hypermethylation of CpG residues. Taking into account the properties of macroH2A toward chromatin structure and dynamics and its role in gene repression our data suggest that the increased expression of macroH2A and the hypermethylation of DNA which occurs upon winter-acclimatization plays a major role for the reorganization of chromatin structure and the regulation of gene expression during the physiological adaptation to a colder environment.     

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.     

Splicing regulates NAD metabolite binding to histone macroH2A

Histone macroH2A is a hallmark of mammalian heterochromatin. Here we show that human macroH2A1.1 binds the SirT1-metabolite O-acetyl-ADP-ribose (OAADPR) through its macro domain. The 1.6-A crystal structure and mutants reveal how the metabolite is recognized. Mutually exclusive exon use in the gene H2AFY produces macroH2A1.2, whose tissue distribution differs. MacroH2A1.2 shows only subtle structural changes but cannot bind nucleotides. Alternative splicing may thus regulate the binding of nicotinamide adenine dinucleotide (NAD) metabolites to chromatin.     

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.