REGULATION OF SMAA PROMOTER BY E BOX ELEMENTS

E BOX 元件对 SMAA 启动子的调节

• 批准号: 2702129
• 负责人: ALLEN D JOHNSON
• 金额: $ 3.31万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2702129
• 关键词: cell differentiation; genetic promoter element; genetic regulation; muscle cells; site directed mutagenesis; southern blotting; transcription factor; transfection; vascular smooth muscle; western blottings; yeast two hybrid system

Vascular injury involves phenotypic changes in smooth muscle cells (SMCs) of the arterial wall, including increased proliferation potential, changes in lipid metabolism, greater extracellular matrix production, and decreased expression of several structural and contractile proteins. To understand how injury decreases contractile protein synthesis, we must first know how SMCs control their synthesis normally. Our laboratory has focused on transcriptional control at the smooth muscle alpha actin (SMalphaA) promoter, both to understand contractile protein regulation, and as a significant component of phenotypic modulation by SMCs. Although other cells also express SMalphaA, there is smooth muscle- specific transcriptional regulation of this promoter, which depends on multiple cis and trans elements (1). Our published experiments found the first 125bp 5' to the start site in the rat SMalphaA promoter were transcriptionally active in multiple cell lines, and that cell-type specific expression required regulator elements lying -125 to -547bp upstream (1). This region includes 3 potential E-box elements (E1, E2, and E3), which are binding sites for basic helix-loop-helix (bHLH) transcriptional factors. Basic HLH homo- and heterodimers play a key role in coordinate control of gene expression during differentiation in skeletal muscle and other tissues. The central focus of this proposal is to determine whether bHLH heterodimers also bind to E-boxes in the SMalphaA promoter. In preliminary experiments, I compared the activity of native and mutated -271bp promoters using CAT reporter assays. Constructs with mutations that disrupted either the E1 or E2 site alone were fully active in SMCs, while double mutations of both E1 and E2 sites abolished activity. In contrast, L6 skeletal myotubes required both E1 and E2 sites for CAT activity. In electrophoretic mobility shift assays (EMSA), smooth muscle and L6 myotube nuclear proteins also bound to DNA oligo probes containing either the native E1 or E2 sites, but not to probes with the same inactivating mutations as CAT assays. The central hypothesis of this proposal is that transcription of the SMalphaA gene requires a single E-box which binds bHLH protein heterodimers, and that one member of the dimer is ubiquitous while the second is SMC restricted. This hypothesis will be tested by addressing two aims. AIM #1 will utilize site-directed mutagenesis and transient transfection assays to characterize the nucleotides within the E1 and E2 sites that are required for transcriptional activity of the -271bp SMalphaA promoter in rat aortic SMCs. Comparison of required nucleotides to the conserved E-box motif, 5'-CAnnTG-3', will determine whether E1 or E2 are functioning as true E-box elements. One to 4bp mutations in both of the putative E-box sites will assayed using CAT reporters, to determine minimal nucleotides required for promoter transcriptional activity. Results of methylation footprinting (MFP) will identify specific nucleotides in or near E1 and E2 sites that are required for SMC nuclear protein binding, and whether they are the same nucleotides that are functionally important in CAT reporter studies. AIM #2 will identify SMC proteins that bind at E1 or E2 sites to regulate transcription by the SMalphaA promoter. Aim #2A will test the hypothesis that the SMalphaA E1 and E2 binding complexes consist of a heterodimer of a known Type I bHLH factor with a smooth muscle restricted or selective bHLH partner. Known bHLH proteins will be identified by EMSA supershifting assays using bHLH-1-specific antibodies, by DNA crosslinking to estimate molecular size, and by comparison of methylation footprints on E1 and E2 by SMCs to footprints of known bHLH-1 proteins. Additional comparisons to E1 and E2 footprints make by rat endothelial cell (ECs), skeletal myoblast and myotube, and fibroblast nuclear proteins will determine whether a smooth muscle specific protein is present in E1 or E2 binding complexes. Aim #2B will be to clone potentially novel smooth muscle-restricted bHLH factors that bind E1 and E2 sites, thereby regulating transcriptional control of SMalphaA promoter. The cloning strategy selected will depend upon data from Aim #2A, but may include the yeast two-hybrid system, using bHLH-1 proteins identified in Aim #2A as "bait." The proposed studies will increase our understanding of tissue-specific regulation by E-box enhancer motifs, by determining whether the bHLH family of transcription factors bind to SMalphaA promoter in SMCs. These data will also enhance our understanding to molecular regulation of SMC differentiation at the level of gene transcription, and lead to further investigations of bHLH-mediated pathways which alter SMalphaA expression during vascular disease. Finally, this study complements my long-term interest in the acute versus stable changes in phenotype that occur in smooth muscle cells in response to injury.

