Function of human & mouse Beta-globin locus control regions

人体的机能

• 批准号: 7982455
• 负责人: MARK T GROUDINE
• 金额: $ 10万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-11-25 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7982455
• 关键词: Address; Award; Binding; Biochemical; Cell Line; Cells; Chromatin; Chromatin Structure; Chromosome Transfer; Chromosomes; Chromosomes, Human, Pair 11; Collaborations; DNA; DNA Ligation; DNA Methylation; Deoxyribonucleases; Development; Embryo; Epigenetic Process; Erythroid; Erythroid Cells; Erythropoiesis; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Globin; Human; Human Chromosomes; Libraries; Ligation; Location; Locus Control Region; Methods; Methylation; Modeling; Modification; Molecular; Molecular Genetics; Mus; Mutant Strains Mice; Mutate; Mutation; Progress Reports; Recording of previous events; Regulation; Regulatory Element; Role; Site; Small Interfering RNA; Somatic Cell; Staging; Structure; System; Testing; Trans-Activators; Transcript; Validation; base; beta Globin; cell type; design; embryonic stem cell; genome wide association study; genome-wide; histone modification; homologous recombination; insight; novel; nuclease; progenitor; stem

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.

描述（由申请人提供）：我们对 β-珠蛋白基因调控的遗传、分子和生化分析产生了新颖且令人惊讶的结果，包括不需要基因座控制区（LCR）来启动或传播 β-珠蛋白基因的“开放”染色质结构。基因座或赋予α样珠蛋白基因的阶段特异性表达。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了解红细胞生成过程中启动和传播 β-珠蛋白位点的染色质和转录状态的分子基础。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 β-珠蛋白基因座活性和沉默状态的启动和传播的候选反式作用因子。我们将检验以下假设：α-珠蛋白“结构域”比以前认为的更大、更动态，并且特定序列、因子和表观遗传修饰参与该基因座的活性和沉默状态的启动和传播。 X-D Fu 博士（加州大学圣地亚哥分校）开发了基于高灵敏度 DNA 选择和连接 (DSL) 阵列的方法，初步研究将重点关注以 LCR 为中心的人类 11 号染色体 (h11) 的 1mb 区域，使用当前可用的数组。我们将设计并构建一个类似的阵列来分析小鼠轨迹。 2.确定顺式作用调节元件、转录和细胞背景对组蛋白修饰、因子结合和α-珠蛋白基因座中的序列共定位的域的作用。我们将使用 ES 细胞衍生的小鼠和我们的 h11 转移系统进行靶向突变分析，以研究有关 p-珠蛋白基因表达调节的几种假设。突变对基因座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，将通过对来自突变小鼠的阶段特异性红细胞以及含有 WT 和修饰 h11s 的细胞系进行 DSL 分析来确定，这些细胞经过不同的细胞背景。这些结果与由初级转录物 FISH、HS 形成以及广义 DNase 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p-珠蛋白基因表达的新基因。我们将在充分表征的 G1E-ER 细胞和 ES 衍生的红系祖细胞 (ES-EP) 系中进行公正的全基因组 siRNA 筛选，以确定参与调节 p-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冗余 siRNA 组组成。我们提出了几种用于候选 siRNA 的验证、识别和优先排序以及已识别基因的生化和功能分析的策略。