Control of chromatin landscapes in effector T cell lineage specifications

1 R 01 AI 077574 - 01 A2; WEAVER，凯西T. 该提案的目标是阐明控制Ifng基因座的谱系特异性表达或抑制的分子机制，作为理解调节效应T细胞谱系特异性的因子的模型。Th 1、Th 2和Th 17细胞虽然来源于常见的幼稚CD 4 + T细胞前体，但在基于独特基因表达表型指定交替适应性免疫功能的不同发育程序的控制下分化。效应T细胞的发育与表观遗传学改变相关，表观遗传学改变建立并维持基因表达的谱系特异性程序，使得能够在细胞内快速有效地产生细胞因子。 回忆回答。虽然Th 2细胞因子基因座，其中包括114，1113和115基因，已成为一个首要的模型，用于了解染色质动态T谱系发展过程中，只有最近才有洞察力调节的Th 1细胞因子基因，Ifng，开始出现。虽然我们开始了解到 与Th 1细胞的Ifng和Th 2细胞的Ifng 114、115和1113的互斥表达相关的染色质结构，详细了解了多个远端调节顺式元件在定义谱系特异性 基因表达有待进一步研究。最近，我们已经鉴定了Ifng基因上游22 kb处的远端保守的非编码序列（CNS）元件（CNS-22），其在体外增强Ifng启动子表达，存在于幼稚T细胞和Th 1和Th 2细胞中的可接近染色质区域，并且当 从Ifng基因座BAC报告基因转基因中条件性缺失，消除了Th 1细胞、CTL和NK细胞中的Ifng表达。据我们所知，这代表了第一次鉴定出基因表达所需的单个远端元件。 在多种谱系中表达免疫细胞细胞因子，为进一步研究奠定基础，以更全面地定义CNS-22可作为协调Ifng基因座的染色质重塑和调节Ifng表达的关键枢纽的机制。因此，在不同T细胞和非T细胞系中与CNS-22相互作用的因子的解剖为调节对效应T谱系特化重要的染色质动力学提供了有吸引力的窗口。虽然CNS-22似乎对Ifng基因表达至关重要，但它仅代表了一种 在Ifng基因座内至少有八个高度保守的远端CNSs，并且不清楚不同的CNSs如何相互作用以控制基因表达。我们假设Ifng基因座中的远端保守非编码序列在整个效应T细胞分化和发育过程中差异性地促进Ifng表达或抑制，并且CNS- 22是在IFN γ产生细胞中促进Ifng基因表达或在非表达谱系中沉默Ifng所需的必需调控元件。这项修订后的提案将测试这一假设的两个相关但独立的方面：第一，CNS-22是Ifng基因谱系特异性表达和抑制所需转录因子组装的中心;第二，CNS-22指导 效应T细胞谱系中Ifng基因座的可及性和重塑。

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.