Understanding how two related mammalian histone acetyl transferase co-activators, SAGA and ATAC, differentially regulate chromatin dynamics and transcription

了解两个相关的组蛋白乙酰转移酶共激活剂，SAGA 和ATAC，差异调节染色质动力学和转录 项目总结 组蛋白翻译后修饰(PTM)及其安装和阅读的复合体，控制 真核生物基因组功能的许多方面，包括转录、DNA修复和复制。由于…… 组蛋白PTM编写者的广泛功能，它们在生物发育、衰老和众多 疾病包括癌症、心脏病，甚至艾滋病毒的整合。大多数组蛋白作家都瞄准了 无序的N-末端尾区。许多组蛋白作家已经被确认并从基因上进行了研究， 从生化和结构上来说。令人惊讶的是，机械论研究主要局限于 组蛋白尾肽和催化亚基，导致在理解组蛋白读取器/写入器如何 在其完整的天然复合体中相互作用并调节染色质结构、动力学、可及性和 抄写。最近，我们发展了纯化内源性人的生化量的方法 SAGA和ATAC复合体，它们是相关的，但在功能上不同的基本转录共激活因子。 这使我们能够定量地研究这两个大型多亚基复合体是如何相对于 它们共同的催化亚基KAT2A单独存在。利用这一点，我们最近发现 (I)内源性SAGA和ATAC复合体对组蛋白八聚体的乙酰化作用比 单独使用KAT2A乙酰转移酶。(Ii)内源性SAGA和ATAC络合物的乙酰化效率为 与未修饰的组蛋白八聚体相比，未修饰的核小体显著抑制。(Iii)传奇和 ATAC HAT模块乙酰化组蛋白H3K9类似于单独相对于KAT2A，但SAGA HAT模块 在未修饰的核小体中乙酰化H3K9比ATAC HAT络合物或KAT2A更有效 独自一人。(Iv)在小鼠ES细胞中，SAGA HAT模块的缺失不会强烈影响全局H3K9 乙酰化，而ATAC HAT模块的删除导致总体H3K9的显著减少 乙酰化。 这些发现使我们研究了组蛋白PTM读取器/写入器以染色质为目标的假设 通过它们的辅助蛋白来动态影响染色质的动力学和可及性。 (1)确定哺乳动物HAT复合体调节读写的染色质特性 传奇故事。 (2)阐明相关哺乳动物HAT复合体的不同亚基，SagA和ATAC， 不同地控制它们的靶向和染色质修饰活性。 (3)确定SAGA和ATAC在小鼠胚胎干细胞中的功能差异。 它们的差异性。 这些目标共同为理解两个关键因素提供了机械和功能的基础 转录共激活因子SAGA和ATAC对染色质动态和转录起着不同的调节作用。

UNDERSTANDING HOW TWO RELATED HISTONE ACETYL TRANSFERASE CO-ACTIVATORS, SAGA AND ATAC, DIFFERENTIALLY REGULATE CHROMATIN DYNAMICS AND TRANSCRIPTION PROJECT SUMMARY Histone post translational modifications (PTMs) and the complexes that install them and read them, control many aspects of eukaryotic genome function including transcription, DNA repair and replication. As a result of histone PTM writer’s broad functions, they are essential in organismal development, aging and numerous diseases including cancer, heart disease, and even HIV integration. The majority of histone writers target the disordered N-terminal tail regions. Numerous histone writers have been identified and studied genetically, biochemically and structurally. Surprisingly, mechanistic studies are primarily limited to interactions between histone tail peptides and catalytic subunits, resulting in key gaps in understanding how histone readers/writers within their full native complexes interact and regulate chromatin structure, dynamics, accessibility and transcription. Recently, we developed the methods to purify biochemical quantities of endogenous human SAGA and ATAC complexes, which are related, but functionally distinct essential transcription co-activators. This allows us to quantitatively investigate how these two large multi-subunit complexes function relative to their common catalytic subunit, KAT2A, alone. Leveraging this, we recently found that (i) The endogenous SAGA and ATAC complexes acetylate histone octamers much more efficiently than the KAT2A acetyltransferase alone. (ii) The acetylation efficiency of endogenous SAGA and ATAC complexes are dramatically inhibited by unmodified nucleosomes relative to unmodified histone octamer. (iii) The SAGA and ATAC HAT modules acetylate histone H3K9 similarly relative to KAT2A alone, but the SAGA HAT module acetylates H3K9 in unmodified nucleosomes much more efficiently than the ATAC HAT complex or KAT2A alone. (iv) In mouse ES cells, deletion of the SAGA HAT module does not strongly affect global H3K9 acetylation, while deletion of the ATAC HAT module results in a significant reduction in overall H3K9 acetylation. These findings have led us to investigate the hypothesis that histone PTM readers/writers target chromatin properties through their accessory proteins to dynamically influence chromatin dynamics and accessibility. (1) Determine the chromatin properties that regulate reading and writing by the mammalian HAT complex SAGA. (2) Elucidate how the different subunits of the related mammalian HAT complexes, SAGA and ATAC, differentially control their targeting and chromatin modifying activity. (3) Determine the functional differences between SAGA and ATAC in mouse embryonic stem cells and during their differentiation. Together these aims provide a mechanistic and functional foundation for understanding of how two key transcriptional co-activators, SAGA and ATAC, differentially regulate chromatin dynamic and transcription.