The contractile properties of the myocardium undergoes profound changes in response to developmental, physiological, pathological and hormonal changes. In order to understand cardiac contractility, it is essential to have an understanding of the mechanisms of regulation which are involved in the production of the functional unit of contraction, that is the sarcomere. We have established that changes in contractility involve induction of an heat shock protein gene, the cellular c-myc and c-fos proto- oncogenes, and reprogramming of the genes coding for the fetal and adult protein isoforms. Our goal is to elucidate at the molecular level the control mechanisms involved in the regulation of these genes. The research proposed here will focus on the cardiac a- and B-myosin heavy chain (MHC) genes, which encode the major functional and structural protein of the sarcomere, because their response to developmental hormonal and physiological stimuli is not only regulated in an antithetic fashion, but is also tissue and species specific. We have characterized the a- and B-MHC gene sequences, examined their activity by transfection assay, and established that several distinct 5' upstream positive and negative regulatory elements contribute to their tissue-specific, developmental and hormonal regulated expression. We will pursue the characterization of these distinct cis-acting regulatory sequences by deletion and mutation analysis using transient expression of the a- and B-MHC reporter gene plasmids. Identification of the DNA binding protein factors that are likely to be involved in the muscle-specific regulation of these MHC genes will be first carried out. The function of these proteins will be evaluated in respect to the interaction with MHC gene sequences, their tissue distribution as well as their interaction with other sarcomeric genes. The information from these experiments will be used to purify and characterize these regulatory nuclear proteins. Using a similar approach, we will study the molecular mechanisms involved in the regulation of MHC gene expression by thyroid hormone (T3) by establishing whether the T3 receptor(s) bind(s) directly to specific sequences in the MHC genes. We will also determine whether negative and positive regulation by T3 involves the interaction of the T3 receptor(s) with the same or different binding domains on the MHC gene sequences. Having isolated three distinct T3 receptor cDNA clones from a cardiac gt10 library, we will study the developmental and tissue specific distribution of the T3 receptor isoforms, their respective interaction with the different MHC gene sequences as well as their T3 binding properties. The fundamentally different pattern of expression of the a- and B- genes in rat and human will be examined by introducing their respective gene sequences into the mouse germ cell line to determine whether species-specificity in the primary structure of the cis-acting regulatory sequences or in the distribution of trans-acting regulatory factors are responsible for this phenomenon. It is hoped that this experiment proposal will eventually lead to the identification, purification and biochemical characterization of the molecules involved in cardiac muscle gene regulation.
| Andres, V; Cervera, M; Mahdavi, V (1995) Determination of the consensus binding site for MEF2 expressed in muscle and brain reveals tissue-specific sequence constraints. J Biol Chem 270:23246-9 |
