) Transcriptional regulators control much of development by regulating cascades of gene expression. Since neoplasia is similar to a disrupted developmental process, it is not surprising that the vast majority of genetic lesions in neoplasia have been aberrant transcriptional regulators. Transcriptional regulators fall into families based on shared amino acid sequences in the DNA binding domains. One such group that has been shown to be important in both development and neoplasia is the winged helix family (formerly called HNF-3/Forkhead) of transcriptional regulators. We have cloned and characterized a novel member of this family, called Genesis, that is important in normal and malignant germ cell development. The hypothesis underlying this proposal is that Genesis regulates the fundamental decision of growth versus differentiation in normal or malignant embryonic stem cells. This proposal will investigate the role of Genesis in germ cell malignancies in four specific aims. First, the biochemistry of transcriptional regulation by Genesis will be investigated using GAL4 and heterologous promoter assays. Second, targets for Genesis interaction will be identified using the two hybrid system and genomic binding site selection. In addition, there are four promoters of transcriptional regulators (HNF-3 beta, Hox A4, Hox B4, and Hox-7) which have potential Genesis binding sites, and are expressed in a manner that indicates possible Genesis regulation. These will be analyzed using co-transfection assays. Third, the phenotypic changes regulated by Genesis in embryonal stem and carcinoma cells will be analyzed in gain-of-function or loss-of-function experiments. The effect of Genesis under-expression (homologous recombination knock-outs) or forced over-expression (retroviral transduction) will be studied. Fourth, since Genesis is located at chromosome 1p31, a region of consistent abnormalities in testicular carcinoma, the structure and expression of Genesis in germ cell neoplasia will be explored. Completion of these aims will provide insight into the function of Genesis in normal versus malignant embryonic stem cell development.
