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

 描述（由申请方提供）：适应性免疫依赖于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库中的主要多样性取决于称为V（D）J重组的基因重排过程，其中V、D和J区段的受控重排在发育中的淋巴细胞中跨越大的基因组距离发生，以产生遗传上独特的BCR或TCR。重要的是，V（D）J重组的失调可以成为免疫缺陷和自身免疫的基础，也可以导致致癌易位。在免疫球蛋白重链（IgH）位点，V（D）J重组受到严格调控。这一规律可以概括为以下四个性质：1.谱系特异性-仅在B细胞而不是T细胞中观察到从VH到DJH重排的完整VHDJH连接2。阶段特异性-在前B细胞中发生轻链重排之前，祖B细胞中的IgH重排。 3.在VH与DJH重排连接之前，两个IgH等位基因上都发生D级到JH重排. VH不连接到未重排的D。 4.反馈调节-一个等位基因上的有效VHDJH重排将产生一条重链，该重链反馈以防止第二个等位基因上的进一步VH至DJH重排，如果它仍然处于DJH构型的话。 Alt实验室已经证明，在100 b IgH VH-D基因间区域内的4kb区域，称为基因间控制区域1（IGCR 1），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节组装是必需的。IGCR 1的这些功能通过防止VH 81 X的过早重排使一抗库多样化。IGCR 1还与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IGCR 1功能的具体机制尚不清楚。因此，我的建议旨在（1）阐明IGCR 1内的CBE和IgH基因座3'端的一组10个CBE在V（D）J重组控制中的单独作用，以及（2）测试转录- 和IGCR 1功能的位置依赖性方面。我提出的项目应该提供新的见解，遗传和表观遗传因素的相互作用，管理V（D）J重组，这也将转化为更广泛的理解基因调控。

 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.