Molecular Characterization of T-bet's Role in Immunity

描述(申请人提供)：谱系受限的转录因子在造血分化过程中起着关键作用，即使是单个因子的解除调控也会导致疾病状态，如白血病、自身免疫和过敏反应。发生这种情况的部分原因是，选择性地丧失一种因子可以极大地改变关键效应细胞群体的发展。因此，识别受这些因素影响的基因表达级联将使我们能够解决导致疾病表型的分子机制。转录因子T-bet似乎在CD4T辅助1细胞的发育中起着重要作用。Th1细胞分泌干扰素-γ，在细胞免疫的发展中起重要作用。过度活跃的Th1反应可能导致自身免疫性疾病，如克罗恩病和1型糖尿病。除了Th1细胞外，T-bet还在免疫系统中的多个谱系中表达，并被认为在每个效应细胞群中发挥细胞特有的功能。在缺乏T-bet的情况下，自然杀伤细胞、树突状细胞和B细胞中会出现细胞特异性缺陷。同样值得注意的是，克罗恩病、哮喘和狼疮的小鼠疾病模型都是通过改变T-bet水平来建立的。因此，人们可以假设T-bet在发育中的免疫反应中发挥重要作用。这项建议的主要目标是解决T-bet在单个造血效应细胞群中所起作用的分子机制。AIM 1的设计目的是在每个细胞群体中识别直接T-bet靶基因。T-bet是以细胞特有的方式定位于基因座，还是在所有细胞环境中与相同的基因相互作用？为了解决这个问题，将采用目标基因识别策略，利用对染色质免疫沉淀程序的修改。这些技术将只识别在特定核环境中直接与T-bet结合的基因。在目标2中，将检查T-bet与选定启动子的关联的功能结果，以确定这种相互作用在不同的细胞环境中是否具有生产力。目标1和目标2中的研究将提供一种解决T-BET小区规格的方法。目标3和4将阐述T-bet在少数选定目标基因的转录调控中所起的作用。T-bet是作为转录激活因子还是作为抑制因子发挥作用？T-bet是否影响染色质事件或帮助RNA聚合酶II向启动子募集？对一些选定的目标基因进行详细的转录调控研究将有助于回答这些问题。

DESCRIPTION (provided by applicant): Lineage-restricted transcription factors play a critical role in the hematopoietic differentiation process and the deregulation of even a single factor can lead to disease states such as leukemia, autoimmunity, and allergic reactions. Part of how this occurs is that the selective loss of a factor can drastically alter the development of critical effector cell populations. Therefore, identifying the gene expression cascades influenced by these factors will allow us to address the molecular mechanisms that contribute to the disease phenotypes. The transcription factor T-bet appears to play a significant role in the development of CD4+ T helper 1 cells. Th1 cells secrete IFNgamma and are important in the development of cell-mediated immunity. A hyperactive Th1 response can contribute to autoimmune diseases such as Crohn's disease and type 1diabetes. In addition to Th1 cells, T-bet is also expressed in multiple lineages in the immune system and is thought to contribute to cell-specific functions in each effector cell population. In the absence of T-bet, cell-specific defects occur in natural killer, dendritic, and B cells. It is also worth noting that murine disease models for Crohn's disease, asthma, and lupus have all been established by altering T-bet levels. Therefore, one may hypothesize that T-bet plays an important role in the developing immune response. The main goal of this proposal is to address the molecular mechanisms behind T-bet's role in the individual hematopoietic effector cell populations. Aim 1 is designed to identify direct T-bet target genes in each cell population. Is T-bet targeted to loci in a cell-specific manner or does it interact with the same genes in all cellular settings? To address this question, target gene identification strategies will be employed that utilize modifications to the chromatin immunoprecipitation procedure. These techniques will only identify genes that are directly bound by T-bet within the context of a given nuclear environment. In Aim 2, the functional consequence of T-bet's association with select promoters will be examined to determine if this interaction is productive in the different cellular settings. The studies in Aim 1 and 2 will provide a means to address T-bet's cell-specifications. Aims 3 and 4 will address the role T-bet plays in the transcriptional regulation of a few select target genes. Does T-bet function as a transcriptional activator or repressor? Does T-bet influence chromatin events or aid in RNA polymerase II recruitment to the promoter? Detailed transcriptional regulation studies of a few select target genes will aid in answering these questions.