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.     

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.     

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     

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.     

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.     

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.     

Structural and functional analysis of an asymmetric bidirectional promoter in Arabidopsis thaliana

Bidirectional promoters are relatively abundant in eukaryotic genomes, suggesting that they have an important biological significance. As yet, few of these promoters have been characterized in detail. Here, using a promoter::GUS transgene approach has revealed that the intergenic region of Arabidopsis thaliana divergent genes At1g71850 and At1g71860 is an asymmetric bidirectional promoter, which exhibits an orientation-dependent expression profile. The strength of the forward promoter was greater than that of the reverse promoter, and their tissue specificities were not identical. Deletion analyses revealed that this bidirectional promoter could be divided into three functional regions. The basal level and tissue specificity of the promoter in the reverse orientation were regulated positively by region II and negatively by region III, whereas promoter activity in the forward orientation was regulated negatively by region II and positively by region I. Thus the 52-bp stretch of Research Artregion II had a dual function, enhancing expression in the reverse orientation and suppressing it in the forward orientation. These results demonstrated that the activity of the At1g71850-At1g71860 bidirectional promoter was modulated by complex interactions between both positive and negative cis-acting elements. These findings will enhance our understanding of the regulatory mechanisms of plant bidirectional promoters.     

Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization

Amyloid-β （Aβ） peptide, the primary constituent of senile plaques in Alzheimer＇s disease （AD）, is generated by β-secretase- and y-secretase-mediated sequential proteolysis of the amyloid precursor protein （APP）. The aspartic protease, β -site APP cleavage enzyme （BACE）, has been identified as the main β-secretase in brain but the regulation of its activity is largely unclear. Here, we demonstrate that both BACE activity and subsequent Aβ production are enhanced after stimulation of receptor tyrosine kinases （RTKs）, such as the receptors for epidermal growth factor （EGF） and nerve growth factor （NGF）, in cultured cells as well as in mouse hippocampus. Furthermore, stimulation of RTKs also induces BACE internalization into endosomes and Golgi apparatus. This enhancement of BACE activity and A β production upon RTK activation could be specifically inhibited by Src family kinase inhibitors and by depletion of endogenous c-Src with RNAi, and could be mimicked by over-expressed c-Src. Moreover, blockage of BACE internalization by a dominant negative form of Rab5 also abolished the enhancement of BACE activity and Aβ production, indicating the requirement of BACE internalization for the enhanced activity. Taken together, our study presents evidence that BACE activity and Aβ production are under the regulation of RTKs and this is achieved via RTK-stimulated BACE internalization, and suggests that an aberration of such regulation might contribute to pathogenic Aβ production.     

Analysis of the essential DNA region for OsEBP-89 promoter in response to methyl jasmonic acid

In rice, the characterization of OsEBP-89 is inducible by various stress- or hormone-stimuli, including ethylene, abscisic acid (ABA), jasmonate acid (JA), drought and cold. Here, we report the investigation of essential DNA region within OsEBP-89 promoter for methyl jasmonic acid (MeJA) induction. PLACE analysis indicates that this promoter sequence contains multiple potential elements in response to various stimuli. First, we fused this promoter with GUS gene and analyzed its expression under MeJA treatment through Agrobacterium infiltration mediating transient expression in tobacco leaves. Our results revealed that this chimeric gene could be inducible by MeJA in tobacco leaves. To further de- termine the crucial sequences responsible for MeJA induction, we generated a series of deletion pro- moters which were fused with GUS reporter gene respectively. The results of transient expression of GUS gene driven by these mutant promoters show that the essential region for MeJA induction is po- sitioned in the region between -1200 and -800 in OsEBP-89 promoter containing a G-box (?1127), which is distinct from the essential region containing ERE (?562) for ACC induction. In all, our finding is helpful in understanding the molecular mechanism of OsEBP-89 expression under different stimuli.     

nifH promoter activity is regulated by DNA supercoiling in Sinorhizobium meliloti

In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown ceils, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S.meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoiling-dependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.