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.
