Histone H2A (H2A.X and H2A.Z) variants in molluscs: molecular characterization and potential implications for chromatin dynamics

Histone variants are used by the cell to build specialized nucleosomes, replacing canonical histones and generating functionally specialized chromatin domains. Among many other processes, the specialization imparted by histone H2A (H2A.X and H2A.Z) variants to the nucleosome core particle constitutes the earliest response to DNA damage in the cell. Consequently, chromatin-based genotoxicity tests have been developed in those cases where enough information pertaining chromatin structure and dynamics is available (i.e., human and mouse). However, detailed chromatin knowledge is almost absent in most organisms, specially protostome animals. Molluscs (which represent sentinel organisms for the study of pollution) are not an exception to this lack of knowledge. In the present work we first identified the existence of functionally differentiated histone H2A.X and H2A.Z variants in the mussel Mytilus galloprovincialis (MgH2A.X and MgH2A.Z), a marine organism widely used in biomonitoring programs. Our results support the functional specialization of these variants based on: a) their active expression in different tissues, as revealed by the isolation of native MgH2A.X and MgH2A.Z proteins in gonad and hepatopancreas; b) the evolutionary conservation of different residues encompassing functional relevance; and c) their ability to confer specialization to nucleosomes, as revealed by nucleosome reconstitution experiments using recombinant MgH2A.X and MgH2A.Z histones. Given the seminal role of these variants in maintaining genomic integrity and regulating gene expression, their preliminary characterization opens up new potential applications for the future development of chromatin-based genotoxicity tests in pollution biomonitoring programs.     

盐离子作用下组蛋白变体H2A.Z增强核小体结构的稳定性

真核生物染色质的基本结构组成单元是核小体,基因组DNA被压缩在染色质中,核小体的存在通常会抑制转录、复制、修复和重组等发生在DNA模板上的生物学过程。组蛋白变体H2A.Z可以调控染色质结构进而影响基因的转录过程,但其详细的调控机制仍未研究清楚。为了比较含有组蛋白变体H2A.Z的核小体和常规核小体在盐离子作用下的稳定性差异,本文采用Förster共振能量转移的方法检测氯化钠、氯化钾、氯化锰、氯化钙、氯化镁等离子对核小体的解聚影响。实验对Widom 601 DNA序列进行双荧光Cy3和Cy5标记,通过荧光信号值的变化来反映核小体的解聚变化。Förster共振能量转移检测结果显示:在氯化钠、氯化钾、氯化锰、氯化钙和氯化镁作用下,含有组蛋白变体H2A.Z的核小体解聚速度相比于常规核小体要慢,且氯化钙、氯化锰和氯化镁的影响更明显。电泳分析结果表明,在75℃条件下含有组蛋白变体H2A.Z的核小体的解聚速率明显低于常规核小体。采用荧光热漂移检测(fluorescence thermal shift analysis , FTS)进一步分析含有组蛋白变体H2A.Z核小体的稳定性,发现两类核小体的荧光信号均呈现2个明显的增长期,含有组蛋白变体H2A.Z核小体的第1个荧光信号增速期所对应的温度明显高于常规核小体,表明核小体中H2A.Z/H2B二聚体的解聚变性温度要高于常规的H2A/H2B二聚体,含有组蛋白变体H2A.Z核小体的热稳定性高。研究结果均表明,含有组蛋白变体H2A.Z的核小体的结构比常规核小体的结构稳定。     

Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome

Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.     

Solution structure of variant H2A.Z.1 nucleosome investigated by small-angle X-ray and neutron scatterings

Solution structures of nucleosomes containing a human histone variant, H2A.Z.1, were measured by small-angle X-ray and neutron scatterings (SAXS and SANS). SAXS revealed that the outer shape, reflecting the DNA shape, of the H2A.Z.1 nucleosome is almost the same as that of the canonical H2A nucleosome. In contrast, SANS employing a contrast variation technique revealed that the histone octamer of the H2A.Z.1 nucleosome is smaller than that of the canonical nucleosome. The DNA within the H2A.Z.1 nucleosome was more susceptible to micrococcal nuclease than that within the canonical nucleosome. These results suggested that the DNA is loosely wrapped around the histone core in the H2A.Z.1 nucleosome.     

