Nucleosome recognition and spacing by chromatin remodelling factor ISW1a

Nucleosomes are actively positioned along DNA by ATP-dependent, chromatin remodelling factors. A structural model for the ISW1a chromatin remodelling factor from Saccharomyces cerevisiae in complex with a dinucleosome substrate was constructed from the X-ray structures of ISW1a (ΔATPase) with and without DNA bound, two different cryo-EM (cryo-electron microscopy) structures of ISW1a (ΔATPase) bound to a nucleosome, and site-directed photo-cross-linking analyses in solution. The X-ray structure of ISW1a (ΔATPase) with DNA bound suggests that DNA sequence may be involved in nucleosome recognition and thereby specificity of promoter interaction. The model suggests how the highly ordered nucleosome arrays observed by mapping nucleosomes in genes and their promoter regions could be generated by a chromatin remodelling factor.     

Specialized RSC: Substrate Specificities for a Conserved Chromatin Remodeler

The remodel the structure of chromatin (RSC) nucleosome remodeling complex is a conserved chromatin regulator with roles in chromatin organization, especially over nucleosome depleted regions therefore functioning in gene expression. Recent reports in Saccharomyces cerevisiae have identified specificities in RSC activity toward certain types of nucleosomes. RSC has now been shown to preferentially evict nucleosomes containing the histone variant H2A.Z in vitro. Furthermore, biochemical activities of distinct RSC complexes has been found to differ when their nucleosome substrate is partially unraveled. Mammalian BAF complexes, the homologs of yeast RSC and SWI/SNF complexes, are also linked to nucleosomes with H2A.Z, but this relationship may be complex and extent of conservation remains to be determined. The interplay of remodelers with specific nucleosome substrates and regulation of remodeler outcomes by nucleosome composition are tantalizing questions given the wave of structural data emerging for RSC and other SWI/SNF family remodelers.     

Deciphering how the chromatin factor RCC1 recognizes the nucleosome: the importance of individuals in the scientific discovery process

The nucleosome repeating unit of chromatin is the target of chromatin enzymes and factors that regulate gene activity in a eukaryotic cell. How the nucleosome is recognized by chromatin enzymes and factors is poorly understood, even though such interaction is fundamental to gene regulation and chromatin biology. My laboratory recently determined the structural basis for how the RCC1 (regulator of chromosome condensation 1) chromatin factor binds to the nucleosome, including the first atomic crystal structure of a chromatin protein complexed with the nucleosome core particle. I describe here how we developed and investigated structural models for RCC1 binding to the nucleosome using biochemical methods and how we crystallized the 300?kDa complex of RCC1 with the nucleosome core particle. This article highlights the contributions made by key laboratory members and explains our thinking and rationale during the discovery process.     

综述: 表观遗传之染色质重塑

真核细胞中的染色质重塑因子种类繁多,多数以蛋白多聚体的形式存在于细胞中．不同的染色质重塑因子在特定时间定位于特定的核小体上,通过改变染色质结构,影响基因转录活性,进而确保细胞内各种生物学过程的正确运行．另外,染色质重塑因子根据所含功能结构域的不同,大致分为SWI/SNF、ISWI、CHD和INO80四大家族,不同的染色质重塑因子之间既有蛋白质结构和酶活性的相似性,各自又有其特异性．本综述的宗旨在于全面概括和总结染色质重塑因子的分类、结构特点以及其在细胞内的生物学功能,为深入研究染色质重塑因子的生物学功能,尤其是在发育和疾病发生中的作用机制提供理论基础．     

Histone H4 K16Q mutation, an acetylation mimic, causes structural disorder of its N-terminal basic patch in the nucleosome

Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K(16)R(17)H(18)R(19) in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K(16)R(17)H(18)R(19).     

基于DNA变形能的核小体定位预测方法研究进展

真核细胞中,作为染色质基本结构单元的核小体参与调控基因的转录、DNA复制、重组以及RNA剪接等诸多生物学过程。阐明核小体定位机制并准确预测核小体在染色体上的位置对解读染色质结构与功能有重要生物学意义。在过去30多年时间里,研究人员发展了多种预测核小体位置的方法。最理想的方法应考虑DNA序列、组蛋白修饰和染色质重塑等影响核小体定位的诸多因素,然而现实中,捕捉主要因素的模型也往往具有很高的鲁棒性和实用价值。DNA序列偏好性是在全基因组尺度上影响核小体定位的最重要因素之一,因此基于DNA序列的核小体定位预测方法也最常见。这种方法可大致分为两类,即基于DNA序列信息的生物信息学模型和基于DNA变形能的生物物理学模型。本文重点介绍生物物理学模型近些年取得的主要进展。     

Nucleosomal anatomy – where are the histones?

The recent surge of discoveries concerning the structural organization of nucleosomes, together with genetic evidence of highly specialized roles for the histones in gene regulation, have brought a renewed need for a detailed understanding of nucleosomal anatomy. Here we review recent structural advances leading to a new level of understanding of the nucleosome and chromatin fibre structure. We discuss the problems and challenges for existing models of chromatin structure and, in particular, consider how linker histones may bind within the nucleosome, together with the implications of their association for the structure of the chromatin fibre.     

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。     

On the role of inter-nucleosomal interactions and intrinsic nucleosome dynamics in chromatin function

Evidence is emerging that many diseases result from defects in gene functions, which, in turn, depend on the local chromatin environment of a gene. However, it still remains not fully clear how chromatin activity code is ‘translated’ to the particular ‘activating’ or ‘repressing’ chromatin structural transition. Commonly, chromatin remodeling in vitro was studied using mononucleosomes as a model. However, recent data suggest that structural reorganization of a single mononucleosome is not equal to remodeling of a nucleosome particle under multinucleosomal content – such as, interaction of nucleosomes via flexible histone termini could significantly alter the mode (and the resulting products) of nucleosome structural transitions. It is becoming evident that a nucleosome array does not constitute just a ‘polymer’ of individual ‘canonical’ nucleosomes due to multiple inter-nucleosomal interactions which affect nucleosome dynamics and structure. It could be hypothesized, that inter-nucleosomal interactions could act in cooperation with nucleosome inherent dynamics to orchestrate DNA-based processes and promote formation and stabilization of highly-dynamic, accessible structure of a nucleosome array. In the proposed paper we would like to discuss the nucleosome dynamics within the chromatin fiber mainly as it pertains to the roles of the structural changes mediated by inter-nucleosomal interactions.