PROTEIN COMPLEX REQUIRED FOR OPTIMAL STAT1A-MEDIATED GBP PROMOTER ACTIVATION

STAT1A 介导的 GBP 启动子最佳激活所需的蛋白质复合物

• 批准号: 7954071
• 负责人: JAMES E DARNELL
• 金额: $ 0.12万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954071
• 关键词: Affect; Affinity; Appearance; Base Pairing; Be++ element; Beryllium; Binding; Binding Sites; Biological Assay; Cell Nucleus; Comparative Study; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Binding; DNA Sequence; Electrophoretic Mobility Shift Assay; Elements; Family; Funding; Gene Activation; Gene Targeting; Genes; Genetic Transcription; Grant; Growth Factor; IRF1 gene; Institution; Interferon Type I; Interferon Type II; Interferons; JAK1 gene; JAK2 gene; Janus kinase; Mediating; Mutation; Nucleic Acid Regulatory Sequences; Nucleoproteins; Nucleotides; Occupations; Oligonucleotides; Pathway interactions; Pattern; Phosphorylation; Preparation; Protein Tyrosine Kinase; Proteins; Reporter; Reporter Genes; Research; Research Personnel; Resources; Response Elements; Role; STAT protein; STAT1 gene; STAT2 gene; Signal Transduction; Site; Source; Specificity; Transactivation; Transcription Coactivator; Transcriptional Activation; Transfection; Tyrosine; United States National Institutes of Health; cytokine; dimer; macromolecule; member; novel; promoter; protein complex; receptor; research study; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Interferons (IFNs) and other cytokines and growth factors activate the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway. In the IFN signaling, type I IFNs (IFN-a/b) upon binding to their receptor, activate intracellular, receptor-associated, tyrosine kinases JAK1 and Tyk2. These activated JAKs, in turn, phosphorylate specific tyrosine residues on latent cytoplasmic transcription factors which subsequently assemble into a complex called ISGF3 (IFN stimulated gene factor 3). This complex is composed of either STAT1a (91kD) or STAT1b (84kD) and STAT2 (113kD), which together constitute ISGF3-a, and a non-STAT protein called ISGF3-g (48kD) which is a member of the interferon regulatory factor (IRF) family. This complex accumulates in the nucleus, binds to a DNA element, ISRE (IFN-a stimulated response element, a 15 base pair non-dyad symmetrical DNA element), and activates transcription of target genes. ISGF3 is not activated by type II IFN, IFN-g. Instead, IFN-g, upon binding to its receptor and consequent activation of JAK1 and JAK2, induces the phosphorylation of STAT1a (or STAT1b), but not STAT2. The phosphorylated STAT1 then dimerizes and binds to a GAS (IFN-g activated sequence), DNA element that is dyad symmetrical 5¿TTN5AA3¿. Although both STAT1a and STAT1b can bind to GAS, only STAT1a forms functional GAF (IFN-g activated factor) that induces transcription from the GAS elements. Ultimately, one of the most crucial determinants affecting inherent transactivation potential of induced STATs may be a particular nucleoprotein microenvironment. Our objective is to discern patterns of cooperativity between activated STATs and other transcription factors, coactivator, and corepressor complexes within the context of the native promoter sequences. Such interactions are probably necessary to explain the role of STATs in gene activation at different IFN inducible genes. We