macroH2A1 Histone Variants Are Depleted on Active Genes but Concentrated on the Inactive X Chromosome

As the primary structural unit of eukaryotic chromatin, the nucleosome represents an important target for modifying chromatin function. One mechanism for modifying nucleosome structure and function is the substitution of variant histones for the major or canonical histones. Histone variants of different primary structures have been described in many organisms (38, 53). Genetic studies showed that histone variants H2A.X, H2A.Z, CENPA, and H3.3 have important functional properties that cannot be provided by their conventional counterparts (reviewed in reference 38). The macroH2A histone variants have an unusual structure consisting of a full-length H2A domain linked to a large nonhistone domain, producing a protein that is nearly three times the size of conventional core histones (40). The H2A domain of macroH2A histones is ∼65% identical to conventional H2As. Most of the nonhistone region appears to be derived from a domain that is found in many contexts in addition to macroH2A including as a domain in proteins involved in the replication of RNA viruses, as domains in some poly(ADP-ribose) polymerases, and as a separate protein in bacteria (4, 41). Recent studies showed that some “macrodomains,” including the one from macroH2A1.1, bind ADP ribose (27). The significance of this binding for macroH2A function is not known. There are three macroH2A variants. macroH2A1.1 and -1.2 are formed by alternate splicing of macroH2A1, and macroH2A2 is encoded by a separate gene (12, 15, 40, 44). The distribution of these three macroH2As is different in different cell types and changes during development (15, 39). The macroH2A1 (-1.1 and -1.2) content of rat liver chromatin, a relatively rich source of macroH2A1, was estimated to be one molecule for every 30 nucleosomes (40). macroH2As are highly conserved among vertebrates. They appear to be absent from most invertebrates (1, 41) but are in the sea urchin genomic database. Several lines of evidence indicate that macroH2As have a role in transcriptional repression. Immunofluorescence studies and studies with green fluorescent fusion proteins indicated that macroH2As are preferentially associated with the inactive X chromosome (12, 14-16), and a recent study indicates that macroH2A1 plays a role in the maintenance of X inactivation (23). Immunofluorescence studies indicate that macroH2As are preferentially associated with other large domains of heterochromatin, including pericentromeric heterochromatin in some cell types (16, 21), the XY body of spermatocytes (24), and transcriptionally silent senescence-associated heterochromatic foci (54). Molecular biological and biochemical studies indicate that both the H2A and the nonhistone domains of macroH2A can contribute to transcriptional repression (5, 13, 42). While immunofluorescence studies indicate that macroH2As are widely but nonrandomly distributed in the chromatin (14), technical limitations of this method make it difficult to probe the distribution of macroH2As on specific sequences. In addition, artifacts associated with antibody accessibility or chromatin density could potentially give a misleading indication of macroH2A distribution. Indeed, it has been asserted that the apparent preferential localization of macroH2A to the inactive X chromosome is due to the increased chromatin concentration of the inactive X and does not reflect a true preferential association with macroH2A (42). In order to directly examine the DNA sequences associated with macroH2A, we developed a biochemical method for purifying macroH2A1-containing chromatin fragments. We applied this technique to mouse liver chromatin and analyzed the distribution of macroH2A1 on active genes, on inactive genes, and on the X chromosome. We also identified some of the proteins that were associated with the purified macroH2A1-containing chromatin fragments.