Hormonal Control of Histone Modifications in ES Cells

描述（由申请人提供）：胚胎干（ES）细胞分化的最早步骤的分子方面仍然知之甚少。我们的初步数据表明，全局组蛋白乙酰化可能是分化的关键第一步。本提案的目标是确定在多种激素诱导的胚胎干细胞分化方法中是否发生全局组蛋白乙酰化和/或甲基化，并使用组蛋白修饰的标准测定法确定这些组蛋白修饰发生的时间框架。我们的模型预测，这些组蛋白修饰的大部分可能发生在启动子区域，我们将利用新的CpG岛阵列来证实这一点。研究旨在确定在未分化胚胎干细胞状态退出过程中发生的全局组蛋白修饰是否与激素信号诱导的高度分化细胞的基因特异性组蛋白修饰有独特的不同。第二个具体目标是通过筛选胚胎干细胞提取物中负责历史共价修饰的各种酶活性，探索在胚胎干细胞分化早期导致这些独特的全局组蛋白修饰的可能机制。第三个特定目的是测试组蛋白修饰在直接调节胚胎干细胞分化中的功能意义。组蛋白去乙酰化酶抑制剂trichostatin A （TSA）将与特定生长因子联合使用，以增加定向到特定承诺细胞命运的细胞的速率和比例。此外，在胚胎干细胞中过度表达旨在抑制组蛋白乙酰转移酶活性或增加组蛋白去乙酰化酶活性的特定基因产物，将被评估对胚胎干细胞分化率和整体细胞命运承诺的影响。这些研究旨在了解早期激素调节的胚胎干细胞分化，并有望在体外显著提高谱系特异性分化的产量。这将极大地促进胚胎干细胞技术的发展，将“纯”细胞群移植到帕金森病、再生障碍性贫血等疾病患者身上。此外，本应用中提出的模型预测，组蛋白去乙酰化酶抑制剂，如丙戊酸（一种已知的致畸剂）和TSA，可能通过改变正常谱系特异性分化所需的非常早期组蛋白乙酰化模式，极大地增强环境化合物的致畸作用。

DESCRIPTION (provided by applicant): The molecular aspects of the earliest steps in embryonic stem (ES) cell differentiation remain poorly understood. Our preliminary data suggest that global histone acetylation may be a critical first step in differentiation. The goals of this proposal are to establish whether global histone acetylation and/or methytation occurs during multiple hormonally induced methods of ES cell differentiation and establish the time frame in which these histone modifications occur using standard assays for histone modifications. Our model predicts that the bulk of these histone modifications probably occur in promoter regions and we will make use of novel CpG island arrays to confirm this. Studies are designed to determine whether the global histone modifications that occur during exit from the undifferentiated ES cell state are uniquely different from the gene-specific histone modifications induced by hormonal signaling to highly differentiated cells. The second specific aim is to explore the possible mechanisms leading to these unique global histone modifications early in ES cell differentiation by screening ES cell extracts for various enzymatic activities responsible for covalent modification of histories. The third specific aim is designed to test the functional significance of histone modifications in directly regulating ES cell differentiation. The histone deacetylase inhibitor, trichostatin A (TSA), will be used in conjunction with specific growth factors to increase the rate and proportion of cells directed to a specific committed cell fate. In addition, overexpression of specific gene products designed to inhibit histone acetyltransferase activity or increase histone deacetylase activity in ES cells will be assessed for effects on the rate of ES cell differentiation and overall cell fate commitment. The proposed studies are designed to understand early hormonally regulated ES cell differentiation with potential application for significantly improving the yield of lineage-specific differentiation in vitro. This would greatly facilitate the development of ES cell technology for potential transplantation of "pure" cell populations into patients with diseases such as Parkinson disease, aplastic anemia, etc. In addition, the model proposed within this application predicts that histone deacetylase inhibitors such as valproic acid (a known teratogen) and TSA may greatly potentiate the teratogenic effects of environmental compounds by altering the very early histone acetylation pattern required for normal lineage-specific differentiation.