Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

Abstract

The regulation of Werner's syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5 upstream region (2.8 kb) of WRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides ؊67 to ؉160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRN promoter-luciferase reporter (WRN-Luc) plasmids that contained the 5-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.Werner's syndrome (WS) is a rare autosomal recessive genetic disorder causing symptoms of premature aging, such as gray hair, baldness, cataracts, and osteoporosis (9, 15, 23), accompanied by rare cancers (14). In vitro studies of fibroblast growth characteristics also suggest that WS may be related to normal aging: the life span of WS fibroblasts as expressed by population doubling levels is much shorter than that of normal fibroblasts (10, 32). The hypermutator phenotype, such as represented by genetic instability, also occurs frequently in WS fibroblasts and lymphoid cells (27,31,33,41).The gene responsible for WS (WRN) has been identified by positional cloning from the 8p11-p12 region (48) and is composed of a total of 35 exons (25, 49) that generate mRNA with 5,189 nucleotide residues. We have recently demonstrated that the gene product of WRN is an active RecQ-type DNA helicase by expressing WRN in insect cells, and we postulated that defective DNA metabolism is involved in a complex process of premature aging in WS patients (40). DNA helicases are enzymes that unwind the energetically stable double-stranded structure of DNA to provide the single-stranded template for important cellular processes such as replication, recombination, and repair (42).Mutations occurring in more than 100 WS patients have been extensively investigated by us and others. The more than 19 diff...

p130/E2F4 Binds to and Represses the cdc2 Promoter in Response to p53

“…The binding of p53 to the SP1 transcriptional activator blocks its ability to bind to promoters (77). This mechanism is responsible for the repression of the Werner helicase gene by p53 (78). Repression of the cyclin B1 promoter by p53 maps to a region between Ϫ287 and Ϫ123, suggesting that the effects of p53 on transcriptional activators bound to this region are involved (28).…”

Transcriptional Repression of Protein Kinase Cα via Sp1 by Wild Type p53 Is Involved in Inhibition of Multidrug Resistance 1 P-Glycoprotein Phosphorylation

“…Sp1 protein is a well known transcription factor that is involved in the control of transcription of many important genes (38). Transcriptional repression of other genes by p53 was shown via p53 binding to Sp1 that prevented Sp1 binding to the target promoter region (39,40). Also, p53 can form a heterocomplex with Sp1 and inhibit Sp1 activity in the TF-1 human erythroleukemia cell line (40).…”

“…Transcriptional repression of other genes by p53 was shown via p53 binding to Sp1 that prevented Sp1 binding to the target promoter region (39,40). Also, p53 can form a heterocomplex with Sp1 and inhibit Sp1 activity in the TF-1 human erythroleukemia cell line (40). Therefore, Sp1 binding site may be responsible for inhibition of PKC␣ by p53.…”

p53-Mediated apoptosis is attenuated in Werner syndrome cells

“…1B, lanes 2-4). The reduced binding to the R249S mutant is interesting, as this particular mutant was not capable of down-regulating the WRN promoter (Yamabe et al 1998).…”

“…The WRN promoter is regulated by two different tumor suppressor gene products. Overexpression of the Rb protein can up-regulate the WRN promoter, whereas overexpression of p53 can down-regulate WRN promoter activity (Yamabe et al 1998).…”