Quantitating chromatin dynamics during erythropoiesis

描述（由申请人提供）：在红细胞生成过程中，细胞经历一系列深刻的形态变化，在此期间，除了有限的基因亚群外，所有基因都被沉默，可能与异染色质的结合有关。与此同时，其他基因，如-珠蛋白，被高度激活。高阶染色质结构调控的机制，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）联系在一起，仍然是未知的。产生具有内源性-珠蛋白位点靶向突变的细胞系和小鼠已被证明是开始将这些过程联系起来的有价值的方法。不幸的是，用于评估广义染色质敏感性的标准凝胶分析是非定量的，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。定量染色质PCR （Quantitative Chromatin PCR， QCP）最近得到了发展，并被证实是一种敏感和特异性筛选基因组大区域dna酶1超敏位点的工具。本提案的目标是通过验证QCP作为定量描述整个基因位点上dna酶1敏感性的连续“轮廓”的工具来扩展这一点，使我们能够将这种染色质景观的变化与细胞形态、亚核定位、组蛋白修饰和终末分化期间的表达联系起来，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1测定进行比较，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。具体来说，我们建议1)开发一个贯穿人类β -珠蛋白位点的染色质敏感性连续图（景观），并将QCP中观察到的模式变化与传统的DNase - 1染色质敏感性测定相关联；2)通过定量定义染色质结构的变化，并确定这些变化如何与旨在解决红细胞生成问题的系统中活性表达的变化相关联，来证明QCP的实用性。这些研究包括在终末分化过程中几个基因的活性表达与染色质结构的相关性，以及确定LCR突变和内源性位点的其他靶向突变如何影响染色质结构。3)利用QCP研究野生型和突变型小鼠原代组织中内源性小鼠β -珠蛋白位点。

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.