组蛋白变体的染色质组装

真核细胞的染色质组装是组蛋白和DNA有序地形成核小体和染色质的过程.通过调节DNA的开放或折叠状态,染色质组装不但影响遗传信息的编码和存储,也决定了遗传信息的提取和解读.作为染色质组装的重要调控因子,组蛋白变体和组蛋白伴侣在与DNA相关的生命活动进程中发挥着至关重要的作用.本文综述了组蛋白变体H2A.Z以及CENP-A进行染色质组装的研究进展,并着重讨论了组蛋白变体和组蛋白伴侣在染色质组装中的重要作用.     

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.     

组蛋白变体的功能与识别机制

组蛋白变体(histone variant)是常规组蛋白的变异体,在染色质的特定位置或特定生物学事件中替换常规组蛋白,调控染色质结构以及相关生物学过程。组蛋白伴侣(histone chaperone)是指可以结合组蛋白,运送组蛋白参与染色质组装和去组装等重要功能的蛋白质。综述了几种主要组蛋白变体在真核生物染色质高级结构的形成及维持、细胞编程与重编程的表观遗传机制等生命进程中发挥的重要作用,以及这些组蛋白变体与其特征伴侣之间特异识别的分子机制。     

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.     

组蛋白变体及组蛋白替换

组蛋白作为核小体的基本组分,是染色质的结构和功能必需的。对于不同状态的染色质,核小体中会组装入相应的组蛋白变体,并且各种组蛋白变体的尾部也能发生多种修饰。这些变体通过改变核小体的空间构象和稳定性,决定基因转录的激活或沉默,DNA的修复,染色体的异染色化等。在组蛋白替换过程中,组蛋白变体是通过相应的染色质重构复合物组装入核小体,不同的变体有着不同的组装途径。对组蛋白变体的研究是近年来表观遗传学新的研究热点,也是对“组蛋白密码”的新的诠释。并且,组蛋白替换揭示了DNA-组蛋白相互作用变化的一种新的机制。

     

技术与方法体外组装含有组蛋白变体H2A.Z及H3.3的核小体

核小体是构成真核生物染色质的基本结构单位,组蛋白变体H2A.Z及H3.3对染色质结构及基因转录过程发挥着重要的调控作用。体内研究核小体及染色质结构受到诸多因素限制,体外重构含有H2A.Z及H3.3的核小体结构是研究与组蛋白变体相关基因表达调控的重要方法之一。实验表达纯化了6种组蛋白,在复性的过程中装配了含有H2A.Z和H3.3的组蛋白八聚体。基于DNA序列10bp周期性及序列模体设计了3条易于形成核小体的DNA序列,通过PCR大量扩增的方法,回收了标记Cy3荧光分子的目的DNA序列。采用盐透析法体外组装了含有H2A.Z和H3.3的核小体结构,利用荧光标记、EB染色及考马斯亮蓝染色检测了含有组蛋白变体的核小体形成效率及形成过程的吉布斯自由能变化。结果发现,设计的3条DNA序列可以有效地组装形成含有组蛋白电梯的核小体结构,而且随着组蛋白八聚体与DNA比例的增加,核小体的形成效率显著提高;采用Cy3荧光标记可以灵敏且定量地计算组装过程的吉布斯自由能。该方法的建立对研究组蛋白变体相关的结构生物学及转录调控等具有一定的意义。     

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。     

植物组蛋白变体生物学功能

组蛋白变体是重要的表观遗传调控因子,能够在染色质特定位置替换常规组蛋白,维持染色质结构进而保证转录激活或抑制的顺利进行.目前,组蛋白变体的调控功能已成为植物学研究领域的一个热点.近年来,随着植物组蛋白变体生物学功能研究的不断深入,发现组蛋白变体能够在植物生长发育和环境应答调控等多个生物学过程中发挥重要作用.该文简要介绍...     

组蛋白伴侣在发育过程中的功能

组蛋白伴侣能够协助组蛋白参与染色质的解凝和组装, 从而调控基因的表达, 对动植物的配子发生、受精、胚胎发育以及生长、衰老等发育过程都具有重要作用。文章主要对目前研究较多的组蛋白伴侣 - 核质蛋白、CAF-1、HIRA、ASF1/CIA及NAP1在发育过程中的相关功能作一综述。     

组蛋白修饰在染色质复制过程中的作用

真核生物中的DNA复制,不但要保证DNA编码的基因组信息高保真复制,也要保证染色质结构所蕴含的表观遗传组稳定传递,这个过程对于维持基因组的完整性和稳定性至关重要。时至今日,人们对DNA复制的机制已经有了深入的认识,但是对染色质复制以及表观遗传信息传递的了解才刚刚开始。组蛋白是染色质结构中最主要的蛋白组成部分,其上面丰富的转录后修饰是表观遗传调控的核心方式之一。从最近几年组蛋白的修饰研究进展入手,主要综述在DNA复制过程中组蛋白修饰如何参与染色质复制的调控。     

A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes

Despite the fundamental role of canonical histones in nucleosome structure, there is no experimental system for higher eukaryotes in which basic questions about histone function can be directly addressed. We developed a new genetic tool for Drosophila melanogaster in which the canonical histone complement can be replaced with multiple copies of experimentally modified histone transgenes. This new histone‐replacement system provides a well‐defined and direct cellular assay system for histone function with which to critically test models in chromatin biology dealing with chromatin assembly, variant histone functions and the biological significance of distinct histone modifications in a multicellular organism.     

