PROTEIN COMPLEX REQUIRED FOR OPTIMAL STAT1A-MEDIATED GBP PROMOTER ACTIVATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 干扰素(IFN)和其他细胞因子和生长因子激活JAK(Janus Kinase)/STAT(信号转导和转录激活因子)途径。在干扰素信号中，I型干扰素(干扰素-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。这些被激活的JAK反过来将潜在的细胞质转录因子上的特定酪氨酸残基磷酸化，然后组装成称为ISGF3(干扰素刺激基因因子3)的复合体。这个复合体由STAT1a(91kD)或STAT1b(84kD)和STAT2(113kD)组成，它们共同构成ISGF3-a，以及一个称为ISGF3-g(48kD)的非STAT蛋白，它是干扰素调节因子(IRF)家族的成员。这种复合体聚集在细胞核中，与DNA元件ISRE(干扰素-α刺激反应元件，一种15碱基对的非二联体对称DNA元件)结合，并激活靶基因的转录。ISGF3不能被II型干扰素、干扰素-γ激活。相反，当干扰素-g与其受体结合并随后激活JAK1和JAK2时，诱导STAT1a(或STAT1b)的磷酸化，但不诱导STAT2的磷酸化。然后，磷酸化的STAT1二聚化并结合到GAS(干扰素-g激活序列)，DNA元件是二倍体对称的5？TTN5AA3？虽然STAT1a和STAT1b都可以与GAS结合，但只有STAT1a形成功能性GAF(干扰素-g激活因子)，诱导GAS元件转录。 最终，影响诱导STAT固有反式激活潜能的最关键的决定因素之一可能是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STATS与其他转录因子、共激活因子和辅阻遏子复合体之间的协同作用模式。这种相互作用可能对于解释STATS在不同干扰素诱导基因的基因激活中的作用是必要的。我们通过对几个STAT反应基因中复杂结合位点的比较研究来解决这个问题。虽然在染色体调控区域中存在STAT结合位点并且很可能是必需的，但在瞬时转染试验中，单个GAS元件本身的诱导作用很小甚至没有。因此，我们从已知的天然GAS元件开始，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对干扰素有反应的最小长度序列。然后，我们确定这种报告诱导性是否与在电泳迁移率变化分析(EMSA)中使用相应的寡核苷酸出现新的条带移动相关。 我们已经在EMSA构成的、低流动性的蛋白质复合体中检测到，通过突变分析表明，这是最佳STAT介导的启动子激活所必需的。特别是，当克隆在异源报告基因前面时，含有分别与STAT1同源二聚体和IRF1结合的完整GAS和ISRE的GBP启动子序列，但不能结合结构性低迁移率复合体的GBP启动子序列是无效的。只有与STAT1、IRF1和构成的低迁移率复合体结合的序列才能在干扰素-g诱导时激活报告基因。对该复合体的DNA亲和力和特异性的研究表明，其DNA结合可能受到GBP启动子的GAS和GAS样位点突变的影响，这表明可能存在与STATS的物理相互作用或STAT位点的占据，这种相互作用在激活的STAT1二聚体出现后解除。在EMSA中观察到的这种复合体的DNA亲和力，与转染实验中相应报告结构的转录激活潜力完全相关。目前，我们正致力于获得更大数量的部分纯化的构造性低迁移率复合体的制备，我们将进一步纯化以鉴定其组成的亚基。

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.