Long-Range Genetic and Epigenetic Control of Igh Gene Assembly

描述(申请人提供)：基因功能的许多方面由表观基因组的变化来协调，包括染色质修饰的动态修改、基因组包装、亚核定位和染色体构象。所有这些表观遗传机制都被发育中的淋巴细胞用来通过V(D)J重组来调节功能性抗原受体基因的产生。这个基因重排的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细胞或T细胞的特征可变区外显子。尽管V(D)J重组对于产生适应性免疫系统至关重要，但基因重组是有风险的。V(D)J重组靶向异常可导致涉及原癌基因的染色体易位。这些易位在绝大多数白血病和淋巴瘤中引发损害。因此，严格的控制机制确保了在淋巴细胞发育过程中重组装置(V(D)J重组酶)的阶段、组织和等位基因特异性靶向。在更局部的水平上，V(D)J重组受到遗传控制元件之间复杂的相互作用的调节，这些遗传控制元件改变了染色质在Ig和TCR基因座的可及性。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，从而使遥远的基因片段进入空间邻近。申请人的实验室已经证明，转录启动子和增强子作为免疫球蛋白重链(IgH)和Tcrb基因座的可及性控制元件(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，以招募共价修饰和重塑核小体的蛋白质，从而产生重组酶可及染色质。与我们对重组酶靶向Tcrb的详细了解相比，我们对控制IgH基因组装的遗传和表观遗传学机制知之甚少，特别是那些促进VH基因片段和重排的DHJH元件之间的远程重排的机制，位于2.5Mb之外。该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了VH染色质的打开，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。为了验证这一假设，申请人建议(I)确定DHJH区域中的已知调控元件是否对初级B细胞前体细胞中的IgH位点收缩至关重要，以及(Ii)确定VH簇中激活其复合基因片段的转录并协调其与DHJH区域的远程相互作用的新的遗传控制元件。总之，这些研究将为指导抗原受体基因组装和防止可能导致致癌易位的异常相互作用的染色体动力学提供一个新的范例。 与公共健康相关：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生成是必不可少的，因为这个组装过程中的缺陷会导致染色体易位，从而导致淋巴样肿瘤。我们建议进行研究，以解决前体B细胞如何控制免疫球蛋白重链基因座空间组织的长期变化，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.