Structure, mechanism, and function of the histone chaperones Spt6 and FACT

组蛋白伴侣 Spt6 和 FACT 的结构、机制和功能

• 批准号: 9265478
• 负责人: Timothy G Formosa
• 金额: $ 29.9万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9265478
• 关键词: Affect; Automobile Driving; Back; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biochemistry; Biological; Biological Assay; Biology; C-terminal; Cell physiology; Charge; Chromatin; Chromatin Structure; Collaborations; Complex; Coupled; Crystallization; DNA; DNA Binding; DNA Repair; DNA biosynthesis; DNA-Directed RNA Polymerase; Data; Dimerization; Disease; Dissection; Equilibrium; Eukaryota; Foundations; Gene Expression Regulation; Genetic; Genetic Transcription; Genomics; Goals; HIV; Health; Histones; Homeostasis; Human; Hydrophobicity; Impairment; Intellectual functioning disability; Kinetics; Maintenance; Malignant Neoplasms; Maps; Modeling; Molecular; Molecular Chaperones; Mutation; N-terminal; Names; Nucleosomes; Pathway interactions; Peptides; Phenotype; Phosphorylation; Physiological; Positioning Attribute; Primary carcinoma of the liver cells; Process; Proteins; Psyche structure; Publishing; RNA; RNA Polymerase II; Reagent; Repression; Role; S Phase; Serine; Site; Structure; Surface; Tail; Testing; Transcription Elongation; Yeasts; dimer; flexibility; in vivo; insight; interdisciplinary approach; mRNA Export; mutant; public health relevance; reconstruction; repaired; stoichiometry; tool; Affect; Automobile Driving; Back; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biochemistry; Biological; Biological Assay; Biology; C-terminal; Cell physiology; Charge; Chromatin; Chromatin Structure; Collaborations; Complex; Coupled; Crystallization; DNA; DNA Binding; DNA Repair; DNA biosynthesis; DNA-Directed RNA Polymerase; Data; Dimerization; Disease; Dissection; Equilibrium; Eukaryota; Foundations; Gene Expression Regulation; Genetic; Genetic Transcription; Genomics; Goals; HIV; Health; Histones; Homeostasis; Human; Hydrophobicity; Impairment; Intellectual functioning disability; Kinetics; Maintenance; Malignant Neoplasms; Maps; Modeling; Molecular; Molecular Chaperones; Mutation; N-terminal; Names; Nucleosomes; Pathway interactions; Peptides; Phenotype; Phosphorylation; Physiological; Positioning Attribute; Primary carcinoma of the liver cells; Process; Proteins; Psyche structure; Publishing; RNA; RNA Polymerase II; Reagent; Repression; Role; S Phase; Serine; Site; Structure; Surface; Tail; Testing; Transcription Elongation; Yeasts; dimer; flexibility; in vivo; insight; interdisciplinary approach; mRNA Export; mutant; public health relevance; reconstruction; repaired; stoichiometry; tool

