Regulation of Dnmt3 Activity at Enhancers of Cell Identity Genes During Differentiation

项目摘要 维持DNA甲基化的正确模式对于细胞身份的完整性至关重要， 导致基因的异常激活，这反过来又调节导致癌症的信号通路， 精神障碍。尽管有大量证据支持异常DNA甲基化在 几种人类疾病的病因、调节靶位点特异性的基本机制 新生DNA MTase，Dnmt 3a和3b，在很大程度上是未知的。基因抑制是一个精心策划的事件， 涉及辅激活因子复合物从调节元件的丢失和染色质状态的变化，包括 获得DNA甲基化，导致稳定的基因抑制。最近的研究列举了en的作用- 在各种癌症中，癌症介导的癌基因调节。其他人则表现出plu的异常表达， 三能性基因介导几种癌症的去分化。增强子染色质状态的变化- 已经显示多能性基因的缺失对于多能性基因的阻遏是关键的， 胚胎干细胞（ESC）分化。DNA甲基化在这一过程中的作用及其机制 在分化过程中将DNA甲基化靶向增强子的方法尚未得到解决。理解 增强子介导的多能性（Pp）建立中涉及的基本表观遗传机制 从长远来看，正常细胞分化过程中的基因抑制将导致治疗性药物的开发。 恢复癌细胞中Pp基因抑制的策略。我们在本申请中的目的是阐明分子- 在Pp基因全基因组增强子处调节Dnmt 3a和3b活性的更大机制 ESC分化作为模型系统。在我们强大的初步数据和最近发表的研究的支持下， 在于Pp基因增强子的染色质状态的动力学，我们将测试我们的假设，Lsd 1- Mi2/BHD活性作为Pp基因增强子的表观遗传开关激活Dnmt 3酶， 位点特异性DNA甲基化和稳定的Pp基因阻遏。我们将进一步阐明染色质的作用 构象在促进Dnmt 3a和/或3b对特定启动子的增强子靶向活性中的作用。 胚胎干细胞分化的早期阶段。为了测试Dnmt 3a和3b是否都受这些机制的调节，我们将绘制 它们在ESC分化期间的全基因组活性。我们进行这些研究的理由是， 完成有望填补差距，在我们的理解如何表观遗传“串扰”机制，dur- 诱导细胞分化，调节Dnmt 3a/3b活性以终止多能性程序， 会导致发育障碍和癌症此外，预期这些研究的结果 提供了新的见解如何相互作用之间的各种表观遗传因素影响基因表达， 表现型变异和跨代遗传。

Project Summary Maintenance of proper patterns of DNA methylation is essential for the integrity of cell identity, failure of which results in aberrant activation of genes, which in turn modulates signaling pathways leading to cancer and de- velopmental disorders. Despite a large body of evidence supporting the role of aberrant DNA methylation in etiology of several human diseases, the fundamental mechanisms that regulate the target site specificity of the de novo DNA MTases, Dnmt3a and 3b, are largely unknown. Gene repression is an orchestrated event that involves loss of coactivator complexes from the regulatory elements and changes in chromatin state including gain of DNA methylation, resulting in stable gene repression. Recent studies have enumerated the role of en- hancer-mediated regulation of oncogenes in various cancers. Others have shown aberrant expression of plu- ripotency genes mediates dedifferentiation in several cancers. Changes in the chromatin state of the enhanc- ers of pluripotency genes have been shown to be critical for the repression of pluripotency genes during mu- rine embryonic stem cell (ESC) differentiation. The role of DNA methylation in this process and the mechanism that targets DNA methylation to the enhancers during differentiation have not been addressed. Understanding the fundamental epigenetic mechanisms involved in the establishment of enhancer-mediated pluripotency (Pp) gene repression during normal cell differentiation will, in the long-term, lead to development of therapeutic strategies to restore Pp gene repression in cancer cells. Our objective in this application is to elucidate molecu- lar mechanism(s) that regulate the activity of Dnmt3a and 3b at enhancers of Pp genes genome-wide using ESC differentiation as a model system. Supported by our strong preliminary data and recently published stud- ies on the dynamics of the chromatin state of Pp gene enhancers, we will test our hypothesis that Lsd1- Mi2/NURD activity acts as an epigenetic switch at Pp gene enhancers to activate Dnmt3 enzymes, causing site-specific DNA methylation and stable Pp gene repression. We will further elucidate the role of chromatin conformation in facilitating enhancer-targeted activity of Dnmt3a and/or 3b to specific promoters during the ear- ly phase of ESC differentiation. To test if both Dnmt3a and 3b are regulated by these mechanisms, we will map their genome-wide activity during ESC differentiation. Our rationale for these studies is that their successful completion is expected to fill the gap in our understanding of how the epigenetic “cross talk” mechanisms, dur- ing cell differentiation, modulate Dnmt3a/3b activity to terminate the pluripotency program, disruption of which could lead to developmental disorders and cancer. Additionally, the outcomes from these studies are expected to provide new insights into how the interplay between various epigenetic factors affects gene expression, con- tributing to phenotypic variation and transgenerational inheritance.