Long-Range Genetic and Epigenetic Control of Tcrb Gene Assembly

描述（由申请人提供）：目前的证据表明，基因功能的许多方面是由表观基因组的变化协调的，包括染色质修饰的动态修订，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用于发育淋巴细胞来调节功能性抗原受体基因的产生。编码免疫球蛋白（Ig）和T细胞受体（TCR）蛋白的基因由前体淋巴细胞通过V(D)J重组过程进行组装，产生了巨大的受体多样性。这些基因组重排必须严格调节，以确保适当的淋巴细胞发育和避免染色体易位导致淋巴样肿瘤。V(D)J重组是由遗传控制元件之间的复杂相互作用控制的，这些控制元件修饰了Ig和Tcr位点染色质对单个V(D)J重组酶复合物的可及性。申请人实验室的研究表明，Tcrb基因组装的激活需要转录增强子（E2）和启动子（PD2）之间的物理相互作用，这在胸腺细胞中形成稳定的全息复合物。PD2/E2全复合体打开与D2和J2基因片段相关的染色质，为重组酶提供通路，促进Tcrb基因组装的第一步（D2 ' J2重组）。与最初的Tcrb激活相比，对于V2基因片段和重排D2J2元件之间必须发生的远程重排事件的遗传和表观遗传控制知之甚少，重排事件位于250 kb以上。新的研究表明，胸腺细胞中的Tcrb位点收缩，使V2基因片段在空间上接近D2J2簇。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V2染色质的打开，并促进这一大群基因片段与远端D2J2区域之间的相互作用。为了验证这一假设，申请人提出(i)确定D2J2区域的已知调控元件是否对原代胸腺细胞中Tcrb位点的收缩至关重要；（ii）在V2集群中识别新的遗传控制元件，激活复合基因片段的转录并协调它们与D2J2区域的远程相互作用。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，防止可能导致与其他染色体易位的异常相互作用。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，因为这种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。我们建议研究前体T淋巴细胞如何控制T细胞受体β位点空间组织的远程变化，以确保它们的适当组装和正常适应性免疫系统的产生。

DESCRIPTION (provided by applicant): Current evidence indicates that many aspects of gene function are coordinated by changes in the epigenome, which includes 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. The genes encoding immunoglobulin (Ig) and T cell receptor (TCR) proteins are assembled by precursor lymphocytes using the process of V(D)J recombination, which generates enormous receptor diversity. These genomic rearrangements must be tightly regulated to ensure proper lymphocyte development and to avoid chromosomal translocations that cause lymphoid tumors. V(D)J recombination is controlled by a complex interplay between genetic control elements that modify the accessibility of chromatin at Ig and Tcr loci to a single V(D)J recombinase complex. Studies in the applicant's laboratory demonstrate that activation of Tcrb gene assembly requires the physical interaction between a transcriptional enhancer (E2) and promoters (PD2), which form a stable holocomplex in thymocytes. The PD2/E2 holocomplex opens chromatin associated with closely-spaced D2 and J2 gene segments, providing access to recombinase and promoting the first step in Tcrb gene assembly (D2`J2 recombination). In contrast to initial Tcrb activation, very little is known about the genetic and epigenetic control of long-range rearrangement events that must occur between V2 gene segments and rearranged D2J2 elements, located more than 250 kb away. Emerging studies indicate that the Tcrb locus contracts in thymocytes to bring V2 gene segments into spatial proximity of the D2J2 cluster. The central hypothesis of the proposed research project is that a series of known and novel genetic control elements mediate the opening of V2 chromatin and facilitate interactions between this large cluster of gene segments and the distant D2J2 region. To test this hypothesis, the applicant proposes to (i) determine whether known regulatory elements in the D2J2 region are essential for Tcrb locus contraction in primary thymocytes and (ii) identify novel genetic control elements within the V2 cluster that activate transcription of the composite gene segments and coordinate their long-range interactions with the D2J2 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 T lymphocytes control long-range changes in the spatial organization of T cell receptor beta loci to ensure their proper assembly and pursuant generation of a normal adaptive immune system.