综述: H2A-H2B类型组蛋白及其伴侣蛋白的研究进展

染色质是真核细胞中遗传物质DNA的载体,染色质结构动态变化与DNA复制、转录、重组、修复等重要生物学事件密切相关.组蛋白是染色质结构的基本组成元件之一,组蛋白变体和组蛋白修饰是两类基本的染色质结构调控因子.在构成核小体的四种核心组蛋白(H2A、H2B、H3、H4)当中,H2A拥有最多的变体类型并在染色质结构调控中发挥重要作用.H2A组蛋白伴侣对H2A组蛋白及其变体的特异识别对于后者的折叠、修饰、传递、转运、组装、移除等生物学功能至关重要.本文着重探讨了组蛋白伴侣特异识别H2A组蛋白的分子机理,二者调控染色质结构的作用机制以及相应的生物学意义.     

组蛋白H3变体H3.3及其在细胞重编程中的作用

组蛋白是真核生物中一类进化上相对保守的蛋白质。由组蛋白八聚体及缠绕其上的DNA构成的核小体是真核生物染色质的基本组成单位。核小体使DNA保持固缩状态,既能维持基因组的稳定性,又能保证DNA序列可以正确地进行复制、转录、重组和修复。核小体调控细胞的生物过程除了通过组蛋白翻译后修饰,还可以通过组蛋白变体替换的方式进行。研究发现,组蛋白H3变体H3.3与常规组蛋白H3尽管仅有几个氨基酸的区别,但H3.3却能由特异的分子伴侣介导,整合进入染色质的特定区域,从而发挥不同的作用。同时,H3.3作为一种母源因子在正常受精和体细胞核移植等细胞重编程过程中也发挥着重要作用。本文总结了H3.3的结构特点和富集情况,探讨了特异的分子伴侣及其在细胞重编程中的作用,以期为提高体细胞重编程效率提供新思路,为体细胞重编程的应用奠定基础。     

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。     

Phosphorylation state of the histone variant H2A.X controls human stem and progenitor cell fate decisions

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组蛋白变体H3.3 L27M突变与胶质瘤发生

组蛋白变体在基因表达等基本细胞过程中发挥重要调节功能。人类有5种H3变体,分别为H3.1、 H3.2、H3.3、着丝粒特异性CENP-A和睾丸特异性H3t。人H3.3有H3F3A和H3F3B两个基因编码。采用DNA全基因组测序的方法在儿童高级别胶质瘤如恶性胶质瘤（GBM）和弥漫性内在脑桥胶质瘤（DIPG）鉴定出高频的H3F3A突变。超过70%DIPG和30%GBM携带H3.3 K27M氨基酸错义突变（27位赖氨酸被甲硫氨酸代替）。H3.3 K27M通过与组蛋白H3K27甲基转移酶EZH2亚基相互作用而抑制多梳抑制复合物2（PRC2）活性并全面减少H3K27me3含量。因此H3.3 K27M突变重塑了表观修饰状态和基因表达模式,从而驱动肿瘤发生。K27M突变可作为分子标志物以更好区分儿童胶质瘤亚型,还可作为特异、敏感的预后标志物。通过抑制组蛋白去甲基化酶如JMJD3活性而增加H3K27甲基化可作为K27M突变胶质瘤治疗的有效策略。本文综述了组蛋白变体H3.3 K27M在胶质瘤中的突变模式、分子机制和临床应用。     

Genome editing a mouse locus encoding a variant histone,H3.3B,to report on its expression in live animals

