The unusual histone variant macroH2A (mH2A) has been associated with repression of transcription, but the molecular mechanisms by which it exerts this function are unknown. Here we have identified a mechanism by which the different domains of mH2A may be involved in the repression of transcription. Evidence is presented that the presence of mH2A in a positioned nucleosome interferes with the binding of the transcription factor NF-kappaB. The nonhistone region of mH2A was identified to be associated with this interference. Importantly, the presence of macroH2A was found to severely impede SWI/SNF nucleosome remodeling and movement to neighboring DNA segments. This property of mH2A was demonstrated to reside only in its H2A-like domain. A hypothesis explaining the role of histone variants in transcriptional regulation is proposed.

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-Å X-ray structure of the nonhistone region reveals an ␣/␤ 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 compaction of DNA into chromatin is an important regulator of DNA accessibility. The nucleosome core particle (NCP), the fundamental repeating unit of chromatin, plays a central role in the regulation of transcription, replication, and repair. An important emerging mechanism to alter the fundamental biochemical composition and characteristics of chromatin is the substitution of major-type core histones with histone variants (18). This may be achieved by structural alterations in the NCP and/or in chromatin higher-order structures that are brought about by the amino acid sequence differences between the histone variants and their corresponding core counterparts (9; for an example, see reference 28). macroH2A1, with a molecular weight of ϳ40 kDa, is almost three times the size of major, replication-dependent H2A and is unique among known histone variants due to its unconventional tripartite structural organization (23). The N-terminal third of its amino acid sequence (amino acids [aa] 1 through 122) is 64% identical to major H2A. A C-terminal nonhistone region (aa 161 through 371) is linked to the histone homology domain via a linker region (aa 123 through 160) (Fig. 1A). The C-terminal nonhistone region in itself exhibits amino acid similarities to members of a functionally highly diverse group of proteins that exist in organisms ranging from bacteria and archaea to eukaryotes, and its function remains unknown (24). macroH2A preferentially localizes at the inactive X-chromosome of mammalian female cells, where it may contribute to the maintenance of transcriptionally silent chromatin (7). Recent studies indicate that macroH2A-containing nucleosomes are repressive toward transcription (4, 25). Here, we have combined X-ray crystallography with biochemical and mutational studies to better understand the biological function of macroH2A. MATERIALS AND METHODSExpression and purification of histone proteins and reconstitution of nucleosomes. All histones were overexpressed in BL21(DE3)-plysS (Stratagene) and purified using previously published protocols (17). The histone domain of macroH2A (aa 1 to 120; macroH2A-HD), together with mouse H2B, H3, and H4, was refolded to a histone octamer (macrooctamer). This was reconstituted onto a 146-bp palindromic DN...

Histone macroH2A, which is a subtype of histone H2A, possesses a histone H2A-like portion fused to a relatively long non-histone portion. MacroH2A has been shown to associate preferentially with the inactive X chromosome [1]. To investigate the specificity of this association, the nuclear distribution of macroH2A was compared with that of regular core histones. In normal human female fibroblasts, all anti-histone antibodies that were tested (including anti-macroH2A antibody) preferentially labeled the inactive X chromosome. Moreover, when expressed as green fluorescent protein (GFP) fusions, both histone H2A and macroH2A were concentrated in the Barr body. These data clearly show the presence of a higher density of nucleosomes in the inactive X chromosome. Accordingly, the specificity of the macroH2A association with the inactive X chromosome should be reconsidered. While investigating the role of macroH2A, we found that the proximity of the non-histone region of macroH2A to a promoter could lead to a specific repression of transcription, suggesting that the incorporation of macroH2A into chromatin might help to establish the stable pattern of gene expression in differentiated cells.