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: 7355085
• 负责人: JAMES E DARNELL
• 金额: $ 0.37万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355085
• 关键词: PROTEIN; COMPLEX; REQUIRED; OPTIMAL; STAT1A

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。干扰素 (IFN) 和其他细胞因子和生长因子激活 JAK（Janus 激酶）/STAT（信号转导器和转录激活剂）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的酪氨酸激酶 JAK1 和 Tyk2。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 ISGF3（IFN 刺激基因因子 3）的复合物。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，ISGF3-g (48kD) 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，与 DNA 元件 ISRE（IFN-a 刺激反应元件，15 碱基对非二元对称 DNA 元件）结合，并激活靶基因的转录。 ISGF3 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 JAK1 和 JAK2 后，诱导 STAT1a（或 STAT1b）磷酸化，但不诱导 STAT2 磷酸化。然后磷酸化的 STAT1 二聚化并与 GAS（IFN-g 激活序列）结合，该 DNA 元件是二元对称的 5'TTN5AA3'。尽管 STAT1a 和 STAT1b 都可以与 GAS 结合，但只有 STAT1a 形成功能性 GAF（IFN-g 激活因子），诱导 GAS 元件转录。 最终，影响诱导 STAT 固有反式激活潜力的最关键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 STAT 与其他转录因子、共激活子和辅阻遏物复合物之间的协同模式。这种相互作用可能对于解释 STAT 在不同 IFN 诱导基因的基因激活中的作用是必要的。我们通过比较研究几个 STAT 响应基因中的复杂结合位点来解决这个问题。虽然 STAT 结合位点存在并且可能是染色体调节区所必需的，但在瞬时转染测定中，单个 GAS 元件本身很少或没有诱导作用。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克隆到报告基因中时具有 IFN 响应性的最小大小序列。然后，我们在电泳迁移率变动分析 (EMSA) 中使用相应的寡核苷酸确定该报告基因诱导性是否与新条带偏移的出现相关。 我们在 EMSA 中检测到了组成型、低迁移率蛋白质复合物，通过突变分析表明该复合物是最佳 STAT 介导的启动子激活所必需的。特别是，含有完整 GAS 和 ISRE 的 GBP 启动子序列（分别结合 STAT1 同二聚体和 IRF1，但不能结合组成型低迁移率复合体）在克隆到异源报告基因之前时处于失活状态。只有结合 STAT1、IRF1 和组成型低迁移率复合物的序列才能在 IFN-g 诱导后激活报告基因。对这种复合物的 DNA 亲和力和特异性的研究表明，其 DNA 结合可能受到 GAS 内的突变以及 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用 STAT 位点，而这种相互作用在出现激活的 STAT1 二聚体后会得到缓解。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，我们正在获取大量部分纯化的组成型低迁移率复合物制剂，我们将对其进行进一步纯化，以鉴定组成亚基。