Chromatin remodeling via incorporation of histone variants plays a key role in the regulation of embryonic development. The histone variant H3.3 has been associated with a number of early events including formation of the paternal pronucleus upon fertilization. The small number of amino acid differences between H3.3 and its canonical counterparts (H3.1 and H3.2) has limited studies of the developmental significance of H3.3 deposition into chromatin due to difficulties in distinguishing the H3 isoforms. To this end, we used zinc‐finger nuclease (ZFN) mediated gene editing to introduce a small C‐terminal hemagglutinin (HA) tag to the endogenous H3.3B locus in mouse embryonic stem cells (ESCs), along with an internal ribosome entry site (IRES) and a separately translated fluorescent reporter of expression. This system will allow detection of expression driven by the reporter in cells, animals, and embryos, and will facilitate investigation of differential roles of paternal and maternal H3.3 protein during embryogenesis that would not be possible using variant‐specific antibodies. Further, the ability to monitor endogenous H3.3 protein in various cell lineages will enhance our understanding of the dynamics of this histone variant over the course of development. genesis 52:959–966, 2014. © 2014 Wiley Periodicals, Inc.     

A Synthetic Approach to Reconstruct the Evolutionary and Functional Innovations of the Plant Histone Variant H2A.W

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Conditional allelic replacement applied to genes encoding the histone variant H3.3 in the mouse

Post‐translational modifications to residues in core histones convey epigenetic information. Their function can be evaluated in amino acid substitution mutants, although to date this method has not been used in mice. To this end, we have evaluated gene targeting vectors designed for Cre recombinase‐mediated conditional allelic replacement at the two unlinked genes encoding the histone variant H3.3. The conditional alleles consist of an uninterrupted wild‐type H3.3 coding sequence upstream of a desired alternative or proxy coding sequence. The arrangement of two loxP sites allows Cre‐mediated replacement of the wild‐type coding sequence with the proxy. To demonstrate proof of principle, at each locus we replaced the wild‐type coding sequence with a fluorescent reporter. This produced null alleles that will be useful to analyse the effects of H3.3 deficiency in development. Each targeting vector can readily be retrofitted with a proxy coding sequence encoding a modified H3.3 protein. Such vectors will allow for the conditional substitution of specific residues in order to dissect the roles of H3.3 post‐translational modifications in development and disease. genesis, 51:142?146, 2013. © 2013 Wiley Periodicals, Inc.     

Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

Although recent advances in broad-scale gene expression analysis have dramatically increased our knowledge of the repertoire of mRNAs present in multiple cell types, it has become increasingly clear that examination of the expression, localization, and associations of the encoded proteins will be critical for determining their functional significance. In particular, many signaling receptors, transducers, and effectors have been proposed to act in higher-order complexes associated with physically distinct areas of the plasma membrane. Adult muscle stem cells (satellite cells) must, upon injury, respond appropriately to a wide range of extracellular stimuli: the role of such signaling scaffolds is therefore a potentially important area of inquiry. To address this question, we first isolated detergent-resistant membrane fractions from primary satellite cells, then analyzed their component proteins using liquid chromatography-tandem mass spectrometry. Transmembrane and juxtamembrane components of adhesion-mediated signaling pathways made up the largest group of identified proteins; in particular, neural cell adhesion molecule (NCAM), a multifunctional cell-surface protein that has previously been associated with muscle regeneration, was significant. Immunohistochemical analysis revealed that not only is NCAM localized to discrete areas of the plasma membrane, it is also a very early marker of commitment to terminal differentiation. Using flow cytometry, we have sorted physically homogeneous myogenic cultures into proliferating and differentiating fractions based solely upon NCAM expression.     

Histone variants as emerging regulators of embryonic stem cell identity

Dynamic regulation of chromatin structure is an important mechanism for balancing the pluripotency and cell fate decision in embryonic stem cells (ESCs). Indeed ESCs are characterized by unusual chromatin packaging, and a wide variety of chromatin regulators have been implicated in control of pluripotency and differentiation. Genome-wide maps of epigenetic factors have revealed a unique epigenetic signature in pluripotent ESCs and have contributed models to explain their plasticity. In addition to the well known epigenetic regulation through DNA methylation, histone posttranslational modifications, chromatin remodeling, and non-coding RNA, histone variants are emerging as important regulators of ESC identity. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone variants in ESC pluripotency and ESC fate, focusing, in particular, on H1 variants, H2A variants H2A.X, H2A.Z and macroH2A and H3 variant H3.3.