The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp~-177bp) upstream of thisgene. In order to elucidate its role, the nuclear ex...The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp~-177bp) upstream of thisgene. In order to elucidate its role, the nuclear extractfrom the human fetal liver has been prepared and the interactions between trans-acting factors and this silencerelement have been examined. By using DNasel footprinting assay, a major protected region from -278bp to -235bpwithin 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 (MW ≈ 32, 28, 26 and 22kD) in the nuclear extractisolated from the human fetal liver, which could specifically bind to this region. Our results suggested that thesetrans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at thefetal stage through the interactions with this silencer.展开更多
The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has ...The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-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 thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.展开更多
The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5’ flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar ...The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5’ flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-кB consensus sequence resides in the negative regulatory element (-3028bp~ -2902bp, termed ε-NRAII) 5’ to the cap site of this gene. NRF DNA fragment (-3010bp~ -2986bp) containing the NF-кB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-кB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-кB motif) could not, suggesting that the binding protein is a member of NF-кB/Rel family. Western blot assay demonstrated that the molecular weight of NF-кB protein in the nuclei of K562 cells is 50ku. We suggested that NF-кB p50 may play an important role in the regulation of human c-globin gene expression.展开更多
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 assay...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 DNasel 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 E-SSF1 might be an erythroid- and developmental stage-specific activator. In addition, we found another nuclear protein factor (termed ε-R1) in the nuclear extract from mouse fetal liver at d 18 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 other 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.展开更多
The human ε-globin gene is expressed in a tissue-specific and developmental stage-specific manner. During the earliest stage of gestation, this gene is expressed in the yolksac, but is silenced completely at the 6th-...The human ε-globin gene is expressed in a tissue-specific and developmental stage-specific manner. During the earliest stage of gestation, this gene is expressed in the yolksac, but is silenced completely at the 6th-8th weeks of gestation. Recently, several studieson the transgenic mice have shown that the 5′-flanking DNA sequences of human展开更多
High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the prese...High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the human e-globin gene promoter (e-promo-ter, -177-+1 bp) has been examined by using both the in vitro nucleosome reconstitution and the electrophoresis mobility shift assay (EMSA). We found that HMG1/2 proteins could bind to the naked e-promoter DNA, however, HMG14/17 could not. Using the in vitro nucleosome reconstitution, we revealed that HMG14/17 could bind to the mononucleosome reconstituted in vitro with E-promoter, while HMG1/2 could not. Those results indicate that the binding of HMG proteins to e-promoter is dynamic as the nucleosome assembling and disassembling. We speculated that this selective binding of HMG proteins to e-promoter might play a critical role in the regulation of e-globin gene expression.展开更多
文摘The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp~-177bp) upstream of thisgene. In order to elucidate its role, the nuclear extractfrom the human fetal liver has been prepared and the interactions between trans-acting factors and this silencerelement have been examined. By using DNasel footprinting assay, a major protected region from -278bp to -235bpwithin 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 (MW ≈ 32, 28, 26 and 22kD) in the nuclear extractisolated from the human fetal liver, which could specifically bind to this region. Our results suggested that thesetrans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at thefetal stage through the interactions with this silencer.
文摘The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-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 thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.
基金This work was supported by National Natural Sciences Foun-dation of China, No. 39893320 and No. 39870378.
文摘The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5’ flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-кB consensus sequence resides in the negative regulatory element (-3028bp~ -2902bp, termed ε-NRAII) 5’ to the cap site of this gene. NRF DNA fragment (-3010bp~ -2986bp) containing the NF-кB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-кB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-кB motif) could not, suggesting that the binding protein is a member of NF-кB/Rel family. Western blot assay demonstrated that the molecular weight of NF-кB protein in the nuclei of K562 cells is 50ku. We suggested that NF-кB p50 may play an important role in the regulation of human c-globin gene expression.
文摘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 DNasel 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 E-SSF1 might be an erythroid- and developmental stage-specific activator. In addition, we found another nuclear protein factor (termed ε-R1) in the nuclear extract from mouse fetal liver at d 18 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 other 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.
基金Project supported by grants from Shanghai Joint Laboratory of Life Sciences, the Chinese Academy of Sciences, and the National Natural Science Foundation of China.
文摘The human ε-globin gene is expressed in a tissue-specific and developmental stage-specific manner. During the earliest stage of gestation, this gene is expressed in the yolksac, but is silenced completely at the 6th-8th weeks of gestation. Recently, several studieson the transgenic mice have shown that the 5′-flanking DNA sequences of human
基金This work was supported by the National Natural Science Foundation of China (Grant No. 39893320)the Foundation of Chinese Academy of Sciences (Grant No. KJ 951 -A 1 -603 (2)).
文摘High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the human e-globin gene promoter (e-promo-ter, -177-+1 bp) has been examined by using both the in vitro nucleosome reconstitution and the electrophoresis mobility shift assay (EMSA). We found that HMG1/2 proteins could bind to the naked e-promoter DNA, however, HMG14/17 could not. Using the in vitro nucleosome reconstitution, we revealed that HMG14/17 could bind to the mononucleosome reconstituted in vitro with E-promoter, while HMG1/2 could not. Those results indicate that the binding of HMG proteins to e-promoter is dynamic as the nucleosome assembling and disassembling. We speculated that this selective binding of HMG proteins to e-promoter might play a critical role in the regulation of e-globin gene expression.