Developmental Control of Enhancer Function

描述(申请人提供)：标记基因谱系特异性表达的最早的发育调节事件仍然知之甚少。然而，这些早期的表观遗传学和DNA占位事件是调控基因表达模式的关键，基因表达模式随后调控发育过程。B细胞发育系统是探索表观遗传调控机制的优秀和易操作的系统，因为广泛的分析导致了特定发育阶段的表型和功能特征，这些表型和功能特征可以很容易地通过细胞表面标记来识别和分离。免疫球蛋白(Ig)基因是一类在B细胞发育过程中受到差异调控的基因，它们利用大规模的、全局的调节机制(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优秀模式基因。我们将利用免疫球蛋白系统来确定在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。我们将利用胚胎干细胞以及体内和体外互补的方法来确定在B细胞发育过程中导致大规模Ig位点收缩、转录激活和Ig基因重排的最早表观遗传学事件。B细胞发育的早期事件是由转录因子PU1启动的，并且严重依赖于转录因子PU1。我们开发了一个表达各种PU.1突变体的PU.1缺失ES细胞系统，该系统将使我们能够结合条件PU.1基因敲除系统来确定对Ig kappa基因染色质结构变化(目标2)和B细胞发育(目标3)重要的PU.1依赖功能。最后，尽管使用了相同的重组机制，但在B细胞发育的早期，IgH和IGL基因座对于重组机制是不同的。我们的初步结果表明，在Pro-B细胞阶段控制Ig kappa基因座不可及的一个重要机制是将转录因子STAT5与Ig kappa 3‘增强子中PU.1中心结合位点以及内含子增强子两侧的位点结合。我们将评估STAT5结合对增强子活性、kappa基因转录和体细胞重排的影响，并将确定竞争置换是否代表了Ig基因功能发育调节的新机制(目标4)。我们预计，我们的研究将专门阐明免疫球蛋白基因的新调控机制：控制其发育表达的基因座，从而调节随后的B细胞发育进程。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控元件介导的发育控制机制的新范式，从而为研究因谱系特异性或发育受限基因的异常表达而导致的发育障碍和/或恶性肿瘤提供见解。 公共卫生相关性：基因表达发育控制的中断会导致多种疾病。这里研究的发育过程中的缺陷可能会导致严重的免疫缺陷，或者导致由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。

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.