Although significant progress has been made in molecular understanding of skeletal muscle differentiation, little is known concerning genes involved in development of the cardiac muscle. Our long-term goal is to develop a novel expression cloning method or to utilize the newly developed """""""" interaction trap"""""""" method in order to identify such cardiac regulatory factors that may not be easily isolated by conventional methods. We first plan to develop a new method of expression cloning in mammalian cells in which expressed cDNA's can be isolated based on their ability to activate expression of reporter gene linked to a cis-acting enhancer sequence. This method may identify factors that do not directly bind to DNA, but act as tissue-specific cofactors that interact with and activate other tissue specific or ubiquitous transcription factors. To achieve this goal, we made a rat neonatal cardiocytes cDNA library in the eukaryotic expression vector containing the SV40 origin of replication, which allows the cDNA's to be expressed a very high levels in SV40 T- antigen transformed cells, such as COS7 cells. We then made two permanently transfected COS7 """"""""reporter"""""""" cell lines (COS-TNC and COS-CA) containing the mouse cardiac troponin C and mouse alpha-cardiac actin promoters linked to a beta-galactosidase reporter gene. Our second expression cloning strategy is to utilize the interaction trap method in yeast. We plan to isolate cDNA clones for proteins that interact with the MADS box-containing protein MEF-3 or the helix-loop- helix proteins E2-2 and Id. The interaction trap method utilizes expression of a """"""""bait"""""""" fusion protein (in this case, MEF-3, E2-2 or Id), in leu-host. A cardiac cDNA library will be made in a vector that allows expression of cDNA encoded proteins fused to an acidic transcription activation domain (""""""""prey"""""""") in yeast. When a cDNA encoded """"""""prey"""""""" is able to associate with a """"""""bait"""""""" fusion protein, the Leu 2 reporter gene is activated, which renders the leu host cells viable in a selection medium. We will use a new and improved version of the interaction trap method developed by Roger Brent. Once novel clones are isolated, we will determine the sequence and pattern of gene expression of theses new factors. We will also determine the mechanism of trans-activation of previously characterized cardiac muscle specific promoters by these factors using various assays including co-transfection, DNA binding, co-immunoprecipitation and site-directed mutagenesis. In addition, in order to examine the role these factors may play in control of the cardiac myogenic program, we will over-express the clone factor in mouse embryonic stem cells that are capable of differentiating into cardiac myocytes in vitro. If time permits, we will create ES cell lines that carry homozygous """"""""knock-out"""""""" of the factor genes and examine their capability to differentiate into cardiac myocytes in vitro. These studies should contribute to our knowledge of the specific mechanisms controlling cardiac myocyte differentiation. Furthermore, the method of expression cloning may have a general applicability for cloning other tissue-specific transcriptional co- factors.
