Control of chromatin landscapes in effector T cell lineage specifications

1R01AI077574-01A2； 这一建议的目的是阐明控制IFNG基因谱系特异性表达或抑制的分子机制，作为了解调节效应器T细胞谱系规范的因素的模型。Th1、Th2和Th17细胞虽然来自常见的原始CD4T细胞前体，但在不同的发育程序的控制下分化，这些程序基于独特的基因表达表型指定另一种适应性免疫功能。效应性T细胞的发育与表观遗传改变有关，表观遗传改变建立和维持特定谱系的基因表达程序，使细胞因子能够在 召回回复。虽然Th2细胞因子基因座包括114、1113和115基因，已经成为了解T细胞系发育过程中染色质动态的主要模型，但直到最近，对Th1细胞因子基因IFNG的调控才开始出现。尽管我们开始理解修改 与Th1细胞和Th2细胞相互排斥表达IFNG相关的染色质结构，以及Th2细胞114、115和1113的染色质结构，详细了解多个末端调控顺式元件在定义线特异性 基因表达有待进一步研究。最近，我们发现了IFNG基因(CNS-22)上游22kb的一个末端保守的非编码序列(CNS-22)元件，它在体外增强了IFNG启动子的表达，位于幼稚T细胞以及Th1和Th2细胞中可获得的染色质区域，当 有条件地从IFNG基因座BAC报告基因中删除，阻断Th1细胞、CTL和NK细胞中IFNG的表达。据我们所知，这代表了基因所需的单一末端元件的首次鉴定。 免疫细胞细胞因子在多个谱系中的表达，为进一步研究CNS-22可能作为协调IFNG基因染色质重塑和调控IFNG表达的关键枢纽的机制奠定了基础。因此，对在不同T细胞和非T细胞中与CNS-22相互作用的因素的剖析为调节染色质动力学提供了一个有吸引力的窗口，而染色质动力学对效应器T谱系的指定非常重要。尽管CNS-22似乎对IFNG基因的表达是必不可少的，但它只代表了 在IFNG基因座中至少有8个高度保守的远端CNS，目前还不清楚不同的CNS如何相互作用来控制基因的表达。我们假设，在整个效应器T细胞的分化和发育过程中，IFNG基因的末端保守的非编码序列对IFNG的表达或抑制起着不同的作用，而且CNS- 特别是，22是在产生IFNY的CEL/S中促进IFNG基因表达或在非表达谱系中沉默IFNG所必需的调控元件。这项修订后的提案将检验这一假设的两个相关但独立的方面：第一，CNS-22是家族特异性表达和抑制IFNG基因所需的转录因子的中心；第二，CNS-22指导 效应器T细胞系中IFNG基因位点的可及性和重塑。

1R01AI077574-01A2; WEAVER, Casey T. The goal of this proposal is to elucidate the molecular mechanisms that control lineage-specific expression or repression of the Ifng locus as a model for understanding factors regulating effector T cell lineage specification. Th1, Th2, and Th17 cells, though derived from common naIve CD4+ T cell precursors, differentiate under the control of distinct developmental programs that specify altemate adaptive immune functions based on unique gene expression phenotypes. Development of effector T cells is associated with epigenetic alterations that establish and maintain lineage-specific programs of gene expression that enable rapid and efficient production of cytokines in recall responses. While the Th2 cytokine locus, which includes the 114, 1113 and 115 genes, has become a premier model for understanding chromatin dynamics during T lineage development, only recently have insights into regulation of the Th1 cytokine gene, Ifng, begun to emerge. Although we are beginning to understand modifications of chromatin structure associated with the mutually exclusive expression of Ifng by Th1 cells, and 114, 115, and 1113 by Th2 cells, a detailed understanding of the interplay of multiple distal regulatory cis-elements in defining lineagespecific gene expression awaits further study. Recently, we have identified a distal conserved, noncoding sequence (CNS) element 22kb upstream of the Ifng gene (CNS-22) that enhances Ifng promoter expression in vitro, resides in an area of accessible chromatin in naIve T cells and in both Th1 and Th2 cells, and when conditionally deleted from a Ifng locus BAC reporter transgene, ablates Ifng expression in Th1 cells, CTLs, and NK cells. To our knowledge, this represents the first identification of a single distal element that is required for gene expression of an immune cell cytokine in multiple lineages, setting the stage for further investigations to more fully define mechanisms by which CNS-22 may act as a critical hub for coordination of chromatin remodeling of the Ifng locus and regulation of Ifng expression. Accordingly, dissection of the factors that interact with CNS-22 in different T cell and non-T-celilineages provides an attractive window to regulation of chromatin dynamics important for effector T lineage specification. Although CNS-22 appears essential for Ifng gene expression, it represents only one of at least eight highly conserved distal CNSs within the Ifng locus, and it is unclear how the different CNSs interact to control gene expression. We hypothesize that distal conserved non-coding sequences in the Ifng locus differentially contribute to Ifng expression or repression throughout effector T cell differentiation and development and that CNS- 22, in particular, is an essenb"al regulatory element required for promob"ng Ifng gene expression in IFNy-producing cel/s, or silencing Ifng in non-expressing lineages. This revised proposal will test two related, but independent, aspects of this hypothesis: one, that CNS-22 is a hub for the assembly of transcription factors required for lineage-specific expression and repression of the Ifng gene; and two, that CNS-22 directs accessibility and remodeling of the Ifng locus in effector T cell lineages.