Control of TCR V Beta Rearrangement & Allelic Exclusion

描述（由申请方提供）：通过V（D）J重组进行的抗原受体基因体细胞组装对于T和B淋巴细胞上产生不同抗原受体库至关重要。T细胞受体（Tcrb）基因座上的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激活和失活。然而，调控Tcrb基因座重组和等位基因排斥的机制仅部分了解。该项目将解决Tcrb基因座调控的未开发的方面是Tcrb基因座V（D）J重组程序的完整性的根本重要性。具体目的我将检验这样的假设，即V（D）J重组的Tcrb基因座染色质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性染色质影响的机制。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或相邻的LINE似乎驱动转录穿过活性染色质结构域。为了评估这些元件对Tcrb基因座调控的重要性，将对LTR/LINE区域进行功能性解剖，以区分有助于染色质屏障和转录活性的组分。将通过使用基因靶向消除屏障功能、启动子活性或两者所需的组分来评估Tcrb基因重组在体内的功能意义。特定目的II将检验以下假设：Tcrb与核纤层的关联以促进异步等位基因重组的方式抑制V至DJ重组，从而为等位基因排斥奠定基础。 将使用四色、三维免疫荧光原位杂交（3D Immuno-FISH）和DamID探测Tcrb等位基因与核纤层之间的相互作用，以评价Tcrb基因座是否在核纤层处采用抑制或允许V至DJ重组的不同构象。然后通过向核层募集通常不含该区室的TCR基因座来直接评估核层对V（D）J重组的抑制作用。这将通过使用基因靶向将lac操纵子（lacO）阵列引入Tcra/Tcrd基因座并通过将lac阻遏物（lacI）表达为与核纤层组分的融合蛋白来实现。这项工作的成功完成将提供对调节D-到-J和V-到-DJ重组的机制的深入了解，以及对等位基因调节的深入了解，这将对理解在其他TCR和IG基因座处的重组程序有价值。一个成功的结果也应该提供基本的见解retroelement和屏障型绝缘子功能，这将影响我们的理解类似的元素，广泛分布在整个基因组中，重要的和广泛适用的见解的作用，核纤层作为基因活性的调节器。 公共卫生相关性：健康相关性V（D）J重组对于功能性免疫系统的发展至关重要。V（D）J重组的失败可导致免疫缺陷，而失调的V（D）J重组可导致自身免疫或染色体易位，从而导致肿瘤发生。这些研究将阐明有助于正常免疫系统发育和免疫缺陷、自身免疫和癌症等病理的基本机制。

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.