GP91-PHOX TRANSCRIPTION FACTORS AND HEMATOPOIESIS

描述：(改编自申请者摘要)本报告的重点 建议对参与干扰素-g-干扰素的转录因子进行研究 诱导表达Gp91-Phox(NADPH氧化酶重链)。 Gp91-Phox的表达仅限于终末分化 吞噬细胞，并被干扰素-γ(干扰素-g)增加。转录 Gp91Phox的表达是干扰素-g的“二次反应”的一部分。Gp91Phox 启动子不含g-活化位点共有序列和干扰素-g 受刺激的gp91Phox转录需要新的蛋白质合成。 参与gl91Phox基因调控的转录因子提供了一个 干扰素-g的“早期反应”与时间遥远之间的联系 核扩散抑制和炎症调解事件。这些链接 目前，事件的特点很差。三种独特的增强剂 Gp91Phox启动子中的元件成瘾地增加了干扰素-g的表达。 这一点已经用gp91Phox 5‘的报告结构进行了演示。 RIA分析中侧翼区域与Hugh表达的偶联(显示 增加了两倍)，在核糖核酸酶保护试验中(显示了十倍 增加)。这三个增强子元素由一个公共 蛋白质复合体(BID1，用于分化过程中诱导的结合)和 在体外，通过分化增加了该复合体的结合。 髓系细胞系。每个增强子元件立即为5‘到一个 抑制物CDP的结合位点已由Dr。 奥金的小组。一种增强子元件结合蛋白(BID1p)已经被 使用对应的寡核苷酸序列鉴定和克隆 与5‘启动子序列之一连接以筛选商业白细胞 Lgt11 CDNA文库。GenBank搜索显示两者没有同源性 BID1p和之前描述的转录因子。几行 大量证据表明，BID1p在gp91的调控中起作用。 PHOX转录：BID1p在髓系细胞系中的过表达 体外破坏CDP与gp91Phox阻遏元件的结合，并 增加gp91Phox消息的丰富性。髓系细胞的治疗 加入IFNG后3h，BID1p基因的表达丰度增加， 提示BID1p可能是IFNG早期反应基因。 因此，本提案建议1)调查BID1p的作用 在干扰素-g反应的髓系基因的调节中。这将是 通过检测BID1p在人类体内过表达的影响来完成 PLB985细胞，并在单独的研究中使用BID1p表达 反义消息表达以确定BID1p表达是否 是gp91Phox转录所必需的。干扰素-g对血管内皮细胞生长的影响 其他蛋白质(如CDP)的转录也将被检测。 这些研究将扩展到调查BID1p在 IL-3依赖的32DC13非恶性造血模型的建立 小鼠细胞BID1p将通过筛选小鼠脾CDNA来克隆 已经有人类cdna的图书馆。第二个目标是 BID1p与gp91Phox增强子结合调控的研究 元素，使用抗BID1p抗血清生成。这 用抗血清检测IFNG对BID1p的影响 蛋白质丰度和亚细胞定位，以确定 Bid1p经历了磷酸化等修饰，并试图 为了从免疫学上证明BID1p存在于凝胶移位 BID1结合实验。干扰素-g对BID1p消息的影响 还将检查转录和消息稳定性是否稳定 和专一性。第三个目标是研究体内的变化 与gp91Phox启动子结合的蛋白质，是In 到目前为止已经用于这一研究的体外足迹方法 项目。

DESCRIPTION: (Adapted from Applicant's Abstract) The focus of this proposal is the study of transcription factors involved in IFN-g- induced expression of gp91-phox (the NADPH oxidase heavy chain). Expression of gp91-phox is restricted to terminally differentiated phagocytes and is increased by interferon gamma (IFN-g). Transcription of gp91phox is part of the "secondary response" to IFN-g. The gp91phox promoter contains no g-activation site consensus sequence and IFN-g stimulated gp91phox transcription requires new protein synthesis. Transcription factors involved in gl91phox gene regulation provide a link between the IFN-g "early responses" and the temporally remote events of proliferation arrest and inflammatory mediation. These linking events are at present poorly characterized. Three unique enhancer elements in the gp91phox promoter addictively increase IFN-g expression. This has been demonstrated using reporter constructs of gp91phox 5' flanking region coupled with hugh expression in an RIA assay (showing a two-fold increase), and in an RNAse protection assay (showing a ten-fold increase). The three enhancer elements are recognized by a common protein complex (BID1, for binding induced during differentiation) and in vitro binding of the complex is increased by differentiation in myeloid cell lines. Each enhancer element is immediately 5' to a binding site for the repressor CDP which had been identified by Dr. Orkin's group. An enhancer element binding protein (BID1p) has been identified and cloned using an oligonucleotide sequence corresponding to one of the 5' promoter sequences to screen a commercial leukocyte lgt11 CDNA library. Genbank search demonstrated no homology between BID1p and previously described transcription factors. Several lines of evidence indicate that BID1p plays a role in regulation of gp91- phox transcription: over-expression of BID1p in myeloid cell lines disrupts in vitro CDP binding to the gp91phox repressor elements and increases the abundance of gp91phox message. Treatment of myeloid cell lines with IFNg increases the abundance of BID1p MRNA at 3 hours, suggesting the possibility that BID1p is an IFNg early response gene. This proposal therefore proposes to 1) investigate the role of BID1p in the regulation of IFN-g responsive myeloid genes. This will be done by examining the effect of overexpression of BID1p in the human PLB985 cells, and in separate studies using expression of BID1p antisense message expression to determine if BID1p expression is necessary for gp91phox transcription. The effect of IFN-g on transcription of other proteins (such as CDP) will also be examined. These studies will be extended to investigate the role of BID1p in a non-malignant hematopoiesis model using the IL3-dependent 32DC13 cells Murine BID1p will be cloned by screening a mouse spleen CDNA library with the human CDNA already in hand. The second aim is to investigation the regulation of BID1p binding to gp91phox enhancer elements, using an anti-BID1p antisera to be generated. This antiserum will be used to examine the effect of IFNg on BID1p protein abundance and subcellular localization, to determine whether Bid1p undergoes modifications such as phosphorylation, and an attempt to demonstrate immunologically that BID1p is present in the gel-shifted BID1 binding experiments. The effect of IFN-g on BID1p message transcription and message stability will also be examined for stability and specificity. The third aim is to investigate the in vivo changes of proteins binding to the gp91phox promoter, an extension of the in vitro footprinting methods that have been utilized to date for this project.