Mechanisms of p300 Activation During Pluripotency and Differentiation.

多能性和分化过程中 p300 激活的机制。

• 批准号: 10685500
• 负责人: Laura Banaszynski
• 金额: $ 42.04万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-17 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685500
• 关键词: Acetate-CoA Ligase; Acetyl Coenzyme A; Acetylation; Acetyltransferase; Amaze; Binding; Binding Proteins; Biological; Biological Assay; Biological Process; Biology; Cell physiology; Cells; Chemicals; Chromatin; Defect; Deposition; Development; Disease; EP300 gene; Embryo; Enhancers; Event; Failure; Family; Gene Expression; Genes; Genetic Enhancer Element; Genetic Models; Genetic Transcription; Genomics; Goals; Histone H3; Histone H3.3; Histones; Infertility; Knock-in; Knowledge; Link; Lysine; Malignant Neoplasms; Mediating; Modeling; Molecular; Mus; Nuclear; Pathogenesis; Pathologic; Phosphorylation; Play; Pre-implantation Embryo Development; Process; Proteins; Proteomics; Regenerative Medicine; Regulatory Element; Role; Specific qualifier value; Syndrome; Testing; Time; Transcription Coactivator; Transcriptional Activation; Variant; Work; cell fate specification; cell type; early pregnancy loss; embryonic stem cell; epigenetic regulation; experimental study; human disease; human model; improved; in vivo; inhibitor; insight; mouse model; novel; p300/CBP-Associated Factor; pluripotency; protein protein interaction; recruit; scaffold; tool; transcription factor

SUMMARY Pluripotency is a critical model for understanding fundamental principles of cell fate specification. This process makes selective use of enhancers, regulatory elements that facilitate the transcription of cell-type specific genes. Although changes in enhancer activity are predicted to have broad developmental and pathological implications, we currently have limited understanding of how enhancers regulate development and disease, in part due to our incomplete understanding of the mechanisms by which enhancers regulate gene expression. Increased understanding of the molecular events that regulate enhancer activity, particularly under developmental contexts, will provide important insights into congenital diseases that occur with their dysregulation. The chromatin state at enhancers is characterized by the presence of the histone variant, H3.3, and recruitment of the CBP/p300 family of transcriptional coactivators. Our recent studies demonstrate that H3.3 deposition at enhancers allows for variant-specific phosphorylation that stimulates p300 acetyltransferase activity towards its substrate histone H3 lysine 27 (H3K27ac) in mouse embryonic stem cells (ESCs). Further, we find that CBP and p300 carry out distinct functions in ESCs, with p300 playing a greater role in maintaining H3K27ac in ESCs. Finally, we find that reduced H3K27ac due to H3.3 or p300 deletion is well tolerated in ESCs, with little correlated change in transcription. However, both H3.3 and p300 are required for differentiation, suggesting that H3.3 deposition and subsequent high levels of H3K27ac may be more important for activating gene transcription than for maintaining ongoing transcription in ESCs. The objective of this proposal is to dissect the molecular and functional mechanisms by which enhancers are activated both in pluripotency and differentiation. In the first aim, we will determine how H3.3 phosphorylation stimulates p300 activity in ESCs. In the second aim, we will determine why H3K27 acetyltransferase activity is restricted to p300 in ESCs. Finally, in the third aim, we will use the tools of chemical biology and novel mouse models that we have generated to ask how H3.3 and p300 function to promote transcription during pre-implantation development. Collectively, our work will explore molecular links between H3.3 and p300 in enhancer activation during the time at which the first lineage specification events occur, with important implications for understanding how enhancer dysregulation contributes to human disease.

概括 多能性是理解细胞命运规范基本原理的关键模型。这个过程 选择性使用增强剂，促进细胞类型转录的调节元素 基因。尽管预计增强剂活性的变化将具有广泛的发育和病理 含义，我们目前对增强剂如何调节发展和疾病的理解有限 部分是由于我们对增强子调节基因表达的机制的不完全理解。 对调节增强剂活性的分子事件的了解增加了，特别是在 发育环境将提供有关其与先天性疾病的重要见解 失调。增强子处的染色质状态的特征是组蛋白变体H3.3， 并招募了CBP/P300转录共激活因子家族。我们最近的研究表明 H3.3在增强子处的沉积允许刺激P300乙酰转移酶的变异特异性磷酸化。 在小鼠胚胎干细胞（ESC）中，其底物组蛋白H3赖氨酸27（H3K27AC）的活性。更远， 我们发现CBP和P300在ESC中发挥了不同的功能，P300在维护中发挥了更大的作用 H3K27AC在Escs中。最后，我们发现由于H3.3或p300删除而减少的H3K27AC在 ESC，转录变化几乎没有相关性。但是，H3.3和P300都需要 分化，表明H3.3沉积和随后的高水平H3K27AC可能更重要 用于激活基因转录，而不是在ESC中保持持续的转录。这个目的 建议是剖析分子和功能机制，通过这些机制激活增强子 多能和分化。在第一个目标中，我们将确定H3.3磷酸化如何刺激p300 ESC的活动。在第二个目标中，我们将确定为什么H3K27乙酰转移酶活性仅限于 p300在ESC中。最后，在第三个目标中，我们将使用化学生物学和新型鼠标模型的工具 我们已经生成询问H3.3和P300如何在植入前促进转录 发展。总的来说，我们的工作将探索H3.3和P300之间的分子链接 在发生第一个血统规范事件的期间，对 了解增强子失调如何导致人类疾病。