Control of TCR V Beta Rearrangement & Allelic Exclusion

描述（由申请人提供）：抗原受体基因通过V(D)J重组的体细胞组装是在T和B淋巴细胞上产生多种抗原受体库所必需的。T细胞受体（Tcrb）位点的V(D)J重组受到严格调控，并在T淋巴细胞发育过程中以促进等位基因排斥的方式被激活和灭活。然而，调控Tcrb基因座重组和等位基因排斥的机制仅部分被了解。该项目将解决Tcrb基因座调控未被探索的方面，这对Tcrb基因座V(D)J重组程序的完整性至关重要。我将验证一个假设，即Tcrb位点染色质在V(D)J重组中的适当发育激活需要打开染色质的机制以及抑制附近抑制染色质影响的机制。逆转录病毒LTR已经定位于抑制性和活性染色质结构域之间的离散边界，并且该LTR或邻近的LINE似乎驱动跨活性染色质结构域的转录。为了评估这些元件对Tcrb位点调控的重要性，将对LTR/LINE区域进行功能解剖，以区分有助于染色质屏障和转录活性的成分。通过使用基因靶向来消除屏障功能、启动子活性或两者所需的成分，来评估体内Tcrb基因重组的功能意义。Specific Aim II将验证Tcrb与核层的关联以促进异步等位基因重组的方式抑制V&#946；到dj &#946；重组的假设，从而为等位基因排斥奠定基础。使用四色、三维免疫荧光原位杂交（3D immune - fish）和DamID检测Tcrb等位基因与核层之间的相互作用，以评估Tcrb位点在核层上是否采用抑制或允许V&#946；到dj &#946；重组的不同构象。然后，通过向核层招募通常不含该区室的TCR位点，可以直接评估核层对V(D)J重组的抑制作用。这将通过使用基因靶向将lac操作符（lacO）阵列引入Tcra/Tcrd位点，并通过表达lac抑制因子（lacI）作为融合蛋白与核层的成分来实现。这项工作的成功完成将有助于深入了解D&#946；到j &#946；和V&#946；到dj &#946；重组的调控机制，以及对等位基因调控的深入了解，这将对理解其他TCR和Ig位点的重组程序有价值。一个成功的结果也应该提供对逆转录因子和屏障型绝缘子功能的基本见解，这将影响我们对广泛分布在基因组中的类似元件的理解，以及对核层作为基因活性调节剂的重要和广泛适用的见解。

DESCRIPTION (provided by applicant): The somatic assembly of antigen receptor genes by V(D)J recombination is essential for the generation of diverse antigen receptor repertoires on T and B lymphocytes. V(D)J recombination at the T cell receptor β (Tcrb) locus is strictly regulated and is activated and inactivated during T lymphocyte development in a manner that promotes allelic exclusion. However the mechanisms that regulate Tcrb locus recombination and allelic exclusion are only partly understood. This project will address unexplored aspects of Tcrb locus regulation that are of fundamental importance for the integrity of the Tcrb locus V(D)J recombination program. Specific Aim I will test the hypothesis that appropriate developmental activation of Tcrb locus chromatin for V(D)J recombination requires mechanisms that open chromatin as well as mechanisms that suppress the influence of nearby repressive chromatin. A retroviral LTR has been localized to a discrete boundary between repressive and active chromatin domains, and this LTR or an adjacent LINE appears to drive transcription across the active chromatin domain. To evaluate the importance of these elements for Tcrb locus regulation, the LTR/LINE region will be functionally dissected to discriminate components contributing to chromatin barrier and transcriptional activity. Functional significance for Tcrb gene recombination in vivo will be assessed by using gene targeting to eliminate components required for barrier function, promoter activity, or both. Specific Aim II will test the hypothesis that Tcrb association with the nuclear lamina suppresses Vβ-to-DJβ recombination in a manner that promotes asynchronous allelic recombination, thereby setting the stage for allelic exclusion. Interactions between Tcrb alleles and the nuclear lamina will be probed using four-color, three dimensional immuno-fluorescence in situ hybridization (3D Immuno-FISH) and DamID to evaluate whether the Tcrb locus adopts distinct conformations at the nuclear lamina that are suppressive or permissive for Vβ-to-DJβ recombination. A suppressive influence of the nuclear lamina on V(D)J recombination will then be directly assessed by recruiting to the nuclear lamina a TCR locus that is normally free of this compartment. This will be accomplished by using gene targeting to introduce a lac operator (lacO) array into the Tcra/Tcrd locus and by expressing lac repressor (lacI) as a fusion protein with a component of the nuclear lamina. Successful completion of this work should provide insights into the mechanisms that regulate both Dβ-to-Jβ and Vβ-to-DJβ recombination and insights into allelic regulation that will be valuable for understanding the recombination programs at other TCR and Ig loci. A successful outcome should also provide fundamental insights into retroelement and barrier-type insulator function that will impact our understanding of similar elements that are widely distributed across the genome, and important and broadly applicable insights into the role of the nuclear lamina as a regulator of gene activity. PUBLIC HEALTH RELEVANCE: Health relatedness V(D)J recombination is essential for the development of a functional immune system. Failure of V(D)J recombination can cause immunodeficiency and disregulated V(D)J recombination can result in autoimmunity or chromosomal translocations that contribute to tumorigenesis. These studies will elucidate basic mechanisms that contribute to normal immune system development and pathologies such as immunodeficiency, autoimmunity and cancer.