Developmental Regulation of MacroH2A1 Chromatin Assembly

哺乳动物体细胞含有相同的DNA含量，但不同细胞类型的基因表达差异很大。体细胞内的DNA被包装成染色质，这是DNA和蛋白质的多样化和动态组合。特殊染色质的组装为建立和维持组织特异性的基因表达模式提供了一种手段。因此，特化的染色质蛋白代表了组织特异性基因表达模式的候选主要调节因子。特殊的组蛋白变体macroH2A1与发育调节的基因沉默事件有关，并且是“严厉”基因集的成员。许多关于macroH2A1的研究都集中在其在X染色体失活中的作用，但macroH2A1在两性中广泛表达，并且在特定的成人器官中表达水平差异很大。我们将在小鼠基因组中精确绘制涉及macroH2A1的染色质重塑事件。初步数据显示，在缺乏小鼠维持胞嘧啶甲基转移酶基因Dnmt 1的胚胎干细胞中，macroH2A1的基因组分布发生了改变。我们将把macroH2A1在野生型和Dnmt - 1突变ES细胞中的基因组分布定位到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，我们将确定并绘制胚胎干细胞在定向分化过程中macroH2A1（染色质重塑）的基因组重分布。我们将使用携带Xist转基因的雌性胚胎干细胞和雄性胚胎干细胞来提供涉及已知X染色体失活事件的内部控制。这项工作将构成一个专门的染色质组件组装在哺乳动物基因组响应发育线索的第一个综合评估。我们还将完成小鼠胚胎发生过程中macroH2A1表达的发育特征，分析细胞衰老，并比较年轻和老年器官中macroH2A1的组织学分布。胚胎干细胞分化为治疗性细胞（干细胞治疗）本质上是一种试图在体外重现发育的尝试。这里提出的研究将为分化生物学提供基本见解，并为未来尝试实现理性引导的胚胎干细胞分化提供框架。

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.