Mechanisms and Role of DNA Methylation in X-chromosome Inactivation

在哺乳动物雌性细胞中，X染色体的一个拷贝变得失活，这是维持正确基因剂量比例所必需的过程。X染色体失活的机制一直是发育遗传学家深入研究的主题-它是基因表达的表观遗传控制的一个迷人的例子。已知该失活过程的主开关是长的非编码RNA基因Xist。Xist RNA包被其表达的染色体，这导致多梳抑制复合物（PRC 1和PRC 2）的募集和异染色质组蛋白修饰的沉积，特别是组蛋白3赖氨酸27三甲基化（H3 K27 me 3）和组蛋白2A赖氨酸119泛素化（H2 AK 119 ub）。这两种标记的补充和沉积机制已被广泛研究，但它们仍远未被完全理解。这是特别困难的，因为PRC 1和PRC 2依赖于相互的反馈机制，并且也是彼此的修改产物的读取器。更有趣的是，组蛋白修饰与DNA修饰紧密交织在一起，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。已知在失活的X染色体上存在升高的启动子甲基化，并且这种甲基化对于维持失活状态可能是重要的。然而，与其他表观遗传修饰相比，DNA甲基化对X失活的贡献知之甚少。 我们的小组已经参与了一些最近的发现，阐明招募DNA甲基转移酶的组蛋白修饰。我们已经证明（温伯格等人，《自然》，2019年），从头甲基转移酶DNMT 3A的PWWP结构域对H3 K36 me 2标记具有很强的亲和力，并且这种修饰是在基因间区域建立DNA甲基化所必需的。最近，我们证明了DNMT 3A有一个额外的结构域，通过它识别H2 AK 119 ub，这种识别在建立非活性基因启动子CpG岛的甲基化中很重要。（温伯格等人，《自然遗传学》，2021）。我们还发现，非活性X染色体上的高水平H2 AK 119 ub为DNMT 3A提供了强靶标。 在这个提议中，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，以及干扰这种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.