A compelling question is how animals develop specialized organs from a single fertilized egg. One facet of this process is the formation of muscle or myogenesis. When myogenesis is duplicated in cell culture, cells turn on numerous genes whose expression is required for muscle function. One such gene is the skeletal muscle actin gene. Genes are turned on and off by interactions between specialized regulatory proteins and specific DNA sequences in their promoter or control regions. Our experiments will help determine specific protein-DNA interactions in the promoter region of the skeletal muscle actin gene which are important in establishing the different levels of actin gene expression observed among cells which have been programmed in different ways to become muscle. We will transfer into different muscle cells a reporter gene driven by the skeletal muscle actin gene promoter and establish at what level the reporter gene is expressed. We will next compare results using the same reporter but with defined deletions in the actin promoter region. We will also use "in vivo footprinting" to determine more specifically where regulatory proteins are bound to the actin promoter in the DNA of the different cell types. The results will provide pieces to the puzzles of gene regulation and development. %%% A compelling question is how multicellular animals such as mice and humans develop specialized organs from a single fertilized egg. We know that every cell, regardless of its location or function in the body, contains for the most part the same set of instructions as every other cell, but that different kinds of cells follow different subsets of these instructions. These instructions are encoded as genes in DNA. This project is designed to help determine how muscle cells know which instructions to follow. The facet of the overall developmental process which results in formation of muscle is called myogenesis. Some steps in myogenesis can be duplicated with mouse pre-muscle cell lines cultured in the laboratory. During myogenesis in culture, cells turn on numerous genes which code for proteins whose expression is required for muscle function. One such gene is the skeletal muscle actin gene. This gene is expressed at different levels in different muscle cell types. While it is known that the activity of genes is regulated by interactions between specialized regulatory proteins and specific control regions in the DNA, the details of these interactions which are important in establishing different levels of expression of the skeletal muscle actin gene are not completely known. Our experiments will use a combination of genetic engineering and biochemical techniques to help determine these specific protein-DNA interactions and how they might differ among cells which have been programmed in different ways to become muscle. The results will provide pieces to the puzzles of gene regulation and development. Solution of this puzzle is intrinsically attractive because of the undeniable elegance of development. Beyond aesthetics, however, understanding the basic science of molecular mechanisms of muscle gene expression and development may one day lead to applications in understanding and managing muscle system disorders which are related to failures in regulated gene expression.
