DNA sequences required for serum-responsive regulation of expression from the mouse thymidine kinase promoter.

We have used site-specific mutagenesis and thymidine kinase (TK) promoter/reporter gene transfection experiments to investigate DNA sequences required for serum-responsive regulation of expression from the mouse thymidine kinase promoter. Mutations were targeted to each of three previously described protein binding domains (MT1, MT2, and MT3) upstream of the TK translation initiation site, as well as to sequences within the TK first exon in order to address each of the following three questions: (a) Do these sequences play any role in regulation? (b) Do all of these sites play the same role? and (c) If any controls are observed, do they act positively or negatively on gene expression? The results of these experiments indicated that, in the wild-type TK promoter, at least some of these sequences do play a role in regulation, that not all of these sites appear to play the same role, and that some of the targeted elements act positively on gene expression, whereas others appear to act negatively. In particular, mutagenesis of the Sp1 site within MT1 virtually eliminated promoter function, whereas mutations in either the MT2 site or the TK first exon rendered reporter gene expression nearly constitutive with respect to serum. Thus, both MT2 and sequences within the TK first exon appear to contain negatively acting elements. In contrast, mutation or deletion of the MT3 site produced a much less pronounced effect on reporter gene regulation. These results support recent observations from our laboratory (Q-P. Dou et al., manuscript in preparation) indicating that although the protein complexes that bind to these various sites are similar, they are not identical.(ABSTRACT TRUNCATED AT 250 WORDS)

Thymidine kinase transcription is regulated at G1/S phase by a complex that contains retinoblastoma-like protein and a cdc2 kinase.

Transcription of the murine thymidine kinase gene, which is coregulated with the G1/S phase transition, is activated by changing the binding of protein complexes Yi1 and Yi2 to three upstream DNA motifs. Yi1 is replaced by Yi2 shortly before S phase. Yi1 contains a protein of 110 kDa that binds to the DNA motif sites and may be an underphosphorylated murine retinoblastoma protein, shown by its molecular mass, timing of its activity, and antibody recognition. An H1 kinase related to cdc2 cofractionates with both complexes. We propose that this kinase phosphorylates the murine retinoblastoma protein, releasing transcriptional inhibitions by Yi1 and permitting cell cycle progression. These results provide a cycle-related molecular target for such complexes. They are based on investigations of cycle control in uninfected cells. The Yi complexes are similar but not identical to complexes that include a cellular protein, E2F, that was originally found to bind to adenovirus DNA.