Cooperative Regulation of Cardiac MHC Genes.

心脏 MHC 基因的协同调节。

• 批准号: 6734178
• 负责人: Kenneth M. Baldwin
• 金额: $ 40.97万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6734178
• 关键词: DNA footprinting; antisense nucleic acid; cardiogenesis; gel mobility shift assay; gene mutation; genetic enhancer element; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; hormone receptor; hormone regulation /control mechanism; laboratory rat; messenger RNA; myosins; precursor mRNA; protein isoforms; protein structure function; thyroid function; transfection

DESCRIPTION (provided by applicant): In preliminary studies we made the novel observation that the expression of the beta myosin heavy chain (MHC) gene is likely under the control of two promoters: 1) a 5'beta promoter (5 'beta) that transcribes pre-mRNA in the sense orientation and 2) a 3' ( promoter (3' betaP) that transcribes pre-mRNA in the antisense direction, with the latter's activity being tightly linked to the transcriptional activity of the (MHC gene (see Figure 1). Further, our findings suggest that the beta antisense pre-mRNA inhibits the processing of ( MHC sense pre-mRNA into mature mRNA and hence protein. The primary goal of this proposal is to ascertain the underlying mechanisms in which the alpha- and beta-MHC gene regulation is coordinated in postnatal development, and in both the euthyroid and hypothyroid states in young adult rats. We hypothesize that this common regulatory mechanism between the alpha sense and the (MHC antisense is mainly responsible for the temporal shift from beta to alpha isoform in the first three weeks of the rodent life; and it is the major site of the antithetical regulation by T3. Also, via selective pre-mRNA analysis, we observed that in the euthyroid adult heart, the 5' beta MHC promoter is more active than previously thought, however, the processing of its product, the (MHC sense pre-mRNA transcript, into mature functional mRNA is apparently inhibited via the (MHC antisense pre-mRNA. This post-transcriptional process leads to the predominance of the (MHC mRNA and protein expression in the normal control euthyroid heart. To further characterize these regulatory mechanisms of the cardiac MHC expression we will study the transcriptional regulation of the 3'(P in the context of the 4 kb intergenic region, and determine the mechanism of cooperative regulation between the (MHC promoter and the 3' beta P. Further, we will study the 5'(MHC promoter to fully characterize the distal and proximal regulatory region and the mechanism of T3 action on this promoter. The intergenic DNA fragment will be isolated and its bi-directional transcriptional activity will be examined using an expression plasmid with two reporter genes placed in the head-to-head direction (see figure 2B). This approach allows studying the cooperative regulation of the two gene promoters under the same experimental condition. These analyses will involve a series of assays including DNase hypersensitive assays, footprinting, deletion and mutational analysis. Collectively these studies will characterize a novel mechanism of cardiac MHC gene regulation and thus enhance our understanding of the complexity of regulatory pathways in mammalian cells.

描述（由申请人提供）：在初步研究中，我们进行了新的观察，即β肌球蛋白重链（MHC）基因的表达可能受到两个启动子的控制：1）5'β启动子（5'beta），以有义方向转录前体 mRNA，2) 3' ( 启动子 (3' betaP) 以反义方向转录前体 mRNA，后者的活性与前体的转录活性紧密相关。 （MHC 基因（见图 1）。此外，我们的研究结果表明，β 反义前体 mRNA 抑制 (MHC 有义前体 mRNA 加工成成熟 mRNA 和蛋白质。该提案的主要目标是确定潜在机制其中 α- 和 β-MHC 基因调节在出生后发育以及年轻成年大鼠的甲状腺功能正常和甲状腺功能减退状态下是协调的，我们假设 α 正义和 (MHC 反义) 之间的这种共同调节机制是。主要负责啮齿动物生命前三周内从 β 异构体到 α 异构体的时间转变；是T3对立调节的主要位点。此外，通过选择性前 mRNA 分析，我们观察到在甲状腺功能正常的成年心脏中，5'β MHC 启动子比之前想象的更活跃，然而，其产物（MHC 正义前 mRNA 转录物）加工成成熟的功能性 mRNA 显然通过 (MHC 反义前 mRNA) 受到抑制。这种转录后过程导致正常对照甲状腺正常心脏中 (MHC mRNA 和蛋白质表达占主导地位。为了进一步表征心脏的这些调节机制MHC 表达我们将研究 4 kb 基因间区域背景下 3'(P) 的转录调控，并确定 (MHC 启动子和 3'β P 之间的协同调节机制。进一步，我们将研究 5 '（MHC 启动子，以充分表征远端和近端调控区以及 T3 对该启动子的作用机制。将分离基因间 DNA 片段，并使用带有两个报告基因的表达质粒检查其双向转录活性这头对头方向（见图 2B）。这种方法允许在相同的实验条件下研究两个基因启动子的协同调控。这些分析将涉及一系列检测，包括 DNase 超敏检测、足迹分析、缺失和突变分析。总的来说，这些研究将描述心脏 MHC 基因调控的新机制，从而增强我们对哺乳动物细胞调控途径复杂性的理解。