Regulation of T cell development by HDAC3

摘要 胸腺细胞的发育受到严格的调控，需要细胞成功地通过几个 胸腺出口前的发育阶段和检查点。胸腺细胞发育的每个检查点， 涉及对一组特定基因的诱导或抑制。基因调控的中断导致 发育停滞，不能产生T细胞和适应性免疫缺陷。基因表达是 由转录激活子、抑制子和染色质修饰物协调。总体而言，组蛋白 乙酰化促进基因表达，而组蛋白去乙酰化导致抑制。我们调查了 组蛋白脱乙酰酶-3(HDAC3)在CD2-ICRE条件性基因敲除T细胞发育中的作用 CKO)小鼠。尽管T细胞在发育过程中共表达几个HDAC家族成员，但这些其他成员 HDAC家族成员不能补偿HDAC3的损失，因为HDAC3-CKO小鼠的T细胞受阻 由于不能进行正选择，细胞在DP阶段发育。T细胞中的阻断 OT-II TCR转基因、Bcl2或Bclxl转基因不能挽救发育。 成功的阳性选择需要下调RoRγt，因为小鼠具有结构性表达 在阳性选择时，RoRγt在T细胞发育中也有类似的阻断作用。在HDAC3-Cko小鼠中，RoRγt是 在DP期未下调TCR刺激，表明阻断了正选择 可能是由于无法下调RoRγt。与此一致，我们观察到增强的RORC 在没有HDAC3的情况下，启动子乙酰化，表明HDAC3可能直接去乙酰化组蛋白 抑制RORC启动子的表达。RoRγt还在DP期控制Bclxl的表达，以 调节DP存活期。因此，要确定RoRγt的持续表达是否对 阳性选择分组：RoRγt-KO BclXL TG HDAC3-CKO(以下简称RB3)小鼠 产生，这恢复了正选择导致正常数量的CD8SP的产生 胸腺细胞(TcRβ+、Runx3+)数。然而，CD4SP胸腺细胞没有产生，这表明 在T细胞发育过程中，HDAC3在谱系选择中的潜在作用，我们认为这是由于 正向选择和世系承诺过程中的CD4表达。以确认改变后的血统 承诺，我们产生了OT-II RB3小鼠，并发现表达OT-II TCR的胸腺细胞是 偏向CD8谱系，而不是像在OT-II转基因中观察到的那样向CD4SP T细胞倾斜(否则 WT)小鼠。这项提案的重点是确定HDAC3在阳性选择和CD4谱系中的作用 发展

Abstract Thymocyte development is tightly regulated, requiring successful transit of cells through several developmental stages and checkpoints prior to thymic egress. Each checkpoint of thymocyte development, involves induction or repression of a particular set of genes. Disruptions in gene regulation leads to developmental arrest, a failure to generate T cells and deficits in adaptive immunity. Gene expression is coordinated by transcriptional activators, repressors, and chromatin modifiers. In general, histone acetylation promotes gene expression while histone deacetylation leads to repression. We investigated the role of histone deacetylase-3 (HDAC3) in T cell development using CD2-icre conditional knockout (HDAC3- cKO) mice. Although T cells co-express several HDAC family members during development, these other HDAC family members cannot compensate for the loss of HDAC3 as HDAC3-cKO mice have a block in T cell development at the DP stage due to an inability to undergo positive selection. The block in T cell development could not be rescued by an OT-II TCR transgene, or by a Bcl-2 or Bcl-xl transgene. Successful positive selection requires down-regulation of RORγt, as mice with constitutive expression of RORγt have a similar block in T cell development at positive selection. In HDAC3-cKO mice, RORγt was not down-regulated upon TCR stimulation at the DP stage, demonstrating that the block in positive selection may be due to an inability to down-regulate RORγt. Consistent with this, we observed enhanced RORC promoter acetylation in the absence of HDAC3, indicating that HDAC3 may directly deacetylate histones at the RORC promoter to inhibit expression. RORγt also controls expression of Bcl-xl at the DP stage to regulate DP survival. Therefore, to determine whether sustained expression of RORγt was responsible for the block in positive selection, RORγt-KO Bcl-xl Tg HDAC3-cKO (hereafter called RB3) mice were generated, which restored positive selection leading to the generation of normal numbers of CD8SP thymocyte (TCRβ+, Runx3+) numbers. However, CD4SP thymocytes were not generated, indicating a potential role for HDAC3 in lineage choice during T cell development, which we believe is due to a defect in CD4 expression during positive selection and lineage commitment. To confirm the altered lineage commitment, we generated OT-II RB3 mice and found that the OT-II TCR-expressing thymocytes are skewed towards the CD8 lineage rather than CD4SP T cells as is observed in OT-II transgenic (otherwise WT) mice. The focus of this proposal is to define the role of HDAC3 in positive selection and CD4 lineage development