Abstract
Human DNA helicase B (HELB/HDHB) regulates DNA replication through association with human DNA polymerase α-primase. In the present study, an 866-base pair (bp) of the 5′-flanking region of the human HELB gene-containing Luciferase (Luc) reporter plasmid, pHDHB-Luc was transfected into various cell lines and Luc activity was analyzed. Deletion analyses revealed that a 121-bp containing the major transcription start site (TSS) was essential for the basal promoter activity in all tested cells. TF-SEARCH analysis indicated that GC-box/Sp1 and duplicated GGAA-motifs containing putative STAT-x and c-ETS binding sites are located close to the TSS. Furthermore, chromatin immunoprecipitation (ChIP) analysis showed that PU.1 and Sp1 bind to the 121-bp region. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analyses showed the HELB gene and protein expression was up-regulated by trans-Resveratrol (Rsv) treatment in HeLa S3 cells. Moreover, transfection experiment indicated that mutations on the GC-boxes and the duplicated GGAA-motif greatly reduced promoter activity and the response to Rsv in HeLa S3 cells. These results suggest that Rsv, which is a natural compound that has been found to elongate the lifespan of various organisms, regulates HELB promoter activity through co-operation of the GC-boxes and the duplicated GGAA-motif in the 121-bp.
Highlights
DNA helicases are DNA-binding proteins that have various functions such as unwinding DNA double-strands or changing chromatin structures
The primer AhDHB-68161was 5′ -end labeled with 32P and hybridized to total RNA isolated from HL-60, and Jurkat cells
Given that Luc activity was not observed in pGL4- HDHBδ 11-transfected cells, it can be assumed that the 74-bp region from − 33 to + 41 is insufficient for promoter function. These results suggest that the minimal core promoter of the human HELB gene is contained in the 252-bp region from −152 to + 100
Summary
DNA helicases are DNA-binding proteins that have various functions such as unwinding DNA double-strands or changing chromatin structures. A temperature-sensitive mutant defective in DNA replication from the mouse mammary carcinoma cell line showed diminished major DNA-dependent ATPase activity during incubation at the non-permissive temperature[4] This resulted from a mutation in the mouse Helb gene encoding a DNA homologous to a bacterial Rec D5. Increases in the HELB gene transcript levels and its encoding protein levels after Rsv treatment were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. Taken together, these data suggest that the GC-boxes and the duplicated GGAA motif in the 121-bp region play important roles in the regulation of HELB promoter activity in response to Rsv
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