MAX/Mga异源二聚体在胚胎干细胞生物学特性维持中的分子机制研究

The capacity of embryonic stem cells for indefinitely unlimited self-renewal and differentiation into virtually all of the cell types building our body has opened up the an entirely new chapter in regenerative medicine. However, the molecular mechanisms underlying the maintenance of pluripotency during proliferative expansion are largely unknown. Recent studies suggest that myc genes are critical for the acquisition (induced pluripotent, iPS cells) and maintenance (mESC and other stem cells) of stem cell properties, but their potential endogenous functions in these processes remain elusive. To address the role of myc gene family in ES cells, we used an independently designed CRISPR-Cas9-based screening to identify genes from this family that are essential for the maintenance of ES cells. We find that While endogenous Myc (including c-myc, N-myc and L-Myc) and MXD are dispensable for ES cell function, MAX and Mga are crucial for the maintenance of pluripotency and self-renewal of ES cells. Deletion of MAX or MGA results in compromised proliferation and abnormal differentiation of ES cells. The overall objective of this study is to comprehensively define the role of MAX/Mga as wells as MAX/Mga-containing PRC1.6 (Polycomb Repressive Complex1.6) in stem cells and embryonic development and to determine their function at the molecular mechanistic level. To address these questions, we take advantage of a multi-disciplinary platform combining molecular, cellular biology, biochemistry, epigenetics as well as the generation and analysis of knockout mouse models. The proposed study will not only enhance our understanding of molecular mechanism governing stem cell pluripotency but also offer basic knowledge that is required for realizing the full 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机体几乎所有的细胞类型的特点。因此，它为再生医学揭开了崭新的篇章。然而，干细胞全能性维持的具体分子机制至今仍未完全阐明。最近人们发现，MYC家族成员在诱导多能干细胞的获得及其各种干细胞的维持中发挥关键调节作用，不过，它们的生理功能还是一个迷。我们利用CRISPR-CAS9技术在胚胎干细胞逐个完全剔除MYC基因超家族的每个成员，结果显示只有MAX和Mga在该细胞的生物学特性维持中起决定性作用。MAX或Mga敲除胚胎干细胞的增殖和分化出现显著异常。蛋白组学研究显示，MAX和Mga是非经典复合物PRC1.6的主要成员。本项研究的目的是运用多学科手段，深入探讨MAX／Mga及其所在PRC1.6在胚胎干细胞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。

多梳蛋白（PcG）家族成员通过形成多蛋白复合物来参与细胞的命运决定，但是，其中的分子机制一直不太清楚。在这项研究中，我们发现PcG成员Pcgf6及Mga在小鼠的胚胎发育过程中非常重要，因为缺失这两个基因的小鼠胚胎分别出现部分和围着床期致死。即使成活的Pcgf6基因剔除小鼠也会出现生育缺陷。有意思的是，Pcgf6基因剔除小鼠的成体组织和细胞中过度表达生殖细胞特有基因。进一步研究发现，Pcgf6通过招募G9a/Glp and Hdac1/2从而实现在正常组织中沉默生殖细胞特有基因的表达。另外，利用CRISPR筛选技术我们在胚胎干细胞发现内胚层基因Gata6的抑制分子，PcG成员Mga。剔除Mga基因的胚胎干细胞出现严重细胞自我更新缺陷，并逐渐自发向胚外内胚层方向分化。总之，我们的研究揭开了PcG在细胞命运决定中的关键作用，突出Mga/Pcgf6分别对内胚层基因及生殖细胞特有基因的抑制是细胞谱系决定的关键所在。

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