Long-Range Genetic and Epigenetic Control of Igh Gene Assembly

描述（由申请人提供）：基因功能的许多方面通过表观基因组的变化来协调，其中包括染色质修饰、基因组包装、亚核定位和染色体构象的动态修订。所有这些表观遗传机制都被发育中的淋巴细胞用于通过V（D）J重组来调节功能性抗原受体基因的产生。这种遗传重排过程融合了随机选择的免疫球蛋白（IG）或T细胞受体（Tcr）基因片段的组合，分别形成每个前体B或T细胞的标志性可变区外显子。虽然V（D）J重组对于产生适应性免疫系统至关重要，但遗传重组是有风险的。V（D）J重组的异常靶向可产生涉及原癌基因的染色体易位。这些易位是绝大多数白血病和淋巴瘤的起始病变。因此，严格的控制机制确保了淋巴细胞发育期间重组装置（V（D）J重组酶）的阶段特异性、组织特异性和等位基因特异性靶向。在更局部化的水平上，V（D）J重组受遗传控制元件之间复杂的相互作用调节，所述遗传控制元件改变染色质在IG和Tcr基因座处的可接近性。在更广泛的水平上，V（D）J重组是由IG和Tcr基因座的主动收缩控制的，以使遥远的基因片段在空间上接近。申请人的实验室已经表明，转录启动子和增强子在IG重链（Igh）和Tcrb基因座内充当可及性控制元件（ACE）。在Tcrb基因座，在远端启动子和增强子元件之间形成稳定的结构，以募集共价修饰和重塑核小体的蛋白质，从而产生重组酶可接近的染色质。与我们对靶向Tcrb的重组酶的详细了解相反，我们对控制Igh基因组装的遗传和表观遗传机制知之甚少，特别是那些促进VH基因片段和重排的DHJH元件之间的长距离重排的机制，位于2.5 Mb之外。拟议研究项目的中心假设是，一系列已知的和新的遗传控制元件介导VH染色质的开放，以及随后这一大簇基因片段与遥远的DHJH区域之间的相互作用。为了检验这一假设，申请人提出（i）确定DHJH区中的已知调控元件是否是原代B细胞前体中Igh基因座收缩所必需的，和（ii）鉴定VH簇内激活其复合基因区段的转录并协调它们与DHJH区的长程相互作用的新遗传控制元件。总之，这些研究将为染色体动力学提供一个新的范例，指导抗原受体基因组装，并防止可能导致致癌易位与其他染色体的异常相互作用。 公共卫生相关性：DNA在细胞核中的包装和空间组织是基因功能的关键决定因素;这些“表观遗传”程序的变化可能是衰老和疾病易感性的核心。此外，表观遗传控制对于抗原受体基因的正确产生是必不可少的，因为该组装过程中的缺陷会导致染色体易位，从而导致淋巴肿瘤。我们建议研究前体B细胞如何控制免疫球蛋白重链基因座空间组织的长程变化，以确保其正确组装并随后产生适应性免疫系统。

DESCRIPTION (provided by applicant): Many aspects of gene function are coordinated by changes in the epigenome, which include dynamic revisions of chromatin modifications, genome packaging, subnuclear localization, and chromosome conformation. All of these epigenetic mechanisms are used by developing lymphocytes to regulate the production of functional antigen receptor genes by V(D)J recombination. This process of genetic rearrangement fuses randomly selected combinations of immunoglobulin (Ig) or T cell receptor (Tcr) gene segments to form a signature variable region exon for each precursor B or T cell, respectively. Although V(D)J recombination is critical for the production of an adaptive immune system, genetic reorganization is risky. Aberrant targeting of V(D)J recombination can produce chromosomal translocations involving proto-oncogenes. These translocations are initiating lesions in the vast majority of leukemias and lymphomas. As such, stringent control mechanisms ensure the stage-, tissue-, and allele-specific targeting of the recombination apparatus (V(D)J recombinase) during lymphocyte development. On a more localized level, V(D)J recombination is regulated by a complex interplay between genetic control elements that modify the accessibility of chromatin at Ig and Tcr loci. At a broader level, V(D)J recombination is controlled by active contraction of Ig and Tcr loci to bring distant gene segments into spatial proximity. The applicant's laboratory has shown that transcriptional promoters and enhancers serve as accessibility control elements (ACEs) within Ig heavy chain (Igh) and Tcrb loci. At the Tcrb locus, a stable structure forms between distant promoter and enhancer elements to recruit proteins that covalently modify and remodel nucleosomes, thus generating recombinase accessible chromatin. In contrast to our detailed understanding of recombinase targeting at Tcrb, little is known about the genetic and epigenetic mechanisms controlling Igh gene assembly, especially those that facilitate long-range rearrangement between VH gene segments and rearranged DHJH elements, located up to 2.5 Mb away. The central hypothesis of the proposed research project is that a series of known and novel genetic control elements mediate the opening of VH chromatin and subsequent interactions between this large cluster of gene segments and the distant DHJH region. To test this hypothesis, the applicant proposes to (i) determine whether known regulatory elements in the DHJH region are essential for Igh locus contraction in primary B cell precursors and (ii) identify novel genetic control elements within the VH cluster that activate transcription of its composite gene segments and coordinate their long-range interactions with the DHJH region. Together, these studies will provide a new paradigm for the chromosomal dynamics that guide antigen receptor gene assembly and prevent aberrant interactions that may lead to oncogenic translocations with other chromosomes. PUBLIC HEALTH RELEVANCE: The packaging and spatial organization of DNA in the nucleus are key determinants of gene function; changes in these "epigenetic" programs may be at the heart of aging and disease susceptibilities. In addition, epigenetic control is essential for the proper generation of antigen receptor genes because defects in this assembly process cause chromosomal translocations that lead to lymphoid tumors. We propose studies to address how precursor B cells control long-range changes in the spatial organization of the immunoglobulin heavy chain locus to ensure its proper assembly and pursuant generation of an adaptive immune system.