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Mammalian centromeres are not defined by a consensus DNA sequence. In all eukaryotes a hallmark of functional centromeres--both normal ones and those formed aberrantly at atypical loci--is the accumulation of centromere protein A (CENP-A), a histone variant that replaces H3 in centromeric nucleosomes. Here we show using deuterium exchange/mass spectrometry coupled with hydrodynamic measures that CENP-A and histone H4 form sub-nucleosomal tetramers that are more compact and conformationally more rigid than the corresponding tetramers of histones H3 and H4. Substitution into histone H3 of the domain of CENP-A responsible for compaction is sufficient to direct it to centromeres. Thus, the centromere-targeting domain of CENP-A confers a unique structural rigidity to the nucleosomes into which it assembles, and is likely to have a role in maintaining centromere identity.

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...

Summary The specialized ribonuclease Dicer plays a central role in eukaryotic gene expression by producing small regulatory RNAs – miRNAs and siRNAs – from larger double stranded RNA (dsRNA) substrates. Although Dicer will cleave both imperfectly base-paired hairpin structures (pre-miRNAs) and perfect duplexes (pre-siRNAs) in vitro, it has not been clear whether these are mechanistically equivalent substrates and how dsRNA binding proteins such as TRBP influence substrate selection and RNA processing efficiency. We show here that human Dicer is much faster at processing a pre-miRNA substrate compared to a pre-siRNA substrate under both single and multiple turnover conditions. Maximal cleavage rates (Vmax) calculated by Michaelis-Menten analysis differed by more than 100-fold under multiple turnover conditions. TRBP was found to enhance dicing of both substrates to similar extents, and this stimulation required the two N-terminal dsRNA binding domains of TRBP. These results demonstrate that multiple factors influence dicing kinetics. While TRBP stimulates dicing by enhancing the stability of Dicer-substrate complexes, Dicer itself generates product RNAs at rates determined at least in part by the structural properties of the substrate.

SUMMARY Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled, due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.