Analysis of sequence-specific binding activity of cis-elements in human thymidine kinase gene promoter during G1/S phase transition

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Abstract
Expression of thymidine kinase (TK) gene in normal human diploid, cells is both cell cycle and age dependent and appears to be transcriptionally regulated. Several studies have indicated that the G1/S control sequence may reside within the region of about 130 bp upstream of the transcription initiation site. We have previously shown that a trans-acting factor, CBP/tk (CCAAT binding protein for TK gene), binds to either one of the two inverted CCAAT boxes in a cell cycle- and age-dependent manner (Pang and Chen, 1993, J. Biol. Chem., 268:2909-2916). An upstream 25 bp fragment (-109/-84), containing both Yi-like and E2F-like binding sites, has recently been proposed to be essential for the G1/S regulation of human TK gene. To assess the contribution of various cis-elements in human TK promoter to the G1/S regulation, we have examined the binding activity of these cis-elements in the nuclear extracts derived from human IMR-90 cells at low passage number. Our results indicated that no binding activity could be detected using either the 25 bp fragment (-109/-94) or the authentic Yi sequence. However, Yi binding activity was observed in SV-40 transformed IMR-90 cells. In contrast, the 28 bp fragment (-91/-64) that contains the distal inverted CCAAT box exhibited a strong binding in serum-stimulated young IMR-90 cells. The binding of CBP/tk to the 28 bp fragment was abolished by a single base mutation in the CCAAT box. The CBP/tk binding of the 28 bp fragment could not be displaced by either the 25 bp fragment or the authentic Yi element. A deletion of the 5'-flanking region of the 28 bp fragment up to 5 bases also abolished the binding activity. The CBP/tk binding in IMR-90 cells was supershifted by antiserum against NF-Ya, but not by antiserum made against p107, pRb, cyclin A, p33cdk2, or p34cdc2. Taken together, our results suggest that the G1/S regulatory cis-element in human TK promoter may be confined only to CBP/tk binding sites.