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.     

The 5‘—flanking cis—acting elements of the human ε—globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5‘-flanking cisacting elements of the human ε-globin gene have been examined.Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII,-446bp- -419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells,indicating that ε-PREII may be an erythroidspecific facultative MAR.In gel mobility shift assay and Southwestern blotting assay,an erythroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (ε-PREII).Furthermore,we demonstrated that the silencer (-392bp- -177bp) upstream of the human ε-globin gene could associate with the nuclear matrices from K562,HEL and Raji cells.In addition,the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer,suggesting that this silencer element might be a constitutive nuclear matrix attachment region(constitutive MAR).Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human ε-globin gene expression.     

Identification of the development stage—specific factors in mouse fetal liver binding to the human β—globin gene promoter

In order to elucidate the molecular mechanisms of globin gene expression during embryonic development,the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared.By using DNase I footprinting and gel mobility shift assays,the binding of protein factors in these extracts to the human β-globin promoter was analyzed.The differences in the binding patterns of protein factors during development were observed.An erythroid-specific and stage-specific nuclear protein in the nuclear extrace from d 18 mouse fetal liver was identified,which can bind to the sequence(from-66bp to-90bp) of human β-globin promoter.We therefore speculate that the function of this cis-acting element may be similar to stage selector element(SSE) in chicken β^A-promoter.     

Proteins binding to the 5‘—flanking regualtory elements of the human β—globin gene

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5‘-flanking regulatory elements of human β-globin gene,two negative control regions(NCR1,-610to-490 bp;NCR2,-338,to-233bp),was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5‘-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in art for the silence of β-glolbin gene in the embryonic and fetal stages.     

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.     

Proteins binding to the 5''''-flanking regulatory elements of the human β-globin gene~1

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human globin gene, two negative control regions(NCR1, -610 to -490 bp; NCR2,-338 to -233bp), was identified. Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2. These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of globin gene and NCR2 might be responsible in part for the silence of globin gene in the embryonic and fetal stages.     

The interaction between the human β-globin locus control region and nuclear matrix

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express humanβ-globin gene. However the molecular mechanisms by which the expression of β-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681- -10971 bp , HS3 core sequence -14991- -14716 bp and HS4 core sequence -18586- -18306 bp) of DNase I hypersensitive sites in the human β-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of humanβ-like globin genes through their interaction with HSs (HS2,HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cel     

Characterization of 5‘—proximal sequence of mouse GABA transporter gene （GAT—1）

The cDNA molecule encoding the mouse GABA transporter gene(GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library.A positive clone,harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G,was fished out from the library,the 5‘ proximal region and intron 1 were sequenced and analysed,and low homology was found in the above region between GAT-1 genes from mouse and human except some short conserved sequences.The DNA-protein interactions between DNA fragments containing the conserved sequences in the 5‘ proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shift assay,and Southern-Western blot.The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.     

cDNA cloning and function analysis of two novel erythroid differentiation related genes

Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen λ-gtll human cDNA expression library of fetal liver in order to obtain the relevant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' functions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a prote