Cooperative Regulation of Cardiac MHC Genes.

描述(申请人提供)：在初步研究中，我们进行了新的观察，发现β-肌球蛋白重链(MHC)基因的表达可能受两个启动子的控制：1)5‘-β启动子(5’-beta)，以正义方向转录前-mRNA；2)3‘(启动子，3’-β)，以反义方向转录前-mRNA，后者的活性与(MHC基因)的转录活性紧密相连(见图1)。此外，我们的发现表明，β反义前-mRNA抑制了(MHC正义前-mRNA)向成熟mRNA的加工，从而抑制了蛋白质的加工。这项建议的主要目标是确定在幼年成年大鼠的正常甲状腺和甲状腺功能低下状态下，α-和β-MHC基因调控在出生后发育中协调的潜在机制。我们推测，在啮齿动物生命的前三周，这种α正义和(MHC)反义之间的共同调控机制主要是导致啮齿动物生命的前三周从β亚型到α亚型的时间转变；它是T3反向调节的主要部位。此外，通过选择性Pre-mRNA分析，我们观察到在正常甲状腺成人心脏中，5‘βMHC启动子比先前认为的更活跃，但其产物(MHC正义Pre-mRNA转录本)通过(MHC反义Pre-mRNA)加工成成熟的功能性mRNA的过程明显受到抑制。这一转录后过程导致了正常对照正常甲状腺心脏中MHC基因和蛋白的优势表达。为了进一步研究心脏MHC表达的这些调控机制，我们将在4kb基因间隔区的背景下研究3‘(P)的转录调控，并确定(MHC启动子和3’βP之间的协同调控机制。此外，我们还将研究5‘(MHC启动子)，以全面表征MHC启动子的远端和近端调控区域以及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载体检测其双向转录活性(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分析，包括DNA酶超敏分析、足迹、缺失和突变分析。总之，这些研究将描述一种新的心脏MHC基因调控机制，从而增强我们对哺乳动物细胞中调控途径的复杂性的理解。

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.