Quantitating chromatin dynamics during erythropoiesis

描述(由申请人提供)：在红细胞生成过程中，细胞经历了一系列深刻的形态变化，在此过程中，除了有限的一部分基因外，所有的基因都被沉默了，这可能与异染色质的整合有关。与此同时，其他基因，如β-珠蛋白，变得高度激活。高阶染色质结构的调控机制，以及它如何与基因激活的其他方面，如亚核定位、表观遗传修饰和表达等联系在一起，目前尚不清楚。用内源性β-珠蛋白基因定点突变产生细胞系和小鼠已被证明是开始将这些过程联系起来的一种有价值的方法。不幸的是，用于评估染色质敏感性的标准凝胶分析方法是非定量的、耗时的、难以重现的、需要大量的组织，并且只能提供有限区域的信息。染色质定量聚合酶链式反应(QCP)是最近被开发和验证的一种工具，可以灵敏和特异地筛选基因组中的DNA酶L超敏部位。这项建议的目标是通过验证QCP作为一种工具来定量描述整个基因座上DNA酶L敏感性的连续“轮廓”，从而使我们能够将染色质景观中的变化与细胞形态、亚核定位、组蛋白修饰和末端分化期间的表达相关联，并确定顺式作用突变的影响。QCP将在几个模型系统中与标准DNA酶L分析进行比较，以同时验证QCP并解决有关红细胞生成过程中染色质结构的问题。具体地说，我们建议1)建立整个人β珠蛋白基因座染色质敏感性的连续图谱(图谱)，并将QCP中观察到的模式变化与传统DNA酶L染色质敏感性分析中观察到的模式变化相关联；2)通过定量定义染色质结构的变化并确定这些变化与用于解决红细胞生成的种子问题的系统中活性表达的变化之间的相关性，来论证QCP的有效性。这些包括在末端分化过程中几个基因的活跃表达和染色质结构的关联，以及确定LCR突变和内源性基因座的额外靶向突变如何影响染色质结构，以及3)采用QCP来研究野生型和突变小鼠初级组织中的内源性小鼠β-珠蛋白基因座。

DESCRIPTION (provided by applicant): During erythropoiesis cells go through a progression of profound morphologic changes during which all but a limited subset of genes are silenced, presumably associated with incorporation into heterochromatin. Meanwhile other genes, such as beta-globin, become highly activated. The mechanisms by which higher order chromatin structure is regulated, and how it is linked to other aspects of gene activation such as subnuclear localization, epigenetic modifications and expression remains unknown. Generating cell lines and mice with targeted mutations of the endogenous beta-globin locus has proven to be a valuable approach to begin to link these processes. Unfortunately, standard gel based assays to assess generalized chromatin sensitivity are non-quantitative, time consuming, difficult to reproduce, require tremendous amounts of tissue, and only provide information on limited areas. Quantitative Chromatin PCR (QCP) has been recently developed and validated as a tool to sensitively and specifically screen large regions of the genome for DNase l hypersensitivity sites. The goal of this proposal is to extend this by validating QCP as a tool to quantitatively describe continuous "profiles" of DNase l sensitivity over entire gene loci allowing us to correlate changes in this chromatin landscape with cellular morphology, sub-nuclear localization, histone modifications and expression during terminal differentiation, and to determine the effect of cis-acting mutations. QCP will be compared with standard DNase l assays in several model systems to simultaneously validate QCP and address questions regarding chromatin structure during erythropoiesis. Specifically we propose to 1) develop a continuous map (landscape) of chromatin sensitivity throughout the human beta globin locus and to correlate changes in patterns observed in QCP with those observed with traditional DNase l chromatin sensitivity assays, 2) demonstrate the utility of QCP by quantitatively defining changes in chromatin structure and determining how these correlate with changes in active expression in systems designed to address seminal questions of erythropoiesis. These include correlating active expression and chromatin structure of several genes during terminal differentiation, and determining how LCR mutations and additional targeted mutations of the endogenous locus affect chromatin structure, and 3) adapt QCP to study the endogenous mouse beta-globin locus in primary tissues of wildtype and mutant mice.