GP91-PHOX TRANSCRIPTION FACTORS AND HEMATOPOIESIS

描述：（改编自申请人的摘要）本发明的重点在于： 建议是研究参与IFN-g的转录因子， 诱导gp 91-phox（NADPH氧化酶重链）的表达。 gp 91-phox的表达仅限于终末分化的 吞噬细胞，并增加干扰素γ（IFN-g）。转录 gp 91 phox的免疫应答是对IFN-γ的“次级应答”的一部分。 gp91phox 启动子不含g-激活位点共有序列， 受刺激的gp 91 phox转录需要新的蛋白质合成。 参与gl 91 phox基因调控的转录因子提供了一个 IFN-g“早期反应”和时间上遥远的 增殖抑制和炎症介导的事件。 这些连接 目前对事件的描述不清楚。三种独特的增强剂 gp 91 phox启动子中的元件加成性地增加IFN-γ表达。 这已经用gp 91 phox 5'端的报告基因构建体证明。 在RIA分析中，与高表达偶联的侧翼区（显示了 在RNA酶保护试验中（显示出10倍增加）， 增加）。 这三个增强子元件由一个共同的 蛋白复合物（BID 1，用于分化期间诱导的结合）和 复合物的体外结合通过分化而增加， 骨髓细胞系 每个增强子元件紧邻5'端， 结合位点的阻遏物CDP已确定博士。 Orkin的团队。 增强子元件结合蛋白（BID 1 p）已被发现。 使用相应的寡核苷酸序列鉴定和克隆 以筛选商业白细胞 lgt 11 cDNA文库。 Genbank搜索表明， BID 1 p和先前描述的转录因子。 几行 的证据表明，BID 1 p在调节gp 91- phox转录：BID 1 p在骨髓细胞系中的过表达 破坏体外CDP与gp 91 phox阻遏元件的结合， 增加了GP 91 PHOX信息的丰度。 骨髓细胞的治疗 具有IFNg的细胞系在3小时时增加了BID 1 p mRNA的丰度， 提示BID 1 p可能是IFNg早期应答基因。 因此，本提案建议1）调查BID 1 p的作用 在调节IFN-γ应答髓样基因中的作用。 这将是 通过检查BID 1 p在人类中的过表达的影响来完成。 PLB 985细胞，以及在使用BID 1 p表达的单独研究中， 反义信息表达，以确定BID 1 p表达是否 gp 91 phox转录所必需的。 IFN-g对 还将检查其他蛋白质（如CDP）的转录。 这些研究将扩展到研究BID 1 p在以下方面的作用： 使用IL 3依赖性32 DC的非恶性造血模型13 通过筛选小鼠脾cDNA克隆小鼠BID 1 p细胞 人类基因组DNA的文库。 第二个目标是 研究BID 1 p与gp 91 phox增强子结合的调节 元件，使用待生成的抗BID 1 p抗血清。 这 抗血清将用于检测IFNg对BID 1 p的影响 蛋白质丰度和亚细胞定位，以确定是否 Bid 1 p经历了磷酸化等修饰， 从免疫学上证明BID 1 p存在于凝胶转移中 BID 1结合实验。 IFN-γ对BID 1 p信息的影响 转录和信息稳定性也将被检查的稳定性 和特异性。 第三个目的是研究体内变化 蛋白质结合到gp 91 phox启动子，一个在 迄今为止，已经使用的体外足迹法 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.