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.     

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.     

Trans—acting factors from the human fetal liver binding to the human ε—globin gene silencer

The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled,in part,by the silencer (-392bp- -177bp) upstream of this gene.In order to elucidate its role,the nuclear extract from the human fetal liver has been prepared and the interactions between trans-acting factors and this silencer element have been examined.By using DNaseI footprinting assay,a major protected region from -278bp to -235bp within this silencer element was identified.Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MV≈32,28,26 and 22kD) in the nuclear extract isolated from the human fetal liver,which could specifically bind to this region.Our results suggested that these trans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at the fetal stage through the interactions with this silencer.     

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.     

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     

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.     

Subcellular Localization Analysis of Bovine Foamy Virus Borfl Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.     

Characterization of transgenic mice expressing EGFP under the control of the monkey 20α-hydroxysteroid dehydrogenase promoter

To directly assess the molecular function of the monkey 20α-hydroxysteroid dehydrogenase(20α-HSD) promoter, we generated transgenic mice(tg) expressing enhanced green fluorescent protein(EGFP) under control of this promoter. We demonstrated that prostaglandin F2α induced 20α-HSD promoter activity in CHO cells in a dose-dependent manner. Furthermore, forskolin treatment markedly reduced 20α-HSD promoter activity, and prolactin exhibited weak inhibitory activity. The transgenic mouse obtained one positive founder male. The transgene was propagated in 10 successive generations without any notable defects to the progeny. EGFP and 20α-HSD in the tg mice were colocalized in the luteal cells of the ovary during late pregnancy. Strong EGFP and 20α-HSD protein signals were also detected in the adult testis. Immunohistochemical analysis revealed high EGFP levels in the seminiferous epithelium, whereas 20α-HSD was expressed in the seminiferous tubules. Our data suggest that the ovaries in monkey and mouse exhibit similar expression patterns of 20α-HSD during pregnancy. However, the expression pattern of EGFP in tg mice testis slightly differed from that of the endogenous 20α-HSD. Further investigation is required to elucidate the functional mechanisms underlying regulation of the monkey 20α-HSD promoter in the tg mice.     

A bacterial artificial chromosome transgenic mouse model for visualization of neurite growth

Class III β-tubulin(Tubb3) is a component of the microtubules in neurons and contributes to microtubule dynamics that are required for axon outgrowth and guidance during neuronal development. We here report a novel bacterial artificial chromosome(BAC) transgenic mouse line that expresses Class III β-tubulin fused to m Cherry, an improved monomeric red fluorescent protein, for the visualization of microtubules during neuronal development. A BAC containing Tubb3 gene was modified by insertion of m Cherry complementary DNA downstream of Tubb3 coding sequence via homologous recombination. m Cherry fusion protein was expressed in the nervous system and testis of the transgenic animal, and the fluorescent signal was observed in the neurons that located in the olfactory bulb, cerebral cortex, hippocampal formation, cerebellum, as well as the retina. Besides, Tubb3-m Cherry fusion protein mainly distributed in neurites and colocalized with endogenous Class III β-tubulin. The fusion protein labels Purkinje cell dendrites during cerebellar circuit formation. Therefore, this transgenic line might be a novel tool for scientific community to study neuronal development both in vitro and in vivo.     

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.     

Cloning of rat sp56,the homologue of mouse sperm ZP3 receptor—sp56

Mouse sp56 is considered as one of the candidates for mouse zona pellucida 3(mZP3)receptor,Up to date,its homologue has only been cloned from guinea pig,namely,AM67.Based on the cDNA sequence of mouse sp56,we designed a pair of primer to amplify its homologue from rat testis cDNA.Using RT-PCR, two tragments of 743 bp and 938 bp were amplified.The PCR products show very high homology to mouse sp56.However,the 743 bp product completely lacks one of the seven Sushi domains of mouse sp56.Using the 743 bp product as the probe to detect the expression profile of sp56 in rat tissues,Northern blot shows that a-2.0kb mRNA expresses specifically in testis.Employed the RACE method,two full cDNA sequences of rat sp56 were obtained.A Mr-42KD band was detected in denatured and non-reducing protein sample of rat testis and sperm with anti-mouse sp56 monoclonal antibody by Western blot method.Rat sp56 was localized on rat sperm head by the indirect immunofluorescence method.Rat sp56 immunoreactivity was detected from the early pachytene spermatocytes and throughout the spermatogenesis.Its cloning will further our understanding of the mechanism of the sperm-egg recognition and binding.