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...

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