A conserved function for the H2A.Z C terminus

Histone H2A variants generate diversity in chromatin structure and functions, as nucleosomes containing variant H2A histones have altered physical, chemical, and biological properties. H2A.Z is an evolutionarily ancient and highly conserved H2A variant that regulates processes ranging from gene expression to the DNA damage response. Here we find that the unstructured portion of the C-terminal tail of H2A.Z is required for the normal functions of this histone variant in budding yeast. We have also identified a novel splice isoform of the human H2A.Z-2 gene that encodes a C-terminally truncated H2A.Z protein that is similar to the truncation mutants we identified in yeast. The short forms of H2A.Z in both yeast and human cells are more loosely associated with chromatin than the full-length proteins, indicating a conserved function for the H2A.Z C-terminal tail in regulating the association of H2A.Z with nucleosomes.     

Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2

Nucleosomes are dynamic entities with wide‐ranging compositional variations. Human histone variants H2A.B and H2A.Z.2.2 play critical roles in multiple biological processes by forming unstable nucleosomes and open chromatin structures, but how H2A.B and H2A.Z.2.2 confer these dynamic features to nucleosomes remains unclear. Here, we report cryo‐EM structures of nucleosome core particles containing human H2A.B (H2A.B‐NCP) at atomic resolution, identifying large‐scale structural rearrangements in the histone octamer in H2A.B‐NCP. H2A.B‐NCP compacts approximately 103 bp of DNA wrapping around the core histones in approximately 1.2 left‐handed superhelical turns, in sharp contrast to canonical nucleosome encompassing approximately 1.7 turns of DNA. Micrococcal nuclease digestion assay reveals that nineteen H2A.B‐specific residues, including a ROF (“regulating‐octamer‐folding”) sequence of six consecutive residues, are responsible for loosening of H2A.B‐NCPs. Unlike H2A.B‐NCP, the H2A.Z.2.2‐containing nucleosome (Z.2.2‐NCP) adopts a less‐extended structure and compacts around 125 bp of DNA. Further investigation uncovers a crucial role for the H2A.Z.2.2‐specific ROF in both H2A.Z.2.2‐NCP opening and SWR1‐dependent histone replacement. Taken together, these first high‐resolution structure of unstable nucleosomes induced by histone H2A variants elucidate specific functions of H2A.B and H2A.Z.2.2 in enhancing chromatin dynamics.     

Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA‐based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z‐nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate‐specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z‐specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome‐wide mapping reveals that PWWP2A binds selectively to H2A.Z‐containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C‐terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z‐specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.     

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.     

A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome

Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the histone octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)(2) tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z.     

致病疫霉组蛋白H2A变异体序列与表达

在真核生物中,DNA缠绕在组蛋白上形成核小体,一个组蛋白分子包括H2A、H2B、H3和H4各2个核心组蛋白亚基。在这4种核心组蛋白中,H2A富含多样化,且在细胞的生物途径中起重要作用的变异体,因此,H2A家族一直是研究热点。致病疫霉是重要的病原菌也是研究卵菌的模式物种之一,目前关于卵菌表观遗传的研究还未见报道。本研究针对致病疫霉组蛋白H2A变异体,利用基因组信息和基因芯片数据,通过序列比对、系统发育分析以及基因表达水平检测,发现在致病疫霉基因组中存在组蛋白H2A变异体H2A.X.1、H2A.X.2和H2A.Z,它们在不同生长发育阶段和侵染过程呈现特异的表达谱。研究结果为进一步研究致病疫霉表观遗传机制奠定了基础。     

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.     

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.