This research is supported by an NSF Career Advancement Award to Women. The work addresses a single aspect of one of the most compelling questions challenging molecular biologists today, that is, how the differentiation processes by which animals develop specialized organs from a single fertilized egg are regulated. One such process is that of muscle formation or myogenesis. During myogenesis, myogenic cells turn on expression of numerous genes whose projects are specific to and necessary for function of muscle. The turning on of muscle specific genes is accompanied by down-regulation of expression of a family of genes which code for proteins whose function appears to be to inhibit differentiation, the Id or Inhibitor of differentiation proteins. Id proteins are not unique to muscle cells and one hypothesis is that Id inhibits the differentiation of cells already commited to a specific developmental pathway until such time in embryogenesis as terminal differentiation is appropriate. In culture, when myoblasts or pre- muscle cells are treated with the drug doxorubicin, and are then placed in medium in which they would normally differentiate into myotubes or muscle cells, they do not differentiate. Instead, the cells produce abnormally high levels of Id mRNA and protein, and the genes which should be turned on during differentiation remain off. Genes are turned on and off by interactions between specialized regulatory proteins and specific DNA sequences in their promoter or control regions. These experiments are designed to determine and compare the sites of specific protein-DNA interactions in the promoter region of the Id gene in cells which are expressing Id at different levels, myoblasts, myotubes, and muscle cells affected by exposure to doxorubicin. "In vivo footprinting" will be used to determine where the regulatory proteins are bound. The nucleotide sequence of the promoter region will be determined, and the occupied sites will be compared to those already known to bind various tissue specific and ubiquitous regulatory proteins. In order for any biological developmental process to occur successfully, it must be carefully regulated. Molecular biologists have been successful at identifying a number of different regulatory proteins which control development. One such protein is called Id, and one of its functions appears to be to keep pre- muscle cells from becoming muscle tissue until exactly the right time in development. The results of this research will increase our appreciation for the intricacies of development, and in so doing may provide insight into some developmental disorders or diseases which occur when molecular controls fail.
