Department of Microbiology, North Carolina State University, Raleigh, NC 27695, USA.
J Immunol. 2012 Mar 1;188(5):2266-75. doi: 10.4049/jimmunol.1002931. Epub 2012 Jan 27.
Activation of germline promoters is central to V(D)J recombinational accessibility, driving chromatin remodeling, nucleosome repositioning, and transcriptional read-through of associated DNA. We have previously shown that of the two TCRβ locus (Tcrb) D segments, Dβ1 is flanked by an upstream promoter that directs its transcription and recombinational accessibility. In contrast, transcription within the DJβ2 segment cluster is initially restricted to the J segments and only redirected upstream of Dβ2 after D-to-J joining. The repression of upstream promoter activity prior to Tcrb assembly correlates with evidence that suggests DJβ2 recombination is less efficient than that of DJβ1. Because inefficient DJβ2 assembly offers the potential for V-to-DJβ2 recombination to rescue frameshifted V-to-DJβ1 joints, we wished to determine how Dβ2 promoter activity is modulated upon Tcrb recombination. In this study, we show that repression of the otherwise transcriptionally primed 5'Dβ2 promoter requires binding of upstream stimulatory factor (USF)-1 to a noncanonical E-box within the Dβ2 12-recombination signal sequence spacer prior to Tcrb recombination. USF binding is lost from both rearranged and germline Dβ2 sites in DNA-dependent protein kinase, catalytic subunit-competent thymocytes. Finally, genotoxic dsDNA breaks lead to rapid loss of USF binding and gain of transcriptionally primed 5'Dβ2 promoter activity in a DNA-dependent protein kinase, catalytic subunit-dependent manner. Together, these data suggest a mechanism by which V(D)J recombination may feed back to regulate local Dβ2 recombinational accessibility during thymocyte development.
种系启动子的激活是 V(D)J 重组可及性的核心，驱动染色质重塑、核小体重定位和相关 DNA 的转录通读。我们之前已经表明，在两个 TCRβ 基因座（Tcrb）D 片段中，Dβ1 被一个上游启动子所包围，该启动子指导其转录和重组可及性。相比之下，DJβ2 片段簇内的转录最初仅限于 J 片段，并且仅在 D 到 J 连接之后才被重定向到 Dβ2 的上游。在 Tcrb 组装之前上游启动子活性的抑制与以下证据相关，表明 DJβ2 重组的效率低于 DJβ1。因为低效的 DJβ2 组装为 V 到 DJβ2 重组提供了挽救移码的 V 到 DJβ1 连接的潜力，所以我们希望确定 Tcrb 重组后如何调节 Dβ2 启动子活性。在这项研究中，我们表明，在 Tcrb 重组之前，转录预启动的 5' Dβ2 启动子的抑制需要上游刺激因子（USF）-1结合到 Dβ2 12 重组信号序列间隔子中的非典型 E 盒中。在依赖 DNA 的蛋白激酶、催化亚基功能齐全的胸腺细胞中，重组和种系 Dβ2 位点的 USF 结合均丢失。最后，双链 DNA 断裂导致 USF 结合迅速丢失，并以依赖 DNA 依赖性蛋白激酶、催化亚基的方式获得转录预启动的 5' Dβ2 启动子活性。总之，这些数据表明了一种机制，即 V(D)J 重组可能反馈调节胸腺细胞发育过程中局部 Dβ2 重组可及性。
