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

 描述(申请人提供)：本提案重点关注Spt6(168 KDa)和FACT，它是Spt16(119 KDa)和Pob3(63 KDa)的异源二聚体。Spt6和FACT是组蛋白伴侣蛋白，在真核生物中是保守的，与HIV潜伏期和其他疾病过程有关，不同于大多数结构特征的组蛋白伴侣蛋白，对生存是必不可少的。Spt6和FACT都在核小体的组装中发挥作用，这是在S阶段复制过程中DNA加倍所必需的，也是取代因重塑或转录过程中RNA聚合酶通过而不可逆转地移位的核小体所必需的。FACT还重组核小体，形成一种松散的结构，促进RNA聚合酶的通过，但保持与每个组分的联系，以便相同的移位的组蛋白分子被合并回重建的核小体。我们的合作以前已经产生了Spt6的多种结构域，Spt6与必需因子Spn1的复合体，以及事实杂二聚体的多种结构域。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，并加深了对机制的理解。我们将继续采取这种多学科方法，这对于在这一具有挑战性的领域取得有意义的进展至关重要。在目标1和目标2中，一个重要的优先事项是从生化和结构上表征与组蛋白的相互作用，这是Spt6和FAX功能的基础。我们支持Spt6和FACT在很大程度上通过覆盖核小体结构中与DNA或其他组蛋白相关的组蛋白表面来发挥作用的模型，因此我们相信，从生物化学的角度了解这些相互作用并验证其生物学意义将揭示Spt6和FACT在松动和组装核小体方面的主要基础。组蛋白是众所周知的具有结合研究挑战性的课题，而我们的多学科方法，包括严格的生物化学，使我们处于追求高影响目标的极佳地位，并已经导致了一个有洞察力的初步事实：H_2A-H_2B晶体结构。目标1和目标2提供的见解产生的特定假设正在推动目标3中对Spt6进行的功能研究，包括Spn1结合伙伴的机制和生理作用。目标4正在推进一个重要但未被充分研究的问题，即基本的组蛋白伴侣蛋白如何与不同的生物途径偶联。具体地说，我们发现Spt6与RNA聚合酶II(RNAPII)和Tom1直接和特异性结合。这些相互作用可能是Spt6的S分别在转录延长和基因输出中扮演不同角色的基础，而Tom1的相互作用进一步涉及Spt6维持组蛋白的动态平衡，并对理解某些形式的肝细胞癌和人类的精神障碍具有重要意义。初步数据包括RNAPII-Spt6复合体的初始EM结构，鉴定了特异性破坏与RNAPII或Tom1结合的Spt6点突变体，以及鉴定了与RNAPII结合的磷酸化依赖的开关。

 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.