Mechanisms and Role of DNA Methylation in X-chromosome Inactivation

DNA 甲基化在 X 染色体失活中的机制和作用

• 批准号: RGPIN-2022-04773
• 负责人: Majewski, Jacek
• 金额: $ 2.91万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=757111
• 关键词: Mechanisms; Role; DNA; Methylation; chromosome

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.

在哺乳动物雌性细胞中，X 染色体的一个副本失活，这是维持正确的基因剂量比例所必需的过程。 X-失活机制一直是发育遗传学家深入研究的课题——它是基因表达表观遗传控制的一个有趣的例子。据了解，这种失活过程的主开关是长非编码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 (H3K27me3) 和组蛋白 2A 赖氨酸 119 泛素化 (H2AK119ub)。这两种标记的招募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（例如胞嘧啶甲基化）紧密相连。 CpG 二核苷酸的甲基化在基因表达沉默中具有明确的作用。众所周知，失活的 X 染色体上启动子甲基化程度升高，并且这种甲基化可能对于维持失活状态很重要。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。 我们小组最近参与了许多阐明组蛋白修饰招募 DNA 甲基转移酶的发现。我们已经证明（Weinberg 等人，Nature 2019），从头甲基转移酶 DNMT3A 的 PWWP 结构域对 H3K36me2 标记具有很强的亲和力，并且这种修饰是在非基因区域建立 DNA 甲基化所必需的。最近，我们证明 DNMT3A 有一个额外的结构域，通过它可以识别 H2AK119ub，并且这种识别对于在失活基因启动子处建立 CpG 岛的甲基化非常重要。 （Weinberg 等人，《自然遗传学》2021 年）。我们还发现，失活 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新发现，以了解 DNA 甲基化是如何在不活跃的 X 上建立的，以及干扰这种甲基化过程的转录后果是什么。