DDB is highly abundant in HeLa cells and binds to multiple forms of DNA damage with high affinity in vitro
, but the question remains as to what purpose this strong DNA damage recognition serves. Moreover, are there independent roles for the individual subunits? In this report we have investigated the basal control of the two DDB genes, and striking similarities and differences in their transcription regulation have been observed. The regions directly upstream of the DDB1 and DDB2 transcription initiation sites are TATA-less and G/C-rich and they each contain important Sp1 and NF-1 elements. These are common characteristics of promoters of housekeeping genes and cell cycle-regulated human promoters (36
Sp1 is a well-investigated factor that regulates transcription through specific sequences in G/C-rich promoter regions and is often critical for transcription initiation of TATA-less promoters (39
). In mammalian cells, promoters lacking TATA boxes generally have several Sp1 elements (40
). Sp1 is a regulator of the cell cycle in G1
). Four Sp1 sequences were identified in the promoter of DDB2 and mutation of Sp1#1 (–29 to –22), which is closest to the transcription initiation site, most drastically reduced transcription activity to 17%. Mutations of the other three Sp1 elements resulted in more modest decreases of activity to 63–80%. Double Sp1 mutations that included Sp1#1 did not decrease activity below that observed with Sp1#1 alone. These findings, combined with the fact that 5′ deletion of the core promoter to –11 produced 15% promoter activity, indicated that Sp1#1 is a critical basal transcription factor element in the DDB2 gene.
In the DDB1 promoter, Sp1 elements were also found to be essential for maximal activity, but less important compared to their role in DDB2 transcriptional activation. Individual mutations of the Sp1 consensus sequences, Sp1#2 (–143 to –135) and Sp1#1 (–123 to –115), reduced promoter activity to 67 and 54%, respectively. A double Sp1 mutation did not reduce transcription below that found for Sp1#1 alone. Similar to the Sp1 elements in DDB2, mutation of the Sp1 site in closest proximity to the initiation site resulted in a greater effect on promoter activity.
The myc family of proto-oncogenes, which includes mainly c-myc, N-myc and L-myc, are central mediators of cell proliferation (42
). In DDB1 the N-myc site (–56 to –51) was identified as the critical promoter element. Mutation of this site drastically reduced activity to 23%. A double mutation with Sp1#1 resulted in a further reduction to 11%, not quite accounting for the loss of activity to 5% with 5′ deletion of the core promoter to –37.
An E2F element was also identified downstream of the putative transcription initiation site in DDB2. E2F elements are often only functional when they are proximal to the transcription initiation site (44
). Mutation of this E2F consensus sequence decreased promoter activity to 74%. A double mutation of Sp1#1 and E2F did not result in any significant further reduction over that seen with Sp1#1 alone. The E2F1–E2F6 family are transcription factors that form a heterodimer complex with DP1–DP3. Overexpression of E2F1–E2F3 activates many proteins which are regulated by cell cycle progression (45
). Sp1 and E2F1 have been observed to form a functional complex in cell cycle-regulated transcription (46
). It also has been demonstrated that DDB2 protein alone can interact with E2F1, though both subunits of DDB are required to stimulate E2F1-activated transcription (23
). Since E2F-controlled transcription appears to be a tightly regulated system, it is feasible that the activation of DDB2 would be regulated by E2F, thereby determining the number of DDB–E2F1 complexes present in the cell.
The mutation (+36 to +39) of the E2F consensus site also deleted a putative p53 element, REhDDB2 (+8 to +38), reported by Tan and Chu (48
). While the search engines that we used did not recognize this p53 site, our laboratory has identified a second putative p53 site. However, no effect of a p53 element would have been detected in the system that we employed to evaluate DDB2 promoter activity because, while HeLa cells do produce p53 mRNA, p53 protein levels are undetectable. HPV18 present in HeLa cells produces active E6 that forms a complex with the ubiquitin ligase, E6AP, and together they target p53 for degradation by proteosome mechanisms (49
). With regard to the putative p53-binding site in the DDB2 promoter which is reported to be active in human cells but not mouse cells, we do not believe that its presence or absence from human cells would affect the results of this study that concerns the basal levels of DDB2 under promoter control. Contrary to misconceptions present in the literature, mouse cells contain the same basal level of DDB activity as do human cells (50
The original intent of this undertaking was to explore the regulation of basal transcription of the DDB genes. Essentially, both the DDB1 and DDB2 promoters are typical of cell cycle-regulated and housekeeping genes: TATA-less and G/C-rich, with active Sp1 and NF-1 elements. In addition, DDB1 contains a critical proto-oncogene N-myc element, again implying that DDB1 is regulated during progression through the cell cycle. Possibly most interesting was the finding that DDB2 has an important E2F-binding site, suggesting a tightly controlled regulation of the number of DDB–E2F1 complexes present in the cell. It has been proposed that in undamaged cells the association of DDB with E2F1 stimulates E2F1 transcription activation of DNA replication genes and progression from G1
to S phase (23
). However, after UV-irradiation DDB2 could be bound to damaged DNA, decreasing the number of DDB complexes formed, resulting in cell cycle arrest at the G1
/S checkpoint. In any event, the apparent regulation of both DDB1 and DDB2 in a cell cycle-dependent manner lends credence to a role for these proteins aside from NER.