Developmental Control of Enhancer Function

描述（由申请人提供）：最早的发育调控事件标志着一个基因的谱系特异性表达仍然知之甚少。然而，这些早期表观遗传和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控机制的优秀且易于处理的系统，因为广泛的分析已经导致特定发育阶段的表型和功能特征，可以很容易地通过细胞表面标记物识别和分离。免疫球蛋白（Ig）基因是在B细胞发育过程中受到差异控制的特征基因，利用大规模的全局调控机制（体细胞重排和Ig基因座收缩）以及更局部的调控机制（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优秀模式基因。我们将利用Ig系统来确定在B细胞发育过程中最初靶向Ig kappa位点的早期表观遗传事件。我们将利用胚胎干细胞以及互补的体内和体外方法来定义在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，并严重依赖于转录因子PU.1。我们开发了一种表达各种PU.1突变体的PU.1缺失ES细胞系统，这将使我们能够与条件PU.1敲除系统一起，确定对Ig kappa位点染色质结构变化（Aim 2）和B细胞发育（Aim 3）重要的PU.1依赖功能。最后，尽管使用相同的重组机制，但在B细胞发育早期，IgH和IgL位点对重组机制的可及性是不同的。我们的初步结果表明，在前b细胞阶段控制Ig kappa基因座不可接近的一个重要机制是转录因子STAT5结合到Ig kappa 3'增强子中与中央PU.1结合位点重叠的位点以及内含子增强子侧翼的位点上。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，并将确定竞争性位移是否代表了Ig基因功能发育调节的新机制（目的4）。我们预计我们的研究将明确阐明控制其发育表达的Ig：基因座的新调控机制，从而调节B细胞发育的后续进展。在更大的全球层面上，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式，从而为谱系特异性或发育限制性基因的异常表达引起的发育障碍和/或恶性肿瘤提供见解。

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.