p53 Negatively Regulates cdc2 Transcription via the CCAAT-binding NF-Y Transcription Factor

Abstract

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We have reported previously that inducible expression of the p53 gene leads to the cell cycle arrest both at G 1 and G 2 /M in association with induction of p21 and reduction of mitotic cyclins (cyclin A and B) and cdc2 mRNA. In this study, we investigated the mechanism by which p53 regulates transcription of the cdc2 gene. Transient transfection analysis showed that wild type p53 represses whereas various dominant negative mutants of p53 increase cdc2 transcription. The cdc2 promoter activity is not repressed in cells transfected with a transactivation mutant, p53 22/23 . An adenovirus oncoprotein, E1B-55K inhibits the p53-mediated repression of the cdc2 promoter, while E1B-19K does not. Since the cdc2 promoter does not contain a TATA sequence, we performed deletion and point mutation analyses and identified the inverted CCAAT sequence located at ؊76 as a cis-acting element for the p53-mediated regulation. We found that a specific DNAprotein complex is formed at the CCAAT sequence and that this complex contains the NF-Y transcription factor. Consistently, a dominant negative mutant of the NF-YA subunit, NF-YAm29, decreases the cdc2 promoter, and p53 does not further decrease the promoter activity in the presence of NF-YAm29. These results suggest that p53 negatively regulates cdc2 transcription and that the NF-Y transcription factor is required for the p53-mediated regulation.Inactivation of p53 tumor suppressor gene occurs in over half of all human tumors, implying that loss of this gene represents a fundamentally important step in genomic instability and susceptibility to malignant transformation (1, 2). The underlying mechanism of tumor suppressor activity of p53 resides in part in its ability to bind DNA in a sequence-specific manner to activate gene transcription (2). It has been reported that a substantial number of genes containing the p53-binding site(s) are activated by p53. These include mdm2 (3, 4), p21/WAF-1 (5), Gadd45 (6), cyclin G (7), bax (8), and an insulin-like growth factor-binding protein (IGF-BP3) (9). p21 and Gadd45 were implicated in the p53-mediated cell cycle regulation (10, 11), while bax and IGF-BP3 were involved in the induction of apoptosis (8,9).In addition to playing a role as a DNA-binding dependent transcription activator, p53 has also been reported to negatively regulate the transcription of a number of genes. These genes include presenilin 1 (12), topoisomerase II␣ (13, 14), map4 (15), O 6 -methylguanine-DNA methyltransferase (16), insulin receptor (17), mdr-1 (18), hsp70 1 (19), interleukin-6 (20), bcl2 (21), c-fos (22), and other viral and cellular promoters (23). In contrast to the transcription activation by p53, no consensus sequence has been found in the promoters that are repressed by p53. It was initially reported that only the promoters containing a TATA box, but not those containing an initiator element, are repressed by p53 (24). This finding...

p53 and its homologues, p63 and p73, induce a replicative senescence through inactivation of NF-Y transcription factor

“…To examine whether p53 family proteins a ect to DNA binding activity of NF-Y, we performed a gel shift assay using CCAAT sequence that is found in the cdk1 promoter. NF-Y/DNA complex was detected as described previously (Yun et al, 1999). DNA binding activity of NF-Y was signi®cantly reduced by the expression of either p53, p63g, or p73b ( Figure 5).…”

“…In addition to transcription activation, p53 has been shown to repress transcription of certain genes (Mack et al, 1993;Seto et al, 1992). We previously reported that p53 represses transcription of cdk1 and cyclin B1, both of whose transcriptions are regulated in a cell cycle dependent manner and irreversibly repressed in senescent cells (Yun et al, 1999;Park et al, 2000). Figure 4 shows that mRNA levels of cdk1 and cyclin B1 were signi®cantly reduced in cells expressing the isoforms of p63 or p73.…”

“…Transcription of the cdk1 and cyclin B genes requires NF-Y transcription factor that binds to CCAAT sequences on the promoters (Yun et al, 1999;Bolognese et al, 1999). NF-Y consists of three di erent subunits, YA, YB and YC, all of which are needed to form a NF-Y/DNA complex (Sinha et al, 1995).…”

Gene expression of cyclin‐dependent kinase subunit Cks2 is repressed by the tumor suppressor p53 but not by the related proteins p63 or p73

“…One prominent example of a transactivated target gene is the cdk inhibitor p21 WAF1/CIP1 [6]. Other target genes such as several regulators of the G 2 /M checkpoint like cyclin B, cdc25C and cdk1 are repressed by p53 [7][8][9][10]. Many investigations concentrated on solving the mechanism of the p53-dependent repression without identifying one mechanism which would yield a general explanation for the regulatory details.…”

Repression of SHP-1 expression by p53 leads to trkA tyrosine phosphorylation and suppression of breast cancer cell proliferation

“…As control for activation and repression, pG13-lu (with 13 tandem wtp53-binding sites) and CDC2 promoter, cotransfected with wtp53, were used. wtp53 activated or repressed expression (el-Deiry et al, 1992;Yun et al, 1999) from pG13-lu or pCDC2-P-lu respectively ( Figure 1A1-2). Co-transfection of wtp53 with pM15-lu (with 15 tandem mutant p53-binding sites) or pGL2 showed very low activity ( Figure 1A1-2).…”