Configuration-specific cofactors of Oct4

项目总结 在同一细胞内，不同的靶基因经常由相同的转录进行不同的调控。 因素。而不同的染色质邻域和共结合因子已知会改变辅因子关联 因此转录输出、DNA结合形式或转录因子构象在这些过程中的作用 流程远没有那么清楚。转录因子Oct4是多能性的主要调节者，在 早期胚胎发育、重新编程和生殖细胞规范。我们对“十四”S认识上的差距 功能限制了我们重新编程效率的能力，限制了我们在体外进行更多造型分化的能力，以及我们的 能够针对不同疾病的Oct4。这一提议的基本假设是转录 Oct4因子以不同的构型与DNA结合，招募不同的辅因子导致不同的 转录输出。Oct4可以根据特定的DNA以不同的构型与DNA结合 序列已识别。使用亲和纯化/质谱学方法，我们鉴定了转录 辅因子，其与Oct4的亲和力被DNA序列改变。其中之一是HBO1-Jade1组蛋白 乙酰基转移酶复合体，优先与与结合元件相关的Oct4同源二聚体结合 被称为mores(更多回文八聚体相关的元素)。我们用纯化的方法概括了这一结果 蛋白质，为我们提供了进行结构/功能研究的机会。更多的站点出现在一个独特的 一类广泛表达的基因，其中Oct4阻止了原本会由 氧化应激，例如与胚泡植入相关的氧化应激。HBO1复合体 乙酰化多个组蛋白H3和H4赖氨酸几乎没有特异性，然而在Oct4结合到 核小体附近的特定DNA位点，我们发现Oct4对HBO1-Jade1的招募导致了特定的 H3K9乙酰化的增强，一个关键的转录激活标志。我们这个项目的目标有三个： 确定Oct4和HBO1复合体之间依赖于配置的相互作用的关键特征 (目标1)，确定多能细胞中HBO1-Jade1是否与含有更多Oct4靶基因相关 (Aim 2)和确定与HBO1-Jade1络合物中更多结合位点的Oct4二聚体的结构(Aim 3)。

PROJECT SUMMARY Within the same cells, different target genes are frequently differentially regulated by the same transcription factor. While distinct chromatin neighborhoods and co-bound factors are known to alter cofactor association and hence transcription output, the role of DNA binding modality or transcription factor conformation in these processes is far less clear. The transcription factor Oct4 is a master regulator of pluripotency with key roles in early embryonic development, reprogramming and germ cell specification. Gaps in our understanding of Oct4’s functions limit our ability to reprogramming efficiency, our ability to more sculpt differentiation in vitro, and our ability to target Oct4 in different diseases. The fundamental hypothesis for this proposal is that the transcription factor Oct4, bound to DNA in different configurations, recruits different cofactors resulting in distinct transcription outputs. Oct4 can bind to DNA in different configurations depending on the specific DNA sequence recognized. Using an affinity purification/mass spectrometry approach, we identified transcription cofactors whose affinity for Oct4 is altered by the DNA sequence. One of these is the HBO1-Jade1 histone acetyltransferase complex, which binds preferentially to Oct4 homodimers associated with binding elements known as a MOREs (More palindromic Octamer-Related Elements). We recapitulated this result using purified proteins, affording us the opportunity to conduct structure/function studies. MORE sites are present in a unique class of broadly expressed genes in which Oct4 prevents inhibition that would otherwise be caused by oxidative stress, e.g. the oxidative stress associated with blastocyst implantation. The HBO1 complex acetylates multiple histone H3 and H4 lysines with little specificity, however in the context of Oct4 bound to a specific DNA site near a nucleosome, we found that Oct4 recruitment of HBO1-Jade1 resulted in specific enhancement of H3K9 acetylation, a key transcription activation mark. Our goals with this project are threefold: to identify the key features of the configuration-dependent interaction between Oct4 and the HBO1 complex (Aim 1), to determine if HBO1-Jade1 is associated with MORE-containing Oct4 target genes in pluripotent cells (Aim 2) and to determine the structure of Oct4 dimers bound to MORE sites in complex with HBO1-Jade1 (Aim 3).