Functional analysis of intracellular signaling molecules in T cell differentiation by IL-7 receptor-deficient mice

IL-7受体（IL-7 R）通过促进T细胞受体（TCR）和IG位点的存活和增殖以及诱导V（D）J重组在淋巴细胞发育中起关键作用。为了确定IL-7 R诱导基因重排的分子机制，我们表征了Stat 5在TCRγ基因生殖系转录中的作用。我们的结果表明IL-7 R激活的Stat 5与Jγ片段5'区的共有基序结合并诱导生殖系转录。我们还发现，Stat 5的组成型活性形式诱导生殖系转录，恢复TCRγ基因的V-J重组，并部分拯救T细胞从IL-7 R-/-T细胞前体发育，特别是有利于γδ T细胞。因此，这些结果揭示了Stat 5在T细胞发育中的潜在作用，并暗示Stat 5可能通过诱导生殖系转录物来控制TCRγ位点的可及性。接下来，为了确定将Stat 5诱导的生殖系转录与TCRγ基因座的可及性联系起来的分子机制，我们表征了TCRγ基因中转录辅激活因子和组蛋白乙酰化的作用。我们证明，细胞因子刺激可快速募集Stat 5和转录辅激活因子至5 'J γ种系启动子，并增加Ba/F3细胞中的组蛋白乙酰化、种系转录和可接近性。我们还发现，在IL-7 R缺陷的胸腺细胞前体中，TCRγ位点的组蛋白乙酰化显著降低，并且活性Stat 5的引入恢复了TCRγ位点的组蛋白乙酰化和可接近性。因此，这些结果表明，Stat 5通过募集转录辅激活因子和诱导组蛋白乙酰化来控制Jγ区域的局部可及性。我们的研究还暗示了Stat 5在TCRγ增强子（Eγ）的全基因座可及性控制中的潜在作用。

IL-7 receptor (IL-7R) plays critical roles in lymphocyte development by promoting survival and proliferation and by inducing V (D) J recombination in TCR and Ig loci. To identify the molecular mechanism of the induction of gene rearrangement by IL-7R, we characterized the role of Stat5 in the germline transcription of TCRγ genes. Our results demonstrated that IL-7R-activated Stat5 binds to consensus motifs in 5' regions of Jγ segments and induces germline transcripts. We also found that a constitutively-active form of Stat5 induces germline transcription, restores V-J recombination of TCRγ genes, and partially rescues T cell development from IL-7R-/-T cell precursors, especially in favor of γδ T cells. Therefore, these results revealed a potential role of Stat5 in T cell development, and implied that Stat5 may control the accessibility of the TCRγ locus by the induction of germline transcripts. Next, to identify the molecular mechanism that links the Stat5-induced germline transcription to the accessibility of the TCRγ locus, we characterized the role of transcriptional coactivators and histone acetylation in TCRγ genes. We demonstrated that cytokine stimulation rapidly recruits Stat5 and transcriptional coactivators to the 5'Jγ germline promoter and increases histone acetylation, germline transcription, and accessibility in Ba/F3 cells. We also show that histone acetylation of the TCRγ locus is significantly reduced in IL-7R-deficient thymocyte precursors and that introduction of active Stat5 restores the histone acetylation and accessibility of the TCRγ locus. Therefore, these results suggested that Stat5 controls local accessibility of the Jγ region by recruiting the transcriptional coactivators and inducing histone acetylation. Our study also implied a potential role of Stat5 in the locus-wide accessibility control by the TCRγ enhancer (Eγ).