Abstract
The Drosophila proliferating cell nuclear antigen (PCNA) gene promoter contains at least three transcriptional regulatory elements, the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. In nuclear extracts of Drosophila Kc cells, we detected a novel protein factor(s), CFDD (common regulatory factor for DNA replication and DREF genes) that appeared to recognize two unique nucleotide sequences (5'-CGATA and 5'-CAATCA) and bind to three sites in the PCNA gene promoter. These sites were located at positions -84 to -77 (site 1), -100 to -93 (site 2) and -134 to -127 (site 3) with respect to the transcription initiation sites. Sites 2 and 3 overlapped with DRE and URE, respectively, and the 5'-CGATA matched with the reported recognition sequence of BEAF-32 (boundary element-associated factor of 32 kDa). Detailed analyses of CFDD recognition sequences and experiments with specific antibodies to DREF (DRE-binding factor) and BEAF-32 suggest that CFDD is different from DREF, UREF (URE-binding factor) and BEAF-32. A UV cross-linking experiment revealed that polypeptides of approximately 76 kDa in the nuclear extract interact directly with the CFDD site 1 sequence. Transient expression assays of chloramphenicol acetyltransferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying mutations in CFDD site 1 and examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying mutations in site 1, introduced into flies by germ line transformation, revealed that CFDD site 1 plays an important role for the promoter activity both in cultured cells and in living flies. In addition to the PCNA gene, multiple CFDD sites were found in promoters of the DNA polymerase alpha and DREF genes, and CFDD binding to the DREF promoter was confirmed. Therefore, CFDD may play important roles in regulation of Drosophila DNA replication-related genes.
Highlights
The proliferating cell nuclear antigen (PCNA),1 an accessory protein of DNA polymerase ␦, is required for replication of
The earlier studies could not exclude the possibility that there is another important regulator; URE, upstream regulatory element; UREF, URE-binding factor; CFDD, common regulatory factor for DNA replication and DRE-binding factor (DREF) genes; BEAF-32, boundary element-associated factor of 32 kDa; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; binding sites (BTS), BEAF-32-binding site(s); GST, glutathione S-transferase; BrdUrd, 5Јbromo-2Ј-deoxyuridine; scs, special chromatin structure
In the work presented here, we identified a novel protein factor(s), CFDD that binds to the region between Ϫ87 and Ϫ62 of the PCNA gene promoter
Summary
Antibodies—Monoclonal antibodies to DREF, Mab-1 and Mab-4, were raised as described previously [15]. A polyclonal antibody that reacts with both BEAF-32A [23] and BEAF-32B [24] was purified from antiserum using E-Z-SEP (Pharmacia Biotech Inc.). Oligonucleotides—The sequences of double-stranded oligonucleotides containing DRE (DRE-P), a 2-base-substituted derivative (DREPM), or other derivatives in the PCNA promoter were as described earlier [25]. The sequences of double-stranded oligonucleotides containing E2F recognition sites 1 and 2 in the PCNA promoter (E2F-P) and E2F recognition sites in the adenovirus E2 gene (AdE2Fwt) were as reported previously [17]. The sequences of double-stranded oligonucleotides containing CFDD-binding sites or their derivatives in the PCNA promoter were defined as follows. Ϫ87/Ϫ62: gatccAGGCGATATCGCCTGTGGCTTTTCACa gTCCGCTATAGCGGACACCGAAAAGTGtctag mut⌬2(Ϫ81Ϫ82): cAGGCG–ATCGCCTGTGGCTTTTCACa gTCCGC–TAGCGGACACCGAAAAGTGt mut⌬6(Ϫ77Ϫ82): cAGGCG——CCTGTGGCTTTTCACa gTCCGC——GGACACCGAAAAGTGt mutIn8(Ϫ81): cAGGCGATcagatctgATCGCCTGTGGCTTTTCACa gTCCGCTAgtctagacTAGCGGACACCGAAAAGTGt
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