Histone variants--the structure behind the function.

In recent years, the chromatin field has witnessed a renewed interest in histone variants as pertaining to their structural role, but mainly because of the functional specificity they impart to chromatin. In this review, I am going to discuss several of the most recent structural studies on core histone (H2A.Bbd, H2A.Z, H2A.X, macroH2A, H3.3, CENP-A) and linker histone variants (histone H1 microheterogeneity) focusing on their role in nucleosome stability and chromatin fibre dynamics with special emphasis on their possible functional implications. The data accumulated to date indicates that histone variability plays an important role in the histone-mediated regulation of chromatin metabolism. Understanding and deciphering the underlying structural amino acid code behind such variability remains one of the most exciting future challenges in chromatin research.     

The Schizosaccharomyces pombe JmjC-Protein,Msc1, Prevents H2A.Z Localization in Centromeric and Subtelomeric Chromatin Domains

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.     

Changes in the nuclear deposition of histone H2A variants during pre-implantation development in mice

Histone H2A has several variants, and changes in chromatin composition associated with their replacement might involve chromatin structure remodeling. We examined the dynamics of the canonical histone H2A and its three variants, H2A.X, H2A.Z and macroH2A, in the mouse during oogenesis and pre-implantation development when genome remodeling occurs. Immunocytochemistry with specific antibodies revealed that, although H2A and all variants were deposited in the nuclei of full-grown oocytes, only histone H2A.X was abundant in the pronuclei of one-cell embryos after fertilization, in contrast with the low abundance of histone H2A and the absence of H2A.Z. The decline in H2A and the depletion of H2A.Z and macroH2A after fertilization were confirmed using Flag epitope-tagged H2A, H2A.Z and macroH2A transgenic mouse lines. Microinjection experiments with mRNA encoding the Flag-tagged proteins revealed a similar pattern of nuclear incorporation of the H2A variants. Fusion protein experiments using H2A, H2A.Z and macroH2A fused with the C-terminal 23 amino acids of H2A.X showed that the C-terminal amino acids of H2A.X function specifically to target this variant histone into chromatin in embryos after fertilization and that the absence of H2A.Z and macroH2A from the chromatin is required for normal development. These results suggest that global changes in the composition of histone H2A variants in chromatin play a role in genome remodeling after fertilization.     

The X and Y chromosomes assemble into H2A.Z-containing [corrected] facultative heterochromatin [corrected] following meiosis

Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1beta and H3 di- and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.     

The evolutionary differentiation of two histone H2A.Z variants in chordates (H2A.Z-1 and H2A.Z-2) is mediated by a stepwise mutation process that affects three amino acid residues

Background  

The histone H2A family encompasses the greatest number of core histone variants of which the replacement variant H2A.Z is currently one of the most heavily studied. No clear mechanism for the functional variability that H2A.Z imparts to chromatin has yet been proposed. While most of the past studies have referred to H2A.Z generically as a single protein, in vertebrates it is a mixture of two protein forms H2A.Z-1 (previously H2A.Z) and H2A.Z-2 (previously H2A.F/Z or H2A.V) that differ by three amino acids.     

H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization

The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.     

Key functional regions in the histone variant H2A.Z C-terminal docking domain

The incorporation of histone variants into nucleosomes represents one way of altering the chromatin structure to accommodate diverse functions. Histone variant H2A.Z has specific roles in gene regulation, heterochromatin boundary formation, and genomic integrity. The precise features required for H2A.Z to function and specify an identity different from canonical H2A remain to be fully explored. Analysis of the C-terminal docking domain of H2A.Z in Saccharomyces cerevisiae using epistatic miniarray profile (E-MAP) uncovered nuanced requirements of the H2A.Z C-terminal region for cell growth when additional genes were compromised. Moreover, the H2A.Z(1-114) truncation, lacking the last 20 amino acids of the protein, did not support regular H2A.Z functions, such as resistance to genotoxic stress, restriction of heterochromatin in its native context, GAL1 gene activation, and chromatin anchoring. The corresponding region of H2A could fully rescue the strong defects caused by loss of this functionally essential region in the C terminus of H2A.Z. Despite the dramatic reduction in function, the H2A.Z(1-114) truncation still bound the H2A.Z deposition complex SWR1-C, the histone chaperone Chz1, and histone H2B. These data are consistent with a model in which retaining the variant in chromatin after its deposition by SWR1-C is a crucial determinant of its function.     

Histone H2A.Z acid patch residues required for deposition and function

The incorporation of histone variants is one mechanism used by the eukaryotic cell to alter the generally repressive chromatin template. However, the exact molecular mechanisms that direct this incorporation are not well understood. The SWR1 chromatin remodeling complex that binds to and directs incorporation of histone variant H2A.Z into chromatin has been characterized, but significantly less information is available concerning the requirements on the H2A.Z target molecule. We performed an unbiased mutagenic screen designed to elucidate the function of H2A.Z in Saccharomyces cerevisiae. The screen identified residues within the conserved acidic patch of H2A.Z as being important for the function of the variant. We characterized single point mutations in the patch that are phenotypically sensitive to a variety of growth conditions and are expressed at lower protein levels, but are functionally defective (htz1-D99A, htz1-D99K, and htz1-E101K). The mutants were significantly less detectable by chromatin immunoprecipitation at PHO5, a gene previously described to be enriched for H2A.Z. These results identify acidic patch residues of H2A.Z that are critical for mediating deposition and function in chromatin, and represent potential candidates for the interaction of H2A.Z with its deposition and/or targeting machinery.     

Reconciling the positive and negative roles of histone H2A.Z in gene transcription

The incorporation of variant histone H2A.Z within chromatin is important for proper gene expression and genome stability. H2A.Z is inserted at discrete loci by the Swr1 or Swr1-like remodeling complexes, although very little is known about the nature of the targeting mechanism involved. Replacement of canonical histone H2A for H2A.Z has been shown to modify nucleosome dynamics, although discrepancies still exist in the literature regarding the mechanisms. Recent experiments have shown that H2A.Z can allow nucleosomes to adopt stable translational positions as compared to H2A, which could influence the accessibility to DNA regulatory proteins. This review provides a brief overview of H2A.Z biology and presents hypotheses that could reconcile contradictory reports that are found in the literature regarding the influence of H2A.Z on nucleosome stability.     

组蛋白变异体H2A.Z的研究进展

H2A.Z是组蛋白H2A的变异体之一,是高度保守的组蛋白变异体,参与保护常染色体,防止形成异染色质;并且与转录调节、抗沉默、沉默和基因组稳定性有关。组蛋白变异体H2A.Z可能与染色体形成独立的结构域,从而调节染色质结构功能。但是,H2A.Z对染色体结构功能的作用机制还不是很清楚。组蛋白变异体H2A.Z和它的表观遗传修饰对染色体动态结构和功能起重要的作用。该文将对组蛋白变异体H2A.Z进行综述。