have approached this problem by comparative study of the complex binding sites in several STAT-responsive genes. While STAT-binding sites exist and are likely required in chromosomal regulatory regions, single GAS elements give very little or no induction on their own in transient transfection assays. Therefore, we started with known native GAS elements and by adding adjacent native nucleotides determined the minimal size sequence that was IFN-responsive when cloned into a reporter gene. Then we determined whether this reporter inducibility correlated with an appearance of a novel band shift using a corresponding oligonucleotide in electrophoretic mobility shift assays (EMSA). We have detected in EMSA constitutive, low-mobility, protein complex that by mutational analysis is shown to be required for optimal STAT-mediated promoter activation. In particular, GBP promoter sequences that contain intact GAS and ISRE that bind STAT1 homodimer and IRF1, respectively, but that cannot bind constitutive low-mobility complex, when cloned in front of the heterologous reporter gene are inactive. Only sequences that bind STAT1, IRF1 and the constitutive low-mobility complex are able to activate a reporter gene upon IFN-g induction. Studies of the DNA affinity and specificity of this complex revealed that its DNA-binding may be affected by mutations within GAS as well as GAS-like site of the GBP promoter, suggesting a possible physical interaction with STATs or occupation of STAT sites that is relieved after appearance of activated STAT1 dimer. DNA affinity of this complex, observed in EMSA, is completely correlated to the transcriptional activation potential of the corresponding reporter constructs in transfection experiments. Currently we are engaged in obtaining larger quantities of partly purified preparation of constitutive low mobility complex which we will further purify in order to identify constituent subunits.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 干扰素（IFN）和其他细胞因子和生长因子激活JAK（JANUS激酶）/STAT（转录的信号换能器和激活因子）途径。在IFN信号传导中，I型IFN（IFN-A/B）与其受体结合后，激活细胞内，受体相关的酪氨酸激酶JAK1和TYK2。这些激活的JAKS反过来磷酸化特异性酪氨酸保留在潜在的细胞质转录因子上，随后将其组装成一种称为ISGF3的复合物（IFN刺激基因因子3）。该复合物由STAT1A（91KD）或STAT1B（84KD）和STAT2（113KD）组成，它们共同构成ISGF3-A，以及称为ISGF3-G（48KD）的非Stat蛋白，该蛋白是Interferon调节因子（IRF）家族的成员。该络合物积聚在细胞核中，与DNA元素结合，ISRE（IFN-A刺激的响应元件，15个碱基对非DYAD非对称DNA元素），并激活靶基因的转录。 ISGF3未通过II型IFN，IFN-G激活。取而代之的是，IFN-G与其受体结合并随之而来的JAK1和JAK2的激活诱导STAT1A（或STAT1B）的磷酸化，但不能诱导STAT2。然后，磷酸化的STAT1二聚并结合与气体（IFN-G活化序列），DNA元件是对称对称的5？ttn5Aa3？尽管STAT1A和Stat1b均可以与气体结合，但仅Stat1a形成函数GAF（IFN-G活化因子），从而影响了从气体元素转移的转录。 最终，影响诱发数据固有的反式激活潜力的最关键的决定剂之一可能是特定的核蛋白微环境。我们的目标是辨别激活统计统计因素和其他转录因子，共激活因子和核心压力复合物之间的协调模式。这种相互作用可能是解释在不同IFN诱导基因上基因激活中Stat在基因激活中的作用的必要条件。我们通过对几个统计响应基因中复杂结合位点的比较研究来解决这个问题。尽管染色体调节区域中存在统计结合位点，并且可能需要单个气体元素，但在瞬时转染测定法中，单个气体元素本身很少或根本没有诱导。因此，我们从已知的天然气体元件开始，并通过添加相邻的天然核苷酸确定了克隆到报告基因基因时具有IFN响应性的最小尺寸序列。然后，我们确定了该报告基因诱导性是否与使用相应的电泳迁移率转移测定法（EMSA）中的寡核苷酸（EMSA）相关的新带移动的外观相关。 我们已经在EMSA组成型，低运动，蛋白质复合物中检测到，通过突变分析被证明是最佳统计介导的启动子激活所必需的。特别是，分别结合STAT1同型二聚体和IRF1的GBP启动子序列，但是当克隆在异源报告基因前面时，这种均二聚体和IRF1的序列分别结合了STAT1同二聚体和IRF1。只有结合STAT1，IRF1和组成型低动作络合物的序列才能在IFN-G诱导后激活报告基因。对该复合物的DNA亲和力和特异性的研究表明，其DNA结合可能会受到气体内部突变以及GBP启动子的类似气体位点的影响，这表明在出现活性stat1二聚体后可能与统计数据或统计位点发生的物理相互作用。在EMSA中观察到的该复合物的DNA亲和力与转化实验中相应的报告基因构建体的转录激活潜力完全相关。目前，我们致力于获得构成低移动性复合物的大量部分纯化的制备，我们将进一步纯化以识别构成亚基。