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

项目摘要 维持适当的DNA甲基化模式对于细胞身份的完整性是必不可少的，而细胞身份的完整性 导致基因异常激活，进而调节导致癌症和细胞死亡的信号通路。 发育障碍。尽管有大量证据支持DNA甲基化异常在 几种人类疾病的病因学，调节靶部位特异性的基本机制 从头DNA MTase，DNMT3a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 涉及调节元件中共激活因子复合体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-2的作用 不同癌症中癌基因的基因调控。其他人则表现出plu-1基因的异常表达。 多能基因在几种癌症中介导去分化。增强的染色质状态的变化- 已证明多能基因的ERs在多能性基因的抑制过程中起着关键作用。 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 在分化过程中以DNA甲基化为靶点的增强子还没有解决。理解 增强子介导的多能性(PP)建立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方法的发展 修复癌细胞中PP基因抑制的策略。我们在这项申请中的目标是阐明分子- 全基因组调控PP基因增强子DNMT3a和3b活性的LAR机制(S) ESC分化作为一个模型系统。在我们强劲的初步数据和最近发表的研究报告的支持下- 关于PP基因增强子染色质状态的动态，我们将检验我们的假设LSD1- Mi2/NuRD活性作为PP基因增强子的表观遗传开关激活Dnmt3酶，导致 位点特异性DNA甲基化和稳定的PP基因抑制。我们将进一步阐明染色质的作用 促进DNMT3a和/或3b对特定启动子的增强子靶向活性的构象 ESC分化的Ly期。为了测试DNMT3a和3b是否都受这些机制的调节，我们将映射 它们在胚胎干细胞分化过程中的全基因组活性。我们进行这些研究的理由是，他们的成功 这项研究的完成有望填补我们在理解表观遗传“串扰”机制方面的空白。 在细胞分化中，调节DNMT3a/3b的活性以终止多能性程序，其中 可能导致发育障碍和癌症。此外，这些研究的结果也是意料之中的 为了对不同表观遗传因素之间的相互作用如何影响基因表达提供新的见解， 归因于表型变异和跨代遗传。

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.