Elucidating the role of cis-regulatory elements in mediating V(D)J recombination

 描述(申请人提供)：获得性免疫依赖于B和T细胞受体(BCR；TCR)识别和结合体内异物的能力。这些BCR和TCR谱系的初级多样性取决于被称为V(D)J重组的基因重排过程，在该过程中，V、D和J片段在发育中的淋巴细胞中跨大基因组距离发生受控重排，从而产生遗传上独特的BCR或TCR。重要的是，V(D)J重组的解除调控可导致免疫缺陷和自身免疫，也可导致致癌易位。在免疫球蛋白重链(IgH)基因上，V(D)J重组受到严格调控。这一调节可概括为以下四个特性：1.谱系特异性--VHDJH从VH到DJH的完整连接重排仅在B细胞中观察到，而T细胞中没有。2.阶段特异性--前体B细胞轻链重排之前的IgH重排，这在前B细胞中发生。3.在连接VH到DJH重排之前，两个IGH等位基因都发生了顺序-D到JH重排。VHS不会加入未重新排列的D。4.反馈调节-一个等位基因上的VHDJH重排将产生一条重链，如果第二个等位基因仍处于DJH构型，则该重链将反馈以防止第二个等位基因上进一步的VH到DJH重排。Alt实验室已经证明，在100bIGH VH-D间隔区内的一个4kb区域，称为基因间控制区1(IGCR1)，包含两个CTCF结合元件(CBE)，这两个元件对于VH、D和JH片段的有序、谱系特异性和反馈调控的组装是必需的。IGCR1的这些功能通过防止VH81X的过早重排而使初级抗体库多样化。IGCR1还与该基因座内的其他增强子和CBE位点形成染色质环，但这些环或其他元件如何参与IGCR1功能的具体机制尚不清楚。因此，我的建议旨在(1)阐明IGCR1内的CBE和一组位于IgH基因3‘端的CBE在V(D)J重组调控中的单独作用，以及(2)转录- 以及IGCR1函数的位置相关方面。我提议的项目应该为控制V(D)J重组的遗传和表观遗传因素之间的相互作用提供新的见解，这也将转化为对基因调控的更广泛的理解。

 DESCRIPTION (provided by applicant): Adaptive immunity is dependent on the ability of B and T cell receptors (BCR; TCR) to recognize and bind foreign substances within the body. Primary diversity in these BCR and TCR repertoires is dependent on the gene rearrangement process termed V(D)J recombination, in which controlled rearrangements of V, D, and J segments occur across large genomic distances in developing lymphocytes to produce genetically unique BCRs or TCRs. Importantly, deregulation of V(D)J recombination can underlie immunodeficiency and autoimmunity, and also lead to oncogenic translocations. At the Immunoglobulin heavy chain (IgH) locus, V(D)J recombination is tightly regulated. This regulation can be summarized into the following four properties: 1. Lineage specificity - Complete VHDJH joins from VH to DJH rearrangements are only observed in B cells and not T cells 2. Stage specificity - IgH rearrangements in progenitor B cells prior to light chain rearrangements, which occur in pre-B cells. 3. Order - D to JH rearrangements occur on both IgH alleles before joining of a VH to DJH rearrangement. VHs do not join to unrearranged Ds. 4. Feedback regulation - A productive VHDJH rearrangement on one allele will produce a heavy chain that feeds back to prevent further VH to DJH rearrangements on the second allele, if it is still in DJH configuration. The Alt lab has demonstrated that a 4kb region within the 100b IgH VH-D intergenic region, termed Intergenic Control Region 1 (IGCR1), contains two CTCF-binding elements (CBEs) that are essential in cis for ordered, lineage-specific, and feedback regulated assembly of VH, D, and JH segments. These functions of IGCR1 diversify primary antibody repertoires by preventing premature rearrangements of VH81X. IGCR1 also forms chromatin loops with other enhancer and CBE sites within the locus, but a specific mechanism for how these loops or other elements may participate in IGCR1 functions is not known. Thus, my proposal aims to (1) elucidate individual roles of the CBEs within IGCR1 and a set of ten CBEs at the 3' end of the IgH locus in V(D)J recombination control, and (2) test for transcription- and position-dependent aspects of IGCR1 functions. My proposed project should provide new insights into the interplay of genetic and epigenetic factors that that govern V(D)J recombination, which also will translate into a broader understanding of gene regulation.