Mechanisms of p300 Activation During Pluripotency and Differentiation.

总结 多能性是理解细胞命运规范的基本原则的关键模型。这个过程 选择性地使用增强子，促进细胞类型特异性转录的调节元件， 基因.尽管增强子活性的变化被预测具有广泛的发育和病理学意义， 影响，我们目前对增强子如何调节发育和疾病的了解有限， 部分原因是我们对增强子调节基因表达的机制还不完全了解。 增加对调节增强子活性的分子事件的理解，特别是在 发展背景下，将提供重要的见解，先天性疾病发生与他们的 失调增强子处的染色质状态的特征在于组蛋白变体H3.3的存在， 以及转录共激活因子CBP/p300家族的募集。我们最近的研究表明， H3.3在增强子上的沉积允许刺激p300乙酰转移酶的变体特异性磷酸化 在小鼠胚胎干细胞（ESC）中，其对底物组蛋白H3赖氨酸27（H3 K27 ac）的活性。此外，本发明还 我们发现CBP和p300在ESCs中执行不同的功能，其中p300在维持ESCs的功能方面发挥更大的作用。 ESC中的H3 K27 ac。最后，我们发现，由于H3.3或p300缺失而导致的H3 K27 ac减少， ESCs，在转录方面几乎没有相关的变化。然而，H3.3和p300都是必需的， 这表明H3.3沉积和随后的高水平H3 K27 ac可能是更重要的 而不是维持胚胎干细胞中正在进行的转录。的目的 建议是剖析增强子被激活的分子和功能机制， 多能性和分化。在第一个目标中，我们将确定H3.3磷酸化如何刺激p300 ESCs中的活性。在第二个目标中，我们将确定为什么H3 K27乙酰转移酶活性仅限于 在ESC中的p300。最后，在第三个目标中，我们将使用化学生物学工具和新的小鼠模型， 我们已经产生的问题是H3.3和p300如何在着床前促进转录 发展总的来说，我们的工作将探索H3.3和p300在增强子激活中的分子联系 在第一个世系规范事件发生的时候， 了解增强子失调如何导致人类疾病。

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.