Developmental Regulation of MacroH2A1 Chromatin Assembly

哺乳动物体细胞含有相同的DNA含量，但基因表达因细胞类型而异。体细胞内的DNA被包装成染色质，染色质是DNA和蛋白质的多样性和动态组合。特化染色质的组装为建立和维持组织特异性基因表达模式提供了一种手段。因此，专门的染色质蛋白代表候选人的主调控基因表达的组织特异性模式。特化组蛋白变体macroH2A1与发育调控的基因沉默事件有关，并且是“干性”基因组的成员。关于macroH2A1的许多工作都集中在它在X染色体失活中的作用，但macroH2A1在两性中广泛表达，并且在特定的成年器官中以截然不同的水平表达。 我们将精确映射小鼠基因组内涉及macroH2A1的染色质重塑事件。 初步数据显示，macroH2A1的基因组分布在缺乏Dnmt 1（小鼠维持胞嘧啶甲基转移酶基因）的胚胎干（ES）细胞中发生改变。我们将绘制macroH2A1在野生型和Dnmt 1突变型ES细胞中的基因组分布到DNA序列水平。使用这些细胞将使我们能够开发和优化哺乳动物染色质研究急需的技术。然后，我们将确定和地图macroH2A1（染色质重塑）在胚胎干细胞的基因组再分布，因为他们进行定向分化。我们将使用携带Xist转基因的雌性ES细胞和雄性ES细胞来提供涉及已知X染色体失活事件的内部对照。这项工作将构成第一次全面评估的组装一个专门的染色质组成部分对哺乳动物基因组的发展线索。我们还将完成小鼠胚胎发育过程中macroH2A1表达的发育特征，分析细胞衰老，并比较macroH2A1在年轻与老年器官中的组织学分布。 胚胎干细胞分化为治疗性细胞（干细胞治疗）本质上是一种尝试重演体外发育。这里提出的研究将提供分化生物学的基本见解，并为未来尝试实现合理指导的ES细胞分化提供一个框架。

Mammalian somatic cells contain identical DNA content, yet gene expression varies widely from cell-type to cell-type. DNA within somatic cells is packaged into chromatin, a diverse and dynamic combination of DNA and proteins. The assembly of specialized chromatin provides a means for the establishment and maintenance of tissue-specific patterns of gene-expression. Therefore, specialized chromatin proteins represent candidate master regulators of tissue-specific patterns of gene expression. The specialized histone variant macroH2A1 has been implicated in developmentally-regulated gene silencing events, and is a member of the "sternness" gene set. Much work on macroH2A1 has focused on its role in X chromosome inactivation, but macroH2A1 is widely expressed in both sexes, and is expressed at vastly different levels in specific adult organs. We will precisely map chromatin remodeling events involving macroH2A1 within the mouse genome. Preliminary data shows that the genomic distribution of macroH2A1 is altered in embryonic stem (ES) cells lacking Dnmt 1, the mouse maintenance cytosine methyltransferase gene. We will map the genomic distribution of macroH2A1 in wild-type and Dnmt 1-mutant ES cells to the level of DNA sequence. Use of these cells will allow us to develop and optimize much-needed technologies for mammalian chromatin research. We will then ascertain and map genomic redistributions of macroH2A1 (chromatin remodeling) in ES cells as they undergo directed differentiation. We will use female ES cells and male ES cells bearing Xist transgenes to provide internal controls involving known X chromosome inactivation events. This work will constitute the first comprehensive assessment of the assembly of a specialized chromatin component upon the mammalian genome in response to developmental cues. We will also complete needed developmental characterization of macroH2A1 expression during mouse embryogenesis, analyze cellular senescence, and compare the histological distribution of macroH2A1 in young versus aged organs. The differentiation of ES cells into therapeutic cells (stem cell therapy) is essentially an attempt to recapitulate development in vitro. Research proposed here will provide basic insights into differentiation biology and provide a framework for future attempts to achieve rationally-guided ES cell differentiation.