Molecular basis for chromatin assembly and modification by multiprotein complexes

Abstract: Epigenetic regulation of the chromatin landscape is often orchestrated through modulation of nucleosomes. Nucleosomes are composed of two copies each of the four core histones, H2A, H2B, H3, and H4, wrapped in ~150 bp of DNA. We focus this review on recent structural studies that further elucidate the mechanisms used by macromolecular complexes to mediate histone modification and nucleosome assembly. Nucleosome assembly, spacing, and variant histone incorporation are coordinated by chromatin remodeler and histone chaperone complexes. Several recent structural studies highlight how disparate families of histone chaperones and chromatin remodelers share similar features that underlie how they interact with their respective histone or nucleosome substrates. Post‐translational modification of histone residues is mediated by enzymatic subunits within large complexes. Until recently, relatively little was known about how association with auxiliary subunits serves to modulate the activity and specificity of the enzymatic subunit. Analysis of several recent structures highlights the different modes that auxiliary subunits use to influence enzymatic activity or direct specificity toward individual histone residues.     

HJURP uses distinct CENP-A surfaces to recognize and to stabilize CENP-A/histone H4 for centromere assembly

Centromeres are defined by the presence of chromatin containing the histone H3 variant, CENP-A, whose assembly into nucleosomes requires the chromatin assembly factor HJURP. We find that whereas surface-exposed residues in the CENP-A targeting domain (CATD) are the primary sequence determinants for HJURP recognition, buried CATD residues that generate rigidity with H4 are also required for efficient incorporation into centromeres. HJURP contact points adjacent to the CATD on the CENP-A surface are not used for binding specificity but rather to transmit stability broadly throughout the histone fold domains of both CENP-A and H4. Furthermore, an intact CENP-A/CENP-A interface is a requirement for stable chromatin incorporation immediately upon HJURP-mediated assembly. These data offer insight into the mechanism by which HJURP discriminates CENP-A from bulk histone complexes and chaperones CENP-A/H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere.     

Xenopus CENP-A assembly into chromatin requires base excision repair proteins

CENP-A is an essential histone H3 variant found in all eukaryotes examined to date. To begin to determine how CENP-A is assembled into chromatin, we developed a binding assay using sperm chromatin in cell-free extract derived from Xenopus eggs. Our data suggest that the catalytic activities of an unidentified deoxycytidine deaminase and UNG2, a uracil DNA glycosylase, are involved in CENP-A assembly. In support of this model, inhibiting deoxycytidine deaminase with zebularine, or uracil DNA glycosylase with Ugi, uracil or UTP results in a lack of detectable CENP-A on sperm DNA. Conversely, inducing DNA damage increases the level of CENP-A detected on sperm chromatin. Our data suggest that base excision repair may be involved in assembly of this histone H3 variant.     

Chromatin assembly: a basic recipe with various flavours

Packaging of eukaryotic genomes into chromatin is a hierarchical mechanism, starting with histone deposition onto DNA to produce nucleosome arrays, which then further fold and ultimately form functional domains. Recent studies provide interesting insight into how nucleosome assembly is coordinated with histone and DNA metabolism and underline the combined contribution of histone chaperones and chromatin remodelers. How these factors operate at a molecular level is a matter of current investigation. New data highlight the importance of histone dimers as deposition entities for de novo nucleosome assembly and identify dedicated machineries involved in histone variant deposition.     

Distinct roles for histone chaperones in the deposition of Htz1 in chromatin

Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1–H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1–H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3–H4)₂ tetramers, H2A–H2B dimers and Htz1–H2B dimers. Nap1 can bind H2A–H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone–DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin.     

Histone H2A‐H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin

The eukaryotic replisome disassembles parental chromatin at DNA replication forks, but then plays a poorly understood role in the re‐deposition of the displaced histone complexes onto nascent DNA. Here, we show that yeast DNA polymerase α contains a histone‐binding motif that is conserved in human Pol α and is specific for histones H2A and H2B. Mutation of this motif in budding yeast cells does not affect DNA synthesis, but instead abrogates gene silencing at telomeres and mating‐type loci. Similar phenotypes are produced not only by mutations that displace Pol α from the replisome, but also by mutation of the previously identified histone‐binding motif in the CMG helicase subunit Mcm2, the human orthologue of which was shown to bind to histones H3 and H4. We show that chromatin‐derived histone complexes can be bound simultaneously by Mcm2, Pol α and the histone chaperone FACT that is also a replisome component. These findings indicate that replisome assembly unites multiple histone‐binding activities, which jointly process parental histones to help preserve silent chromatin during the process of chromosome duplication.