Function of human & mouse Beta-globin locus control regions

描述（由申请人提供）：我们对？-珠蛋白基因调控已经产生了新的和令人惊讶的结果，包括基因座控制区（LCR）不需要启动或繁殖基因座的“开放”染色质结构或赋予β-珠蛋白的阶段特异性表达。比如珠蛋白基因在其天然位置，LCR的主要作用是增强从基础转录到激活转录的转变。我们现在建议进一步深入了解启动和传播的染色质和转录状态的分子基础？红细胞生成过程中的珠蛋白位点。我们的具体目标包括：1.定义表观遗传特征，包括组蛋白修饰，序列共定位，DNA甲基化和参与启动和传播的活性和沉默状态的候选反式作用因子？珠蛋白基因座我们将测试这个假设，？-珠蛋白“结构域”比以前认识到的更大且更动态，并且特定序列、因子和表观遗传修饰涉及该基因座的活性和沉默状态的起始和传播。最初的研究，与X-D Fu博士（UCSD）合作，开发了高灵敏度的DNA选择和连接（DSL）基于阵列的方法，将集中在人类染色体11（h11）的1 mb区域，以LCR为中心，使用目前可用的阵列。我们将设计并构建一个类似的阵列，用于分析小鼠基因座。2.确定顺式作用调控元件的作用，转录和细胞背景对组蛋白修饰，因子结合和序列共定位在？珠蛋白基因座我们将使用ES细胞衍生的小鼠和我们的h11转移系统中的靶向突变分析来研究关于β-珠蛋白基因表达调控的几种假设。突变对基因座的表观遗传状态的影响，包括因子结合、组蛋白修饰和序列相互作用，将通过DSL分析在来自突变小鼠的阶段特异性红系细胞和通过不同细胞背景的含有WT和修饰的h11的细胞系中确定。这些结果与由初级转录本FISH、HS形成和广义DNA酶敏感性的程度和范围定义的激活状态的相关性将解决p-珠蛋白基因调控的几种模型。3.鉴定参与红细胞成熟和β-珠蛋白基因表达的新基因。我们将在充分表征的G1 E-ER细胞和ES衍生的红系祖细胞（ES-EP）系中进行无偏倚的全基因组siRNA筛选，以鉴定参与调节β-珠蛋白基因表达和红系成熟的新因子。该筛选利用慢病毒siRNA文库，该文库由每个已知小鼠基因的siRNA冗余组组成。我们提出了几种策略的验证，鉴定和优先级的候选siRNA，以及生化和功能分析的基因鉴定。

DESCRIPTION (provided by applicant): Our genetic, molecular and biochemical analyses of ?-globin gene regulation have yielded novel and surprising results, including that the locus control region (LCR) is not required to initiate or propagate the "open" chromatin structure of the locus or confer stage-specific expression of the ?-like globin genes. In its native location, the primary role of the LCR is to enhance the transition from basal to activated transcription. We now propose to gain further insights into the molecular basis for initiating and propagating the chromatin and transcription states of the ?-globin locus during erythropoiesis. Our Specific Aims include: 1. Define epigenetic features including histone modifications, sequence co-localization, DNA methylation and candidate trans-acting factors involved in the initiation and propagation of active and silent states of the ?-globin locus. We will test the hypothesis that the ?-globin "domain" is larger and more dynamic than formerly appreciated, and that specific sequences, factors and epigenetic modifications are involved in the initiation and propagation of active and silent states of the locus. Initial studies, in collaboration with Dr. X-D Fu (UCSD) who developed the highly sensitive DNA Selection and Ligation (DSL) array-based method, will focus on a 1mb region of human chromosome 11 (h11) centered on the LCR, using a currently available array. We will design and build a similar array for analysis of the mouse locus. 2. Determine the role of cis- acting regulatory elements, transcription and cellular background on domains of histone modifications, factor binding and sequence co-localization in the ?-globin locus. We will use targeted mutation analyses in ES cell derived mice and our h11 transfer system to investigate several hypotheses regarding the regulation of p-globin gene expression. The effects of mutations on the epigenetic states of the locus, including factor binding, histone modifications and sequence interactions, will be determined by DSL analyses in stage-specific erythroid cells from mutant mice and in cell lines containing WT and modified h11s passed through different cellular backgrounds. Correlation of these results with activation state defined by primary transcript FISH, HS formation and the degree and extent of generalized DNase sensitivity will address several models of p-globin gene regulation. 3. Identify novel genes involved in erythroid maturation and p-globin gene expression. We will perform unbiased, genome-wide siRNA screens in the well- characterized G1E-ER cells and in ES derived erythroid progenitor (ES-EP) lines to identify novel factors involved in regulating p-globin gene expression and erythroid maturation. The screen makes use of a lentiviral siRNA library comprised of a redundant set of siRNAs for each known mouse gene. We propose several strategies for the validation, identification and prioritization of candidate siRNAs, as well as for the biochemical and functional analysis of genes identified.