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）：最早的发育调节事件标志着谱系特异性表达的基因仍然很少了解。但是，这些早期的表观遗传和DNA占用事件是调节随后调节发育过程的基因表达模式的关键。 B细胞发育系统是一个探索表观遗传调节机制的出色且可进行的系统，因为广泛的分析导致了特定发育阶段的表型和功能表征，可以通过细胞表面标记很容易鉴定和隔离。免疫球蛋白（IG）基因是在B细胞发育过程中受差异性控制的良好表征的基因，并且利用大规模的，全球调节机制（体细胞重排和IG基因座收缩）以及更局部的调节机制（增强器激活，可诱导的转录）。因此，它们是探索调节基因表达的分子事件的杰出模型基因。我们将利用IG系统来确定最初针对B细胞发育过程中Ig Kappa基因座的早期表观遗传事件。我们将利用胚胎茎（ES）细胞以及体内互补和体内方法来定义最早导致B细胞发育过程中大规模IG基因座收缩，转录激活和IG基因重排的大规模IG基因座的收缩。 B细胞发育中的早期事件由转录因子PU.1启动，并且至关依赖于1。我们开发了一种表达各种PU.1突变体的PU.1-NULL ES细胞系统，该突变体将使我们与条件PU.1敲除系统结合使用，以确定PU.1-依赖性功能对于IG Kappa基因座（AIM 2）和B细胞发育的染色质结构的变化很重要（AIM 3）。最后，尽管利用了相同的重组机制，但在早期B细胞开发过程中，IGH和IGL基因座在重组机制方面仍可差异化。我们的初步结果表明，在Pro-B细胞阶段控制IG KAPPA基因座的无法访问的重要机制是转录因子STAT5与IG KAPPA 3'增强子中的中央PU.1结合位点与内含子增强子的位点重叠的位点结合。我们将评估STAT5结合对增强子活性，KAPPA基因座转录和躯体重排的后果，并将确定竞争性位移是否代表了Ig基因功能发育调控的新机制（AIM 4）。我们预计我们的研究将特别阐明Ig：控制其发育表达的IG：基因座的新型调节机制，从而调节随之而来的B细胞发育的进展。在更全球的水平上，我们预测，这些研究将揭示通过单个增强子调节元素介导的发育控制机制的新范式，从而提供了有关谱系特异性或发育受限制基因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：基因表达的发育控制中的破坏导致许多疾病。此处研究的发育过程中的缺陷可能导致严重的免疫缺陷，也可能导致因有缺陷的转录因子功能引起的恶性肿瘤发展。因此，了解这些过程直接与公共卫生有关。