Mechanism of Transcriptional Regulation of Th2 Cell Development

项目摘要 在胸腺中发育的不同Th 1、Th 2、Th 17和Treg亚型的CD 4 + T辅助（Th）细胞具有重要的 在适应性免疫中发挥作用。它们的失调也与疾病有关，如Th 2- 相关的炎性和过敏性疾病，包括哮喘、克罗恩病和特应性皮炎。Th2 幼稚CD 4 + T细胞的谱系特异性分化受到细胞因子（IL-4，5，13）的严格控制， 由一组转录因子和调节因子指导的高度有序的亚型特异性基因转录， 相反的活性以促进（YY 1，Gata 3，Stat 6）或抑制（Foxp 3）Th 2细胞分化。但 潜在的分子机制仍然是难以捉摸的。BRD 4是布罗莫结构域的关键成员， 末端（BET）蛋白家族，因其在基因转录激活中的作用而众所周知，但它是否以及如何 以细胞类型特异性方式在基因转录抑制中的功能仍然是难以捉摸的。最近我们 发现Brd 4与Polycomb抑制复合物2（PRC 2）一起抑制转录表达 Th 2细胞谱系特异性分化中Th 2负调控因子Foxp 3和E3-泛素连接酶Fbxw 7的表达 来自小鼠原代幼稚CD 4 + T细胞。Brd 4通过其第二布罗莫结构域（BD 2）结合赖氨酸。 PRC 2复合物的乙酰化EED亚基通过组蛋白H3赖氨酸27控制靶基因阻遏 三甲基化（H3 K27 me 3）。我们发现Foxp 3抑制Th 2特异性转录因子的转录， Gata 3，而Fbxw 7通过泛素化介导的蛋白降解控制Gata 3蛋白的稳定性，这在 反过来确保Gata 3介导的Th 2细胞因子（包括IL 4、IL 5和IL 13）的转录激活。化学 BRD 4 BD 2的抑制诱导Foxp 3和Fbxw 7的转录去抑制，以及转录下调。 调节IL 4、IL 5和IL 13，导致抑制Th 2细胞分化。我们的研究揭示了以前 未被识别的BRD 4在指导Th 2特异性基因转录抑制以保护Th 2细胞中的功能 谱系分化在我们有希望的发现的基础上，在这项研究中，我们试图定义机械的 BRD 4在Th 2细胞的时间调节中的转录抑制功能的细节，开发和使用 新的BRD 4-BD 2选择性抑制剂，用于研究Th 2细胞分化的转录调节，和 研究BRD 4-BD 2抑制作为新的Th 2免疫调节以阻断Th 2- 哮喘小鼠模型中相关的过敏性炎症。

PROJECT SUMMARY CD4+ T helper (Th) cells of different Th1, Th2, Th17 and Treg subtypes, developed in thymus, have important functions in adaptive immunity. Their dysregulation has also been implicated in diseases such as Th2- associated inflammatory and allergic diseases including asthma, Crohn’s disease and atopic dermatitis. Th2 lineage-specific differentiation from naïve CD4+ T cells is tightly controlled by cytokines (IL-4, 5, 13) through highly ordered subtype-specific gene transcription directed by a set of transcription factors and regulators with opposing activities to promote (YY1, Gata3, Stat6) or inhibit (Foxp3) Th2 cell differentiation. However, the underlying molecular mechanism remains elusive. BRD4, a key member of the bromodomain and extra- terminal (BET) protein family, is well known for its role in gene transcriptional activation, but whether and how it functions in gene transcriptional repression in a cell-type specific manner has remained elusive. Recently, we discovered that Brd4 works with Polycomb repressive complex 2 (PRC2) to repress transcriptional expression of Th2-negative regulators Foxp3 and E3-ubiqutin ligase Fbxw7 in lineage-specific differentiation of Th2 cells from mouse primary naïve CD4+ T cells. Brd4 through its second bromodomain (BD2) binds to lysine- acetylated-EED subunit of the PRC2 complex to control target gene repression through histone H3 lysine 27 trimethylation (H3K27me3). We found that Foxp3 represses transcription of Th2-specific transcription factor Gata3, while Fbxw7 controls Gata3 protein stability via ubiquitination-mediated protein degradation, which in turn ensures Gata3-mediated transcriptional activation of Th2 cytokines including Il4, Il5 and Il13. Chemical inhibition of BRD4 BD2 induces transcriptional de-repression of Foxp3 and Fbxw7, and transcriptional down- regulation of Il4, Il5 and Il13, resulting in inhibition of Th2 cell differentiation. Our study uncovers previously unappreciated BRD4 functions in directing Th2-specific gene transcriptional repression to safeguard Th2 cell lineage differentiation. Building on our promising findings, in this study, we seek to define the mechanistic details of BRD4’s transcriptional repression function in the temporal regulation of Th2 cells, develop and use new BRD4-BD2 selective inhibitors to study the transcriptional regulation of Th2 cell differentiation, and investigate the therapeutic potential of BRD4-BD2 inhibition as new Th2 immunomodulation to block Th2- associated allergic inflammation in mouse models of asthma.