血管损伤涉及平滑肌细胞的表型变化 动脉壁（SMC），包括增殖增加 潜力、脂质代谢的变化、更大的细胞外基质 产生，并减少一些结构和表达的表达 收缩蛋白。 了解损伤如何降低收缩力 蛋白质的合成，首先要知道SMCs是如何控制其合成的 通常情况下。 我们的实验室专注于转录控制 平滑肌α肌动蛋白（SMalphaA）启动子，两者都了解 收缩蛋白调节，并作为重要组成部分 SMC 的表型调节。 虽然其他细胞也表达 SMalphaA，但平滑肌- 该启动子的特异性转录调控取决于 多个顺式和反式元件 (1)。 我们发表的实验发现 大鼠 SMalphaA 启动子中起始位点 5' 的第一个 125bp 是 在多种细胞系中具有转录活性，并且该细胞类型 特定表达所需的调节元件位于 -125 至 -547bp 上游 (1)。该区域包括 3 个潜在的 E-box 元素（E1、E2、 和 E3)，它们是基本螺旋-环-螺旋 (bHLH) 的结合位点 转录因子。 基本 HLH 同二聚体和异二聚体发挥着关键作用 分化过程中基因表达协调控制的作用 骨骼肌和其他组织。 本提案的核心焦点 是为了确定 bHLH 异源二聚体是否也与 E-box 结合 SMalphaA 启动子。 在初步实验中，我比较了本地和 使用 CAT 报告基因检测突变 -271bp 启动子。 构造与 单独破坏 E1 或 E2 位点的突变完全被 在 SMC 中活跃，同时 E1 和 E2 位点的双突变被废除 活动。相比之下，L6 骨骼肌管需要 E1 和 E2 CAT 活动的网站。 在电泳迁移率变动分析中 (EMSA)，平滑肌和 L6 肌管核蛋白也与 DNA 结合 寡核苷酸探针包含天然 E1 或 E2 位点，但不包含 具有与 CAT 检测相同的失活突变的探针。 该提案的中心假设是转录 SMalphaA 基因需要一个结合 bHLH 蛋白的 E-box 异二聚体，并且二聚体的一个成员是普遍存在的，而 其次是SMC限制。 该假设将通过解决 两个目标。 AIM #1 将利用定点突变和瞬时突变 转染测定来表征 E1 内的核苷酸和 -271bp 转录活性所需的 E2 位点 大鼠主动脉 SMC 中的 SMalphaA 启动子。 所需比较 保守的 E-box 基序 5'-CAnnTG-3' 的核苷酸将确定 E1 或 E2 是否充当真正的 E-box 元件。 1 至 4bp 两个假定的 E-box 位点的突变将使用 CAT 进行分析 报告基因，以确定启动子所需的最小核苷酸 转录活性。 甲基化足迹（MFP）结果 将识别 E1 和 E2 位点内或附近的特定核苷酸 SMC核蛋白结合所需的物质，以及它们是否相同 在 CAT 报告基因研究中具有重要功能的核苷酸。 AIM #2 将识别在 E1 或 E2 位点结合的 SMC 蛋白 通过 SMalphaA 启动子调节转录。 目标 #2A 将测试 假设 SMalphaA E1 和 E2 结合复合物由 已知 I 型 bHLH 因子与平滑肌的异二聚体 限制性或选择性 bHLH 伴侣。 已知的 bHLH 蛋白是 通过使用 bHLH-1 特异性的 EMSA 超移测定进行鉴定 抗体，通过 DNA 交联来估计分子大小，并通过 SMC 在 E1 和 E2 上的甲基化足迹与足迹的比较 已知的 bHLH-1 蛋白。 与 E1 和 E2 的其他比较 大鼠内皮细胞（EC）、骨骼肌细胞和 肌管和成纤维细胞核蛋白将决定是否平滑 肌肉特异性蛋白存在于 E1 或 E2 结合复合物中。 目的 #2B 将克隆潜在的新型平滑肌限制性 bHLH 结合 E1 和 E2 位点的因子，从而调节转录 SMalphaA 启动子的控制。 选择的克隆策略将取决于 根据目标 #2A 的数据，但可能包括酵母双杂交系统， 使用 Aim #2A 中鉴定的 bHLH-1 蛋白作为“诱饵”。 拟议的研究将增加我们对组织特异性的理解 通过E-box增强子基序的调节，通过确定bHLH是否 转录因子家族与 SMC 中的 SMalphaA 启动子结合。 这些数据也将增强我们对分子调控的理解 SMC 在基因转录水平上的分化，并导致 进一步研究 bHLH 介导的改变 SMalphaA 的途径 血管疾病期间的表达。 最后，这项研究补充了我的 对表型的急性与稳定变化的长期兴趣 发生在平滑肌细胞对损伤的反应中。