The INO80 Chromatin Remodeling Complex Functions in Sister Chromatid Cohesion

Here we identify a defect in sister chromatid cohesion in the Saccharomyces serevisiae arp8 mutant, which impairs the chromatin remodeling activity of the INO80 complex, and we report the direct association of Ino80 with centromeres and cohesin-associated regions. In early S phase, Ino80 is recruited to replication forks along with Ctf18 and PCNA, both of which are involved in the establishment of sister chromatid cohesion. The arp8 mutation perturbs the association of Ctf18 and PCNA but not of cohesin with replication forks. We propose that the INO80 complex is required for the proper establishment of sister chromatid cohesion.     

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding

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INO80 Chromatin Remodeling Coordinates Metabolic Homeostasis with Cell Division

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Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex''s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.     

Beyond the Nucleosome: Nucleosome-Protein Interactions and Higher Order Chromatin Structure

The regulation of chromatin biology ultimately depends on the manipulation of its smallest subunit, the nucleosome. The proteins that bind and operate on the nucleosome do so, while their substrate is part of a polymer embedded in the dense nuclear environment. Their molecular interactions must in some way be tuned to deal with this complexity. Due to the rapid increase in the number of high-resolution structures of nucleosome-protein complexes and the increasing understanding of the cellular chromatin structure, it is starting to become clearer how chromatin factors operate in this complex environment. In this review, we analyze the current literature on the interplay between nucleosome-protein interactions and higher-order chromatin structure. We examine in what way nucleosomes-protein interactions can affect and can be affected by chromatin organization at the oligonucleosomal level. In addition, we review the characteristics of nucleosome-protein interactions that can cause phase separation of chromatin. Throughout, we hope to illustrate the exciting challenges in characterizing nucleosome-protein interactions beyond the nucleosome.     

Structure of the Human Core Centromeric Nucleosome Complex

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Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.     

核小体及染色质修饰的基因组分布模式和染色质状态

染色质是真核DNA的存在方式,可以通过影响DNA的可及性调节基因转录,其基本单元为核小体,系由约147 bp的DNA缠绕在组蛋白八联体上形成的结构,核小体之间以连接DNA相连．核小体组蛋白上能发生甲基化和乙酰化等化学修饰．核小体位置、DNA的甲基化和组蛋白的修饰等对染色质状态(常染色质或异染色质)及基因组之间的长程相互作用有重要影响．近年,基于高通量测序技术,核小体位置和染色质修饰在多种细胞中的基因组分布已被测定．结果显示,这些标记的分布模式具有位点特异、动态变化、相互偶联和高度复杂的特征．本文详细回顾并评述了核小体位置和染色质修饰的分布模式、对应生物学功能、修饰之间的关联、实验测定技术、染色质状态的计算分析等内容．该工作对于深入认识和理解染色质的表观遗传调节机制有重要意义．