L3mbtl2在胚胎干细胞自我更新和多能性维持中的分子机制研究

Embryonic stem cells can be grown indefinitely in culture while maintaining the capacity to differentiate into any cell type in the body and therefore hold tremendous promise for the future of regenerative medicine. However, a detailed understanding of the molecular mechanisms that regulate the pluripotent state is currently lacking. Recently, we show that L3mbtl2, an mbt family member, is critical for early embryo development as well aspluripotencymaintenance in embryonic stem (ES) cells. Deletion of L3mbtl2 results in embryonic lethality with failure of gastrulation and accordingly this correlates with compromised proliferation and abnormal differentiation of L3mbtl2-deficient ES cells. In ES cells, L3mbtl2 establishes an atypical Polycomb repressive complex 1(PRC1) that includes Oct4, G9A and several components of the E2F6 and NuRD repressor complexes. Intriguingly, the majority of genes bound and repressed by L3mbtl2 in ESCs are not occupied by canonical PRC1 and PRC2.The main objective of this study is to comprehensively establish the role of L3mbtl2 as wells as L3mbtl2-containing atypical PRC1 in stem cells and embryonic developmentand to define its function at a molecular, mechanistic level. In addressing these research questions we use a multi-disciplinary platform that encompassesbiochemistry, molecular, cellular biology,epigeneticsand the generation and analysis of knockout mouse models.The success of our study will not only contribute to uncovering novel and essential molecular mechanism for governing stem cell pluripotency but also provide basic knowledge that in the long term is required for realizing the therapeutic potential of stem cells.

胚胎干细胞具有体外培养无限增殖、同时保持被诱导分化为体内几乎所有的细胞类型的特性。因此，它在再生医学方面具有广泛而诱人的应用前景。然而，有关干细胞全能性的具体分子机制至今仍不清楚。我们最近的研究发现，MBT家族成员L3mbtl2在早期胚胎发育和胚胎干细胞多能性维持中发挥关键调节作用。完全剔除L3mbtl2可导致小鼠胚胎不能完成原肠胚发育而致死的结果; 与此相应的是，L3mbtl2敲除胚胎干细胞的增殖和分化出现异常。蛋白组学及全基因组的研究显示，L3mbtl2通过招募非经典PRC1相关的复合物来调节基因表达。本项研究的主要目的是运用包括小鼠遗传学、生物化学、基因组学和蛋白组学等多学科手段，深入探讨L3mbtl2及其介导的非典型PRC1复合物在胚胎干细胞增殖与分化和胚胎发育中的作用及其分子机制。本课题的顺利开展不仅能进一步揭示干细胞全能性的分子机制，而且能为将来实现干细胞的临床应用提供理论依据

尽管我们先前的研究揭示多梳蛋白（Polycomb）家族成员L3mbtl2及其Pcgf6作为核心表观调控因子在胚胎干细胞的多能性维持中具有关键作用，但是它们的生理功能以及作用的具体分子机制仍是未解之谜。在这项研究中，通过对L3mbtl2及Pcgf6的结构功能分析，我们发现L3mbtl2的第一及二个MBT结构域对它们所在的复合物PRC1.6的靶基因招募中具有重要作用。非常有趣的是，这个在进化上高度保守的重要区域也存在于果蝇的Sfmbt中，并能形成Pho结合口袋；这个口袋中关键氨基酸残基的突变能使L3mbtl2完全丧失它和PRC1.6复合物其它成员以及靶基因的结合。另外，染色质沉淀技术显示， 在Pcgf6, L3mbtl2, 及Max 分别剔除的细胞中， PRC1.6复合物在靶基因上富集都有显著下降。总之，我们的研究揭开了多梳蛋白复合物在染色质上富集的招募机制，突出L3mbtl2/Pcgf6及其它成员的相互结合是招募的关键所在，颠覆了长期以来统治多梳蛋白领域的单因子作用假说。