TRANSCRIPTIONAL CONTROL OF INTERFERON-GAMMA IN T AND NK CELLS

描述(申请人提供)：干扰素-β，一种细胞因子，主要由NK细胞和Th1、CD4和CD8 T细胞产生，在细胞内病原体感染的免疫反应中发挥核心作用。相反，对细胞外后生动物的保护依赖于Th2、CD4T细胞及其产生的细胞因子，包括IL-4、IL-13和IL-5。干扰素-？的表达而Th2细胞因子的表达主要在转录水平上进行调节。而控制干扰素-？的转录因子？近些年来，表达已被阐明得相当详细，但关于它们在哪里起作用以及如何起作用，人们知之甚少。与干扰素-β不同的是，在哺乳动物中，Th2细胞因子IL-4、IL-13和IL-5聚集在一个~150kb的位点上，现在对它们的调控已经有了相当详细的分子基础。在本申请中，我们建议获得关于干扰素-？的类似水平的知识。通过全面识别管理干扰素-？的调控要素。通过三个目标表达和确定IFNG轨迹的功能边界： 目的1：确定干扰素-？的表达调控元件。通过对小鼠IFNG基因座进行全面的染色质分析。假设：DNA酶高敏感部位和富含转录有利的组蛋白修饰的区域延伸到小鼠IFNG基因的上游和下游约50kb，并确定控制干扰素的调控元件。表情。 目的2：利用BAG转基因来补充干扰素基因缺陷，确定IFNG基因的功能边界。基因敲除(GKO)小鼠。假设：含有小鼠IFNG基因的BAG转基因并延伸-gt；=50kb，5‘和3’将重组适当的干扰素？表达，从而弥补GKO小鼠的免疫缺陷。 目的3：确定IFNG基因内转录调控元件影响干扰素-β的机制。表达和在这些位点起作用的关键转录因子。假说：T-bet、STAT4、NFAT、NF-βB和AP-1不仅与IFNG启动子结合，而且还与额外的上下游调控元件结合，从而影响干扰素？表达：Ifng5‘CNS2和3和/或Ifng3’CNS3有助于使IFNG基因座免受周围基因的影响。

DESCRIPTION (provided by applicant): IFN-?, a cytokine produced primarily by NK cells and by Th1 CD4 and CD8 T cells, plays a central role in the immune response to infection with intracellular pathogens. Conversely, protection against extracellular metazoans is dependent on Th2 CD4 T cells and the cytokines they produce, which include IL-4, IL-13 and IL-5. Expression of IFN-? and of Th2 cytokines is primarily regulated at the level of transcription. While transcription factors that govern IFN-? expression have in recent years been elucidated in considerable detail, very little is known regarding where and how they act. By contrast to IFN-?, regulation of the Th2 cytokines IL-4, IL-13 and IL-5, which are clustered into a single ~150 kb locus in mammals, is now understood in considerable molecular detail. In this application, we propose to gain a similar level of knowledge regarding IFN-? by comprehensively identifying the regulatory elements governing IFN-? expression and the functional boundaries of the Ifng locus through 3 Aims: Aim 1: Identify regulatory elements governing the expression of IFN-? through comprehensive chromatin profiling of the murine Ifng locus. Hypothesis: DNase hypersensitive sites and regions enriched in transcriptionally favorable histone modifications extend ~50 kb upstream and downstream of the murine Ifng gene and identify regulatory elements governing IFN-? expression. Aim 2: Define the functional boundaries of the Ifng locus using BAG transgenes to complement the defect in IFN-? knockout (GKO) mice. Hypothesis: BAG transgenes containing the murine Ifng gene and extending >= 50 kb 5' and 3' will reconstitute proper IFN-? expression and thereby complement the immunological defect in GKO mice. Aim 3: Identify the mechanisms bv which the transcriptional regulatory elements within the Ifng locus influence IFN-? expression and the key transcription factors that act at these sites. Hypotheses: T-bet, STAT4, NFAT, NF-?B and AP-1 bind not only to the Ifng promoter but to additional upstream and downstream regulatory elements, thereby affecting IFN-? expression; Ifng5'CNS 2 and 3 and/or Ifng3'CNS3 help to insulate the Ifng locus from the influence of surrounding genes.