PROTEIN COMPLEX REQUIRED FOR OPTIMAL STAT1A-MEDIATED GBP PROMOTER ACTIVATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 干扰素（IFN）和其他细胞因子和生长因子激活JAK（Janus激酶）/STAT（信号转导和转录激活因子）途径。在IFN信号传导中，I型IFN（IFN-a/B）在与其受体结合后激活细胞内受体相关酪氨酸激酶JAK 1和Tyk 2。这些激活的JAK反过来磷酸化潜在细胞质转录因子上的特定酪氨酸残基，随后组装成称为ISGF 3（IFN刺激基因因子3）的复合物。该复合物由STAT 1a（91 kD）或STAT 1b（84 kD）和STAT 2（113 kD）组成，它们共同构成ISGF 3-a，以及称为ISGF 3-g（48 kD）的非STAT蛋白，其是干扰素调节因子（IRF）家族的成员。该复合物在细胞核中积累，结合DNA元件ISRE（IFN-α刺激反应元件，15个碱基对的非二联体对称DNA元件），并激活靶基因的转录。ISGF 3不被II型IFN、IFN-g激活。相反，IFN-g在与其受体结合并随后激活JAK 1和JAK 2后，诱导STAT 1a（或STAT 1b）磷酸化，但不诱导STAT 2磷酸化。磷酸化的STAT 1然后二聚化并结合到GAS（IFN-g激活序列），DNA元件是二分体对称的5 <$TTN5AA 3 <$。虽然STAT 1a和STAT 1b都可以与GAS结合，但只有STAT 1a形成功能性GAF（IFN-g激活因子），诱导GAS元件的转录。 最终，影响诱导STAT的固有反式激活潜力的最关键的决定因素之一可能是一个特定的核蛋白微环境。我们的目标是在天然启动子序列的背景下辨别激活的STAT与其他转录因子、辅激活因子和辅抑制因子复合物之间的协同性模式。这种相互作用可能是必要的，以解释在不同的IFN诱导基因的基因激活的STAT的作用。我们已经接近这个问题的比较研究的复杂的结合位点在几个STAT反应基因。虽然STAT结合位点存在并且可能在染色体调控区中是必需的，但是在瞬时转染测定中，单个GAS元件自身产生非常少的诱导或不产生诱导。因此，我们从已知的天然GAS元件开始，并通过添加相邻的天然核苷酸来确定当克隆到报告基因中时是IFN响应性的最小大小的序列。然后，我们确定是否这个报告诱导相关的一个新的条带移动的外观使用相应的寡核苷酸在电泳迁移率变动测定（EMSA）。 我们已经在EMSA组成型低迁移率蛋白复合物中检测到，通过突变分析表明，该蛋白复合物是最佳STAT介导的启动子激活所需的。特别地，当克隆在异源报告基因之前时，含有分别结合STAT 1同源二聚体和IRF 1但不能结合组成型低迁移率复合物的完整GAS和ISRE的GBP启动子序列是无活性的。只有结合STAT 1、IRF 1和组成性低迁移率复合物的序列才能在IFN-γ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结合可能受到GAS内突变以及GBP启动子的GAS样位点的影响，这表明可能与STAT发生物理相互作用或占据STAT位点，在活化的STAT 1二聚体出现后缓解。在EMSA中观察到的该复合物的DNA亲和力与转染实验中相应报告构建体的转录激活潜力完全相关。目前，我们正致力于获得大量的组成性低迁移率复合物的部分纯化制剂，我们将进一步纯化，以确定组成亚基。

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.