Mechanisms and Role of DNA Methylation in X-chromosome Inactivation

在哺乳动物雌性细胞中，X染色体的一个拷贝失活，这是维持正确的基因剂量比例所必需的过程。x失活的机制一直是发育遗传学家深入研究的课题——它是基因表达的表观遗传控制的一个引人入胜的例子。众所周知，这种失活过程的主开关是长链非编码RNA基因Xist。Xist RNA包裹在其表达的染色体上，导致多梳抑制复合物（PRC1和PRC2）的募集和异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）。这两种标记的吸收和沉积机制已被广泛研究，但仍远未完全了解。这是特别困难的，因为PRC1和PRC2依赖于相互反馈机制，并且也是彼此修改产品的读者。更有趣的是，组蛋白修饰与DNA修饰紧密交织在一起，比如胞嘧啶甲基化。CpG二核苷酸的甲基化在基因表达沉默中起着重要作用。众所周知，在无活性的X染色体上存在升高的启动子甲基化，并且这种甲基化可能对维持无活性状态很重要。然而，与其他表观遗传修饰相比，DNA甲基化对x失活的贡献知之甚少。我们的团队参与了许多最近的发现，阐明了通过组蛋白修饰来募集DNA甲基转移酶。我们已经证明（Weinberg等人）。Nature 2019)发现，新甲基转移酶DNMT3A的PWWP结构域与H3K36me2标记具有很强的亲和力，并且这种修饰是在基因间区建立DNA甲基化所必需的。最近，我们证明DNMT3A有一个额外的结构域，它通过该结构域识别H2AK119ub，并且这种识别对于在非活性基因启动子上建立CpG岛的甲基化是重要的。（Weinberg et al.）自然遗传学2021)。我们还发现，在失活的X染色体上高水平的H2AK119ub是DNMT3A的强靶标。在本提案中，我们将进一步探索这些最近的发现，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。

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.