Characterization of the 5′-flanking region of the human DNA helicase B (HELB) gene and its response to trans-Resveratrol

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.

Characterization of the human zinc finger nfx‑1‑type containing 1 encoding ZNFX1 gene and its response to 12‑O‑tetradecanoyl‑13‑acetate in HL‑60 cells

“…The supernatant was stored at -80˚C. The Luc assay was performed with a Luciferase Assay system (Promega Corporation), and relative Luc activities were normalized by the protein concentration and calculated as described previously (10,11).…”

“…Proteins were separated via 8/15% SDS-PAGE. Western blot analysis was carried out as previously described (10,11) with antibodies against ZNFX1 (Abcam, cat no. ab179452) and β-actin (Sigma-Aldrich; Merck KGaA, cat no.…”

“…RT-qPCR. RT-qPCR was carried out as described previously (9)(10)(11). Total RNAs were extracted by GeneElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), according to the manufacturer's protocols.…”

“…Surveillance of the human genomic DNA database revealed that duplicated GGAA (TTCC) motifs, which are frequently contained in the 5' flanking regions of the DNA repair, apoptosis, anti-viral response and mitochondrial function-associated factor encoding genes (6,7), are present in the 5' flanking regions of several ZNF family genes (8). Interestingly, the duplicated GGAA motif responds to interferon (IFN) β (9) and trans-resveratrol (10)(11)(12). In the ataxia telangiectasia and Rad3-related, xeroderma pigmentosum type B, and retinoblastoma (RB1) promoters, the motif notably responds to 12-O-tetra decanoylphorbol-13-acetate (TPA) in HL-60 cells (13).…”

Introductory Chapter: Gene Expression Controlling System and Its Application to Medical Sciences

“…Tannic acid, coumarins, and chalcones suppress 12-O-tetra decanoylphorbol-13-acetate (TPA)-induced HIV promoter activity in human Jurkat cells [53]. Rsv induces promoter activities of DNA-repair factor-encoding genes, including TP53, WRN, TERT, and HELB [54][55][56]. The GGAA-containing motif and the GC-box have been suggested to play an essential role in the response.…”