Developmental Control of Enhancer Function

增强子功能的发育控制

• 批准号: 8056649
• 负责人: Michael Lee Atchison
• 金额: $ 30.89万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-12 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8056649
• 关键词: Address; Automobile Driving; B cell differentiation; B-Cell Development; B-Lymphocytes; Binding; Binding Sites; Cell surface; Cells; Chromatin Structure; Control Locus; DNA; DNA Binding; DNA Sequence Rearrangement; Data; Defect; Development; Developmental Process; Disease; ES Cell Line; Embryo; Enhancers; Epigenetic Process; Event; Gene Expression; Gene Expression Regulation; Genes; Genetic Recombination; Genetic Transcription; Human; IGK@ gene cluster; Immune; Immunoglobulin Gene Rearrangement; Immunoglobulin Genes; Immunoglobulins; Introns; Knock-out; Knowledge; Lead; Malignant Neoplasms; Mediating; Modeling; Molecular; Mus; Nucleic Acid Regulatory Sequences; Pattern; Process; Proto-Oncogene Protein c-kit; Public Health; Publishing; Regulation; Regulatory Element; Role; STAT5A gene; Site; Staging; System; Testing; Transcriptional Activation; Variant; Work; base; chromatin remodeling; developmental disease; embryonic stem cell; gene function; in vivo; insight; kappa-Chain Immunoglobulins; mutant; novel; proto-oncogene protein Spi-1; public health relevance; stem; transcription factor; translational study

DESCRIPTION (provided by applicant): The earliest developmentally regulated events that mark a gene for lineage-specific expression is still poorly understood. However, these early epigenetic and DNA occupancy events are key for regulating gene expression patterns that subsequently regulate developmental processes. The B cell developmental system is an excellent and tractable system to explore epigenetic regulatory mechanisms, as extensive analysis has led to the phenotypic and functional characterization of specific developmental stages that can be readily identified and isolated by cell surface markers. The immunoglobulin (Ig) genes are very well characterized genes that are differentially controlled during B cell development, and that utilize large scale, global regulatory mechanisms (somatic rearrangement and Ig locus contraction), as well as more localized regulatory mechanisms (enhancer activation, inducible transcription). Therefore, they are outstanding model genes to explore the molecular events regulating gene expression. We will utilize the Ig system to determine the early epigenetic events that initially target the Ig kappa locus during B cell development. We will utilize embryonic stem (ES) cell as well as complementary in vivo and ex vivo approaches to define the earliest epigenetic events that lead to large-scale Ig locus contraction, transcriptional activation, and Ig gene rearrangement during B cell development. Early events in B cell development are initiated by, and are critically dependent upon, the transcription factor PU.1. We developed a PU.1-null ES cell system expressing various PU.1 mutants that will enable us, in conjunction with conditional PU.1 knockout systems, to determine the PU.1- dependent functions important for changes in chromatin structure at the Ig kappa locus (Aim 2) and for B cell development (Aim 3). Finally, despite utilizing the same recombination machinery, the IgH and IgL loci are differentially accessible to the recombination machinery during early B cell development. Our preliminary results suggest that an important mechanism for controlling inaccessibility of the Ig kappa locus at the pro-B cell stage is binding of transcription factor STAT5 to a site that overlaps the central PU.1 binding site in the Ig kappa 3' enhancer as well as to sites flanking the intron enhancer. We will assess the consequences of STAT5 binding on enhancer activity, kappa locus transcription, and somatic rearrangement and will determine if competitive displacement represents a novel mechanism for developmental regulation of Ig gene function (Aim 4). We anticipate our studies will specifically elucidate novel regulatory mechanisms at the Ig: locus that control its developmental expression, thereby regulating the consequent progression of B cell development. On a more global level, we predict that these studies will reveal new paradigms for developmental control mechanisms mediated through a single enhancer regulatory element, thereby providing insights into the developmental disorders and/or malignancies caused by aberrant expression of lineage-specific or developmentally restricted genes. PUBLIC HEALTH RELEVANCE: Disruptions in the developmental control of gene expression result in numerous diseases. Defects in the developmental processes studied here can result in either severe immune defects, or in the development of malignancies caused by defective transcription factor function. Understanding these processes therefore directly relates to public health.

描述（由申请人提供）：标记基因谱系特异性表达的最早的发育调控事件仍然知之甚少。然而，这些早期表观遗传和 DNA 占据事件对于调节随后调节发育过程的基因表达模式至关重要。 B 细胞发育系统是探索表观遗传调控机制的优秀且易于处理的系统，因为广泛的分析已得出特定发育阶段的表型和功能特征，这些特征可以通过细胞表面标记轻松识别和分离。免疫球蛋白 (Ig) 基因是特征非常明确的基因，在 B 细胞发育过程中受到差异性控制，并且利用大规模的全局调节机制（体细胞重排和 Ig 位点收缩）以及更局部的调节机制（增强子激活、诱导转录）。因此，它们是探索调控基因表达的分子事件的杰出模型基因。我们将利用 Ig 系统来确定 B 细胞发育过程中最初针对 Ig kappa 基因座的早期表观遗传事件。我们将利用胚胎干 (ES) 细胞以及互补的体内和离体方法来定义 B 细胞发育过程中导致大规模 Ig 基因座收缩、转录激活和 Ig 基因重排的最早表观遗传事件。 B 细胞发育的早期事件由转录因子 PU.1 启动，并且严重依赖于转录因子 PU.1。我们开发了表达各种 PU.1 突变体的 PU.1-null ES 细胞系统，这将使我们能够与条件性 PU.1 敲除系统相结合，确定对 Ig 染色质结构变化重要的 PU.1 依赖性功能kappa 位点（目标 2）和 B 细胞发育（目标 3）。最后，尽管使用相同的重组机制，但在早期 B 细胞发育过程中，重组机制对 IgH 和 IgL 基因座的可及性存在差异。我们的初步结果表明，在原 B 细胞阶段控制 Ig kappa 位点不可接近的一个重要机制是转录因子 STAT5 与 Ig kappa 3' 增强子中的中心 PU.1 结合位点重叠的位点的结合至于内含子增强子侧翼的位点。我们将评估 STAT5 结合对增强子活性、kappa 位点转录和体细胞重排的影响，并将确定竞争性置换是否代表 Ig 基因功能发育调控的新机制（目标 4）。我们预计我们的研究将专门阐明 Ig：控制其发育表达的基因座的新调节机制，从而调节 B 细胞发育的后续进展。在更全球的层面上，我们预测这些研究将揭示通过单一增强子调控元件介导的发育控制机制的新范例，从而提供对由谱系特异性或发育限制基因的异常表达引起的发育障碍和/或恶性肿瘤的见解。 公共卫生相关性：基因表达发育控制的破坏会导致多种疾病。这里研究的发育过程中的缺陷可能导致严重的免疫缺陷，或者导致转录因子功能缺陷引起的恶性肿瘤的发展。因此，了解这些过程直接关系到公共卫生。