ANALYSIS OF SEQUENCE-SPECIFIC BINDING-ACTIVITY OF CIS-ELEMENTS IN HUMAN THYMIDINE KINASE GENE PROMOTER DURING G1 S PHASE-TRANSITION/

Expression of thymidine kinase (TK) gene in normal human diploid cells is both cell cycle and age dependent and appears to be transcriptiona lly regulated. Several studies have indicated that the G1/S control se quence may reside within the region of about 130 bp upstream of the tr anscription initiation site. We have previously shown that a trans-act ing factor, CBP/tk (CCAAT binding protein for TK gene), binds to eithe r one of the two inverted CCAAT boxes in a cell cycle- and age-depende nt manner (Pang and Chen, 1993, J. Biol. Chem., 268:2909-2916). An ups tream 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 ci s-elements in human TK promoter to the G1/S regulation, we have examin ed the binding activity of these cis-elements in the nuclear extracts derived from human IMR-90 cells at low passage number. Our results ind icated that no binding activity could be detected using either the 25 bp fragment (-109/-94) or the authentic Yi sequence. However, Yi bindi ng activity was observed in SV-40 transformed IMR-90 cells. In contras t, the 28 bp fragment (-91/-64) that contains the distal inverted CCAA T box exhibited a strong binding in serum-stimulated young IMR-90 cell s. The binding of CBP/tk to the 28 bp fragment was abolished by a sing le base mutation in the CCAAT box. The CBP/tk binding of the 28 bp fra gment could not be displaced by either the 25 bp fragment or the authe ntic Yi element. A deletion of the 5'-flanking region of the 28 bp fra gment up to 5 bases also abolished the binding activity. The CBP/tk bi nding in IMR-90 cells was supershifted by antiserum against NF-Ya, but not by antiserum made against p107, pRb, cyclin A, p33(cdk2) or p34(c dk2). Taken together, our results suggest that the G1/S regulatory cis -element in human TK promoter may be confined only to CBP/tk binding s ites. (C) 1995 Wiley-Liss, Inc.