Mechanisms and Role of DNA Methylation in X-chromosome Inactivation

在哺乳动物的雌性细胞中，X染色体的一个拷贝会失活，这是保持正确的基因剂量比例所必需的过程。X基因失活的机制一直是发育遗传学家深入研究的主题--这是表观遗传控制基因表达的一个有趣的例子。已知这一失活过程的主开关是非编码的长链RNA基因Xist。Xist RNA覆盖在表达Xist的染色体上，导致多梳抑制复合体(PRC1和PRC2)的招募和异染色组蛋白修饰的沉积，特别是组蛋白3赖氨酸27三甲基化(H3K27me3)和组蛋白2A赖氨酸119泛素化(H2AK119ub)。这两个标记的招募和沉积机制已经得到了广泛的研究，但它们仍然远未完全被理解。这尤其困难，因为PRC1和PRC2依赖于相互的反馈机制，并且也是彼此的修改产品的读取器。更有趣的是，组蛋白修饰与DNA修饰紧密交织在一起，例如胞嘧啶甲基化。CpG二核苷酸的甲基化在基因表达沉默中具有公认的作用。已知在不活跃的X染色体上存在高水平的启动子甲基化，并且这种甲基化可能对维持不活跃状态很重要。然而，与其他表观遗传修饰相比，DNA甲基化对X-失活的贡献还知之甚少。我们小组参与了一些最近的发现，阐明了组蛋白修饰对DNA甲基转移酶的招募。我们已经证明(Weinberg等人)。自然2019年)，从头甲基转移酶DNMT3A的PWWP结构域与H3K36me2标记有很强的亲和力，并且这种修饰是建立异源区域DNA甲基化所必需的。最近，我们证明了DNMT3A有一个额外的结构域，它通过这个结构域识别H2AK119ub，并且这种识别在建立非活性基因启动子上的CpG岛的甲基化方面是重要的。(温伯格等人)自然遗传学2021)。我们还发现，非活动X染色体上高水平的H2AK119ub是DNMT3A的强烈靶点。在这个提案中，我们将进一步探索这些最新的发现，以了解DNA甲基化是如何在非活性X上建立的，以及干扰这一甲基化过程的转录后果是什么。

In mammalian female cells, one of the copies of the X chromosome becomes inactivated, a process that is necessary to maintain correct gene dosage proportions. The mechanisms of X-inactivation have been a subject of deep scrutiny by developmental geneticists - it is a fascinating example of epigenetic control of gene expression. It is known that the master switch of this inactivation process is the long non-coding RNA gene, Xist. The Xist RNA coats the chromosome from which it is expressed which leads to the recruitment of polycomb repressive complexes (PRC1 and PRC2) and deposition of heterochromatic histone modifications, specifically Histone 3 Lysine 27 trimethylation (H3K27me3) and Histone 2A Lysine 119 ubiquitylation (H2AK119ub). The mechanisms of recruitment and deposition of those two marks have been extensively studied, but they are still far from being completely understood. This is particularly difficult, since PRC1 and PRC2 depend on mutual feedback mechanisms and are also readers of each other's modification products. Even more interestingly, histone modifications are tightly intertwined with DNA modifications, such as Cytosine methylation. Methylation of CpG dinucleotides has a well-established role in silencing of gene expression. It is known that there is elevated promoter methylation on the inactive X chromosome, and that this methylation may be important for maintenance of the inactive state. However, compared to other epigenetic modifications, the contribution of DNA methylation to X-inactivation is very poorly understood. Our group has been involved in a number of recent discoveries elucidating the recruitment of DNA methyltransferases by histone modifications. We have shown (Weinberg et al. Nature 2019) that the PWWP domain of the de novo methyltransferase DNMT3A has a strong affinity for the H3K36me2 mark, and that this modification is required for establishing DNA methylation in inetergenic regions. More recently, we demonstrated that DNMT3A has an additional domain through which it recognizes H2AK119ub, and that this recognition is important in establishing methylation of CpG islands at inactive gene promoters. (Weinberg et al. Nature Genetics 2021). We have also found that the high levels of H2AK119ub on the inactive X chromosome present a strong target for DNMT3A. In this proposal, we will further explore those recent findings in order to understand how DNA methylation is established on the inactive X, and what are the transcriptional consequences of disturbing this methylation process.