Cooperative Regulation of Cardiac MHC Genes.

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）基因的表达可能在两个启动子的控制下：1）以有义方向转录前mRNA的5 'β启动子（5 ' β）和2）3'β启动子（5 ' β），在反义方向转录前mRNA的β启动子（3 ′ β P），后者的活性与β MHC基因的转录活性紧密相关（见图1）。此外，我们的研究结果表明，β反义前mRNA抑制了β MHC正义前mRNA加工成成熟mRNA，从而抑制了蛋白质。该提案的主要目标是确定α-和β-MHC基因调节在产后发育以及年轻成年大鼠甲状腺功能正常和甲状腺功能减退状态下协调的潜在机制。我们推测，这种α正义和α MHC反义之间的共同调节机制主要负责啮齿动物生命的前三周内从β亚型到α亚型的时间转变;并且它是T3的反向调节的主要位点。此外，通过选择性前体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启动子比以前认为的更活跃，然而，其产物（MHC正义前体mRNA转录物）加工成成熟的功能性mRNA明显受到（MHC反义前体mRNA）的抑制。这种转录后过程导致在正常对照甲状腺功能正常的心脏中MHC mRNA和蛋白表达占优势。为了进一步表征心脏MHC表达的这些调节机制，我们将研究在4kb基因间区域的背景下3 'β P的转录调节，并确定MHC启动子和3' β P之间的协同调节机制。我们将研究5 '端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，并使用具有以头对头方向放置的两个报告基因的表达质粒检查其双向转录活性（参见图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测定，包括DNA酶超敏测定、足迹法、缺失和突变分析。总的来说，这些研究将表征心脏MHC基因调控的新机制，从而增强我们对哺乳动物细胞中调控途径复杂性的理解。

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.