Structural Characterization of the Histone Variant macroH2A

Abstract

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...

The histone variant macroH2A1 marks repressed autosomal chromatin, but protects a subset of its target genes from silencing

“…However, it is interesting to hypothesize that the large macrodomain-containing nonhistone region of macroH2A1 plays a role in this process. This domain represents a platform that can (1) recruit chromatin-dependent transcriptional regulators (e.g., HP1b, HDAC1, and PARP-1) (Chakravarthy et al 2005;Changolkar and Pehrson 2006;Ouararhni et al 2006;Nusinow et al 2007), (2) provide a novel platform for covalent modifications (e.g., S137 phosphorylation) (Bernstein et al 2008), and (3) bind small molecule ligands (e.g., the NAD + metabolites poly[ADP-ribose], ADP-ribose, and O-acetyl-ADP-ribose, in the case of macroH2A1.1) (Kustatscher et al 2005;Timinszky et al 2009). All of these features may locally regulate the function of macroH2A1-containing nucleosomes and thereby affect the expression of a subset of the genes to which it is bound.…”

“…Nevertheless, the structure of homotypic and heterotypic macroH2A-containing nucleosomes is largely similar to nonvariant histone nucleosomes (Chakravarthy et al 2005). While more salt-stable, macroH2A1-containing nucleosomes organize the same amount of DNA (147 base pairs [bp]) as their canonical counterparts (Abbott et al 2004).…”

“…The macrodomain of macroH2A provides a unique platform for the recruitment of protein factors to chromatin, and a handful of such factors have been identified (e.g., PARP-1, HDAC1) (Chakravarthy et al 2005;HernandezMunoz et al 2005;Ouararhni et al 2006;Nusinow et al 2007). Several macrodomains, including the macrodomain of one of the splice variants of macroH2A1 (macroH2A1.1), have been shown to bind NAD + metabolites, including poly(ADP-ribose), ADP-ribose and O-acetyl-ADP-ribose, with high affinity (Kustatscher et al 2005;Ahel et al 2009;Gottschalk et al 2009;Kraus 2009;Timinszky et al 2009).…”

Histone variants: dynamic punctuation in transcription

“…The macro domain on the C terminus is connected through an unstructured linker to a histone fold domain that is ;60% identical to canonical H2A, resulting in a histone protein that is approximately three times the size of H2A (Chakravarthy et al 2005). Macro domain-containing proteins are found in many organisms; however, macroH2A is restricted to vertebrates and a few invertebrates (Talbert and Henikoff 2010).…”

ATRX-mediated chromatin association of histone variant macroH2A1 regulates α-globin expression

“…mH2A is abundant in heterochromatin, including senescence-associated heterochromatic foci (SAHF) and the inactivated X chromosome (Xi) (Costanzi and Pehrson 1998;Zhang et al 2005). In vitro studies suggest that the macro domain sterically hinders access of transcription factors to DNA, while mH2A's L1 loop produces inflexible nucleosomes (Angelov et al 2003;Chakravarthy et al 2005).Our group has recently demonstrated a role for mH2A isoforms in suppressing melanoma progression, and others have linked mH2A expression or its splice patterns to breast and lung cancer (Sporn et al 2009;Kapoor et al 2010;Novikov et al 2011). However, the factors that regulate the association of mH2A with chromatin remain obscure.…”