Configuration-specific cofactors of Oct4

Oct4 的配置特定辅因子

• 批准号: 10713592
• 负责人: DEAN TANTIN
• 金额: $ 15.4万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10713592
• 关键词: Acetylation; Affinity; Affinity Chromatography; Amino Acids; Beckwith-Wiedemann Syndrome; Binding; Binding Sites; Biochemistry; Biological Assay; Biological Testing; C-terminal; Cell Reprogramming; Cells; ChIP-seq; Chromatin; Complex; Cryoelectron Microscopy; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Development; Disease; Elements; Embryonic Development; Ewings sarcoma; Funding; Genes; Genetic Transcription; Germ Cells; Germ cell tumor; Goals; Health; Histone H3; Histone H4; Human; In Vitro; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Modality; Molecular Conformation; Mutation; Neighborhoods; Nucleosomes; Outcome; Output; Oxidative Stress; POU domain factors; Pharmacologic Substance; Phosphorylation; Process; Protein Isoforms; Proteins; Regenerative Medicine; Research; Role; Site; Specific qualifier value; Specificity; Structure; Transcriptional Activation; Western Blotting; Work; biological adaptation to stress; cofactor; dimer; embryonic stem cell; histone acetyltransferase; human disease; insight; natural Blastocyst Implantation; pluripotency; prevent; primary ovarian insufficiency; protein purification; public health relevance; recruit; regeneration potential; response; transcription factor

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).

项目摘要 在同一细胞内，不同的靶基因通常会通过同一转录对不同的调节 因素。虽然已知不同的染色质邻域和联合因子会改变辅助因子关联 因此，转录输出，DNA结合方式或转录因子构象的作用 流程远不清楚。转录因子OCT4是多能性的主调节器，在 早期胚胎发育，重编程和生殖细胞规范。我们对Oct4的理解的差距 功能限制了我们重新编程效率的能力，我们在体外进行更多雕刻分化的能力以及我们的 在不同疾病中靶向OCT4的能力。该提议的基本假设是转录 因子Oct4，以不同的配置与DNA结合，报告不同的辅助因子，导致不同的辅助因子 转录输出。 OCT4可以根据特定DNA的不同构型与DNA结合 序列已识别。使用亲和力纯化/质谱方法，我们确定了转录 DNA序列改变了对OCT4的亲和力的辅助因子。其中之一是HBO1-JADE1组蛋白 乙酰基转移酶配合物，它优先与与结合元件相关的OCT4同型二聚体结合 被称为mores（更倾向于八聚体相关的元素）。我们使用纯化概括了此结果 蛋白质，为我们提供了进行结构/功能研究的机会。更多的网站以独特的形式存在 一类广泛表达的基因，其中OCT4阻止抑制作用，否则 氧化应激，例如与胚泡植入有关的氧化应激。 HBO1复合物 乙酰是多种组蛋白H3和H4赖氨酸，但在OCT4的背景下与A结合 特定的DNA位点附近的核小体附近，我们发现OCT4募集HBO1-Jade1导致了特定 H3K9乙酰化的增强，一个关键的转录激活标记。我们对这个项目的目标是三重： 识别Oct4和HBO1复合物之间构型相互作用的关键特征 （AIM 1），确定HBO1-JADE1是否与多能细胞中更含有更含有的OCT4靶基因有关 （AIM 2）并确定与HBO1-JADE1相关的更多位点的Oct4二聚体的结构（AIM 3）。