Control of TCR V Beta Rearrangement & Allelic Exclusion

描述(由申请人提供)：通过V(D)J重组对抗原受体基因的体细胞组装是在T和B淋巴细胞上产生不同的抗原受体谱系所必需的。T细胞受体(Tcrb)基因上的V(D)J重组受到严格的调控，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失活。然而，调控Tcrb基因座重组和等位基因排斥的机制还只有部分了解。该项目将解决Tcrb基因座调控的未探索方面，这些方面对Tcrb基因座V(D)J重组计划的完整性至关重要。具体目的我将验证这样一种假设，即对于V(D)J重组，Tcrb基因座染色质的适当发育激活需要开放染色质的机制以及抑制附近抑制性染色质的影响的机制。逆转录病毒LTR被定位在抑制性染色质结构域和活性染色质结构域之间的离散边界上，这一LTR或相邻的线似乎推动了活性染色质结构域的转录。为了评估这些元件对Tcrb基因座调控的重要性，将从功能上解剖LTR/LINE区域，以区分与染色质屏障和转录活性有关的成分。体内Tcrb基因重组的功能意义将通过使用基因打靶来消除屏障功能、启动子活性或两者兼而有之所需的成分来评估。SPARTIAM II将测试这一假设，即Tcrb与核层的关联以一种促进非同步等位基因重组的方式抑制了V&to-DJ；重组，从而为等位基因排除奠定了基础。Tcrb等位基因与核层之间的相互作用将用四色、三维免疫荧光原位杂交和DamID来探测，以评估Tcrb基因座在核层是否采用了抑制或允许V&to-DJ重组的独特构象。核板对V(D)J重组的抑制影响将通过向核板招募一个通常没有这个间隔的TCR基因座来直接评估。这将通过使用基因打靶将lac操纵子(LACO)阵列引入TCRA/Tcrd基因座，并通过将lac抑制因子(LacI)表达为与核层的一个组成部分的融合蛋白来实现。这项工作的成功完成将提供对调控D&T-J和V&D-DJ重组的机制的洞察，以及对等位基因调控的洞察，这将对理解其他TCR和Ig基因座的重组程序有价值。一个成功的结果还应该提供对逆转录元件和障碍型绝缘体功能的基本见解，这将影响我们对广泛分布于基因组中的类似元件的理解，以及对核层作为基因活动调节因子的作用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.