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，染色质是DNA和蛋白质的多样化和动态组合。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，特殊的染色质蛋白代表组织特异性基因表达模式的候选主要调节者。特殊的组蛋白变异体宏H_2A1与发育调节的基因沉默事件有关，是“严厉”基因组的成员。许多关于宏H_2A1的工作都集中在它在X染色体失活中的作用上，但是宏H_2A1在两性中广泛表达，并且在特定的成体器官中的表达水平差异很大。我们将精确地定位小鼠基因组中涉及宏H_2A1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DNMT1的胚胎干细胞中，宏H_2A1的基因组分布发生了变化。我们将在DNA序列水平上定位野生型和Dnmt1-突变型ES细胞中宏H_2A1的基因组分布。这些细胞的使用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，我们将确定并绘制在ES细胞进行定向分化时宏H_2A1(染色质重塑)的基因组重新分布。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。这项工作将构成对哺乳动物基因组上专门的染色质成分对发育线索的反应的第一次全面评估。我们还将完成小鼠胚胎发育过程中宏H_2A1表达所需的发育特征，分析细胞衰老，并比较宏H_2A1在年轻和老年器官中的组织分布。胚胎干细胞向治疗性细胞的分化(干细胞治疗)本质上是一种重述体外发育的尝试。这里提出的研究将提供对分化生物学的基本见解，并为未来实现合理指导的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.