This project seeks to understand the mechanisms which regulate cardiac muscle gene expression and the commitment of embryonic mesodermal cells to the cardiac muscle cell lineage. Our studies will focus on analysis of cis- and trans-regulatory components of the mouse M-creatine kinase (MCK) gene and on the isolation of cardiac muscle determination factors.
The specific aims are: [1.] Identification of elements which control MCK gene expression in cardiac muscle. Initial studies will focus on a 5'-enhancer that is active in cardiac muscle and that binds skeletal muscle determination factors. Related studies will identify control elements in other MCK gene regions, and will search for locus control regions. Transgenic mouse studies will determine whether different MCK control elements exhibit temporal expression differences during heart development. [2.] Analysis of growth factor-mediated mechanisms which control MCK gene expression in cardiac muscle. Fibroblast growth factor response elements in the MCK gene will be identified and studied with respect to their cardiac muscle DNA-binding factors. [3.] Isolation and characterization of cardiac factors that bind MCK gene control elements. DNA-binding factors interacting with elements identified in Aims 1 & 2 will be characterized via cDNA library screening and analyzed for tissue specificity and developmental expression. [4.] Identification of cardiac muscle determination factors. Initial studies will develop cell culture assays for detecting cardiac muscle determination factors. These will be used to screen cDNAs of the factors identified in Aims 2 & 3. If this screen is negative, model precursor cell cultures and heart cells at various stages of development will be used to generate cDNA libraries that will be screened for cardiac determination factors. [5.] Identification of the mechanisms by which cardiac determination factors and their genes are regulated during embryonic development. Results from this project should be directly applicable to gene therapy for heart muscle diseases; e.g. highly active cardiac-specific MCK regulatory regions could be used to express proteins that were deficient in certain heart muscle disease states. If the goal of isolating cardiac muscle determination factors is realized, determination factor cDNAs could potentially be applied to reconstructive heart surgery; e.g. a patient's non-cardiac mesodermal cells could be transformed into heart muscle (with cardiac determination factors) and then used as homografts.
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