Cell-cycle and Growth Regulation of the Murine Thymidine Kinase Promoter by Transcription Factors of the E2F-Family

E2F is a family of heterodimeric transcription factors which regulate the expression of genes, whose products are essential for progression through the mammalian cell cycle. Moreover, it integrates cell-cycle progression with transcription through its cyclical interactions with important growth and cell cycle regulators, such as the retinoblastoma-tumor-suppressor-gene product (pRB), cyclins and cyclin dependent kinases. The activity of the E2F transcription factors is regulated by a multitude of mechanisms. Most of these mechanisms seem to involve protein-protein interactions with cell cycle regulators like pRB or with other transcription factors. Deregulated expression of E2F-1 the first cloned member of the family can be oncogenic. Contrary to the expectations, inactivation of the E2F-1 gene in the mouse germline resulted in viable and fertile mice showing hyperplasia and neoplasia during aging. This indicates a high degree of redundancy among the E2F proteins. Studies dealing with the role of individual members of the E2F family were done mainly with mobility shift assays utilizing oligonucleotides comprising an isolated E2F site. A caveat with this experiments is that the environment for the binding reaction is rather different to the native situation. In the nucleus a transcription factor has to interact with nucleosomal organized DNA in the context of a promoter. This could mean that a mobility shift assay merely reflects the amount of individual E2F proteins and not the actual interaction with a given promoter. We have shown in an earlier study that binding and regulation of the murine thymidine kinase (TH) promoter by E2F depends not only on a functional E2F binding site but also on an adjacent GC box (binding proteins of the Sp- family) at a certain distance. This is mediated by protein-protein interaction of Sp1 (and possibly other Sp members) with E2F-1, -2, and -3 (but not E2F-4 and -5). Preliminary experiments indicate that the amino-terminal region of E2F-1, -2, and -3, which mediates the interaction with Sp1, is able to bind several other cellular factors. Interaction with these factors might define specific tasks of E2F-1, -2 or -3. Aim of this project is to determine what specifies the role of individual E2F proteins in the regulation of cellular promoters. The murine thymidine kinase promoter with its rather simple architecture will serve as a model system . Studying the effect of a factor binding in the vicinity of an E2F motif will be done by mobility shift assays. To find out which E2F proteins bind to a given promoter, a system will be established which allows immunoprecipitation of chromatin-bound transcription factors. E2F factors actually binding to the murine TK promoter will be ascertained by analyses of the co-precipitated DNA by southern blotting or PCR. To identify cellular proteins interacting with the aminoterminal regions of E2F-1, -2, or -3 these domains will be fused to the DNA binding domain of the GAL4 transcription factor and used for an interaction screen against a library of cDNAs fused to the GAL4 activation domain in a yeast two hybrid system. The role of these proteins in defining the regulation of E2F will be investigated.