 DESCRIPTION (provided by applicant): This proposal focuses on Spt6 (168 kDa) and FACT, which is a heterodimer of Spt16 (119 kDa) and Pob3 (63 kDa). Spt6 and FACT are histone chaperones that are conserved throughout eukaryotes, implicated in HIV latency and other disease processes, and unlike most structurally characterized histone chaperones, are essential for viability. Both Spt6 and FACT function in the assembly of nucleosomes, which is required as the DNA is doubled during replication in S phase and also to replace nucleosomes that become irretrievably displaced by remodeling or by passage of an RNA polymerase during transcription. FACT also reorganizes nucleosomes, forming a loosened structure that promotes passage of RNA polymerase but maintains contacts with each of the components so that the same displaced histone molecules are incorporated back into the reconstructed nucleosome. Our collaboration has previously produced multiple structures of domains of Spt6, a complex of Spt6 with the essential factor Spn1, and multiple structures of domains of the FACT heterodimer. We also performed genetic and binding studies that validated the biological importance of these structures and advanced understanding of mechanisms. We will continue this multidisciplinary approach, which is essential for making meaningful progress in this challenging field. An important priority in Aims 1 and 2 is to biochemically and structurally characterize interactions with histones, which are fundamental to Spt6 and FACT function. We favor the model that Spt6 and FACT function in large part by covering histone surfaces that otherwise associate with DNA or other histones in the nucleosome structure, and we therefore believe that biochemically understanding these interactions and verifying their biological importance will reveal the primary basis of Spt6 and FACT activities in loosening and assembling nucleosomes. Histones are notoriously challenging subjects for binding studies, and our multidisciplinary approach, which includes rigorous biochemistry, places us in an excellent position to pursue high impact goals and has already led to an insightful preliminary FACT:H2A-H2B crystal structure. Specific hypotheses generated from the insights provided by Aims 1 and 2 are driving functional studies that are being pursued for Spt6 in Aim 3, including the mechanistic and physiological role of the Spn1 binding partner. Aim 4 is advancing the important but understudied question of how the essential histone chaperones are coupled to diverse biological pathways. Specifically, we have discovered that Spt6 binds directly and specifically with RNA polymerase II (RNAPII) and with Tom1. These interactions presumably underlie Spt6's distinct roles in transcription elongation and mRNA export, respectively, and the Tom1 interaction further implicates Spt6 in the maintenance of histone homeostasis and has ramifications for understanding certain forms of hepatocellular carcinoma and mental impairment in humans. Preliminary data include an initial EM structure of the RNAPII-Spt6 complex, identification of Spt6 point mutants that specifically disrupt binding to RNAPII or to Tom1, and identification of a phosphorylation- dependent switch for binding to RNAPII.

 描述（由适用提供）：该提案侧重于SPT6（168 kDa）和事实，这是SPT16（119 kDa）和POB3（63 kDa）的异二聚体。 SPT6和事实是整个真核生物中保守的组蛋白链酮，在HIV潜伏期和其他疾病过程中施加，并且与大多数结构表征的组蛋白链酮不同，对于生存能力至关重要。 SPT6和事实功能在核组组的组装中都需要，因为在S期复制过程中DNA会加倍，并替代通过重塑或通过转录过程中RNA聚合酶的通过而变化的核体。事实还重组核组，形成松散的结构，促进RNA聚合酶的通过，但与每个组件保持接触，从而使相同的移位组蛋白分子被掺入重建的核病室中。我们的合作以前已经生成了SPT6域的多个结构，SPT6的复合物具有SPN1的基本因素，以及事实异二聚体的多个结构。我们还进行了遗传和结合研究，验证了这些结构的生物学重要性以及对机制的高级理解。我们将继续这种多学科方法，这对于在这个挑战性领域取得有意义的进步至关重要。目标1和2中的一个重要优先级是在生化和结构上表征与组蛋白的相互作用，这是SPT6和事实功能的基础。我们赞成SPT6和事实在很大程度上发挥作用的模型，该模型覆盖了核体结构中与DNA或其他组蛋白相关的组蛋白表面，因此我们相信，我们相信生物化学从生物化学上理解这些相互作用并验证其生物学重要性将揭示SPT6的主要基础，以及在松动和组装核渗透器中的事实活动。众所周知，组蛋白是对具有约束力研究的挑战，我们的多学科方法包括严格的生物化学，处于实现高影响目标的极好位置，并且已经导致了有见地的初步事实：H2A-H2B晶体结构。 AIMS 1和2提供的见解产生的特定假设是驱动AIM 3中SPT6的功能研究，包括SPN1结合伙伴的机械和身体作用。 AIM 4正在推进重要但了解的问题，即基本组蛋白链酮如何与潜水员生物学途径耦合。具体而言，我们发现SPT6直接与RNA聚合酶II（RNAPII）和TOM1直接结合。这些相互作用大概是SPT6在转录伸长和mRNA输出中的独特作用的基础，而TOM1相互作用进一步暗示了SPT6在维持组蛋白稳态中的影响，并且在理解某些形式的肝细胞癌和人类精神障碍的影响方面具有影响。初步数据包括RNAPII-SPT6复合物的初始EM结构，SPT6点突变体的鉴定，该突变体特异性破坏了与RNAPII或TOM1的结合，以及鉴定磷酸化依赖性开关以结合与RNAPII。