A stage—specific protein factor binding to a CACCC motif in both human β—globin gene promoter and 5‘—HS2 region

The DNaseI hypersensitive site 2 (HS2) of human β-globin locus control region(LCR) is required for the high level expression of human β-globin genes.In the present study,a stage-specific protein factor (LPF-β) was identified in the nuclear extract prepared from mouse fetal liver at d 18 of gestation,which could bind to the HS2 region of human β-globin LCR.We also found that the shift band of LPF-β factor could be competed by human β-globin promoter.However,it couldn‘t be competed by human ε-globin promoter or by human ^Aγ-globin promoter.Furthermore,our data demonstrated that the binding-sequence of LPF-β factor is 5‘CACACCCTA 3‘,which is located at the HS2 region of β-LCR(from-10845 to-10853 bp)and human β-globin promoter(from-92 to -84 bp).We speculated that these regions containing the CACCC box in both the human β-globin promoter and HS2 might function as stage selector elements in the regulation of human β-globin switching and the LPF-β factor might be a stage-specific protein factor involved in the regulation of human β-globin gene expression.     

Studies on DNA—protein interactions in the upstream regulatory region of the human ε—globin gene promoter

The erythroid- and developmental stage-specific expression of the human ε-globin gene is controlled,in part,by the 5‘-flanking DNA sequence of this gene.In the present study,we have used DNA-protein binding assays to identify trans-acting factors which regulate the temporal expression of the human ε-globin gene during development.Using gel mobility shift assays and DNaseI footprinting assays,a nuclear protein factor (termed ε-SSF1) in the nuclear extracts from mouse haematopoietic tissues at d 11 and d 13 of gestation was identified.It could specifically bind to the positive control region (between-535 and -453bp) of the human ε-globin gene.We speculated that the ε-SSF1 might be an erythroid-and developmental stage-specific activator.In addition,we found another nuclear protein factor (terned ε-R1) in the nuclear extract from mouse fetal liver at d18 of gestation,which could strongly bind to the silencer region (between-392 and -177bp) of this gene.Therefore,we speculated that the ε-R1 might be an erythroid-and developmental stagespecific repressor.Our data suggest that both ε-SSF1 and ε-R1 might play important roles in developmental regulation of the human ε-globin gene expression during the early embryonic life.On the hand,we observed that the binding patterns of nuclear proteins from three cell lines (K562,HEL and Raji) to these regulatory regions were partially different.These results suggest that different trans-acting factors in K562,HEL and Raji cells might be responsible for activating or silencing the human ε-globin gene in three different cell lines.     

Activation of STAT3 stimulates AHSP expression in K562 cells

Studies on the chaperone proteinα-hemoglobin stabilizing protein(AHSP)reveal that abundant AHSP in erythroid cells enhance the cells’tolerance to oxidative stress imposed by excessα-hemoglobin in pathological conditions.However,the potential intracellular modulation of AHSP expression itself in response to oxidative stress is still unknown.The present study examined the effect and molecular mechanism of STAT3,an oxidative regulator,on the expression of AHSP.AHSP expression increased in K562 cells upon cytokine IL-6-induced STAT3 activation and decreased in STAT3 knock-down K562 cells.Regulation of AHSP in oxidative circumstance was then examined inα-globin-overloaded K562 cells,and real-time PCR showed strengthened expression of both AHSP and STAT3.ChIP analysis showed binding of STAT3 to AHSP promoter and binding was significantly augmented with IL6 stimulation and uponα-globin overexpression.Dual luciferase reporter assays of the wildtype and mutated SB3 element,an IL-6RE site,in the AHSP promoter in K562 cells highlighted the direct regulatory effect of STAT3 on AHSP gene.Finally,direct binding of STAT3 to SB3 site of AHSP promoter was confirmed with EMSA assays.Our work reveals an adaptive AHSP regulation mediated by the redox-sensitive STAT3 signaling pathway,and provides clues to the therapeutic strategy for AHSP enhancement.     

Treatment of hepatitis C virus core-positive hepatocytes with the transfer of recombinant caspase-3 using the 2~,5~-oligoadenylate synthetase gene promoter

Hepatitis C virus （HCV） infection is a leading cause of liver-related morbidity and mortality throughout the world. There is no vaccine available and current therapy is only partially effective. Since HCV infects only a minority of hepatocytes, we hypothesized that induction of apoptosis might be a promising approach for the treatment of hepatitis C. In the present study, recombinant caspase-3 gene （re-caspase-3） was used because it has the ability to induce apoptosis that is independent of the initiator caspases. An HCV-specific promoter is required to regulate the cytotoxic caspase-3 expression in HCV-infected cells. It has been reported that HCV core protein can specifically activate the 21,5~-oligoadenylate synthetase （OAS） gene promoter in human hepatocytes. Therefore, we constructed an expression vector consisting of the re-caspase-3 under the OAS gene promoter （pGL3-OAS-re-caspase-3） and then investigated its effect on HCV core-positive liver cells. It was found that the pGL3-OAS-re-caspase-3 construct induced apoptosis in HCV core-positive liver ceils, but not in normal liver ceils. These results strongly suggested that the transfer of the re-caspase-3 gene under the OAS promoter was a novel targeting approach for the treatment of HCV infection.