Regulation Mechanism Of Human SNF2L Gene Expression

ISWI is a member of the SWI2/SNF2 superfamily of ATP-dependent chromatin remodelers,which are defined by the presence of SANT and SLIDE domains in addition to highly conserved ATPase domains.The ISWI proteins are involved in multiple nuclear functions,including transcriptional regulation,replication,and chromatin assembly.The ISWI gene was originally identified in Drosophila,and later shown to be highly conserved in a number of species from yeast to humans.Drosophila Iswi is the ATP-hydrolyzing component of three separate protein complexes:the nucleosome-remodeling factor(NURF),ATP-utilizing chromatin-assembly and -remodeling factor(ACF)and the chromatin-accessibility complex(CHRAC).The NURF complex has a role in transcriptional regulation,whereas the ACF and CHRAC complexes are involved in nucleosome assembly and spacing during replication.Mammalian genomes encode two ISWI orthlogs,SNF2H and SNF2L.Similarly, the mammalian ISWI complexes can be divided broadly into two functional categories.The complexes containing Snf2h as the ATP-dependent motor mediate nucleosome assembly and spacing during replication,whereas the complexes containing Snf21 have gene-specific effects on transcription during terminal differentiation. Consistent with their distinct and separate functions,developmental expression studies have shown their differential expression pattern.In mouse,Snf2h is expressed ubiquitously,whereas Snf2l expression is restricted to the central nervous system and gonadal tissue.Unlike mouse Snf2l,human SNF2L is expressed ubiquitously,and regulation is mediated by isoform variation.In human,there are two isoform SNF2L, SNF2L and SNF2L+13.SNF2L+13 isoform lacks both ATPase and chromatin remodeling activities.The inactive human SNF2L+13 isoform is predominant in non-neuronal tissues,whereas the active human SNF2L isoform is expressed in neurons.Moreover,two proteins also exhibit temporal differences in their expression. Snf2h is the predominant ISWI in progenitor cells,whereas Snf21 is found at greater levels in differentiated tissues.Although much attention has been focused on functional aspects of the SNF2L protein,the promoter elements and transacting factors directly involved in SNF2L gene transcription have not been identified.The purpose of this study is to determine and characterize the promoter of the human SNF2L gene.PART ONEIdentification and Characterization of human SNF2L PromoterAs the first step to understand the mechanism of regulation,we focused on the region near transcription start site of human SNF2L gene.To isolate the human SNF2L promoter we performed a BLAST search of human genome sequence using the human SNF2L cDNA(GenBank Accession No.NM₀03069)as a query.The 5' sequence of the SNF2L cDNA perfectly matched to a genomic sequence(GenBank Accession No.NW₉27721).Computer-based analysis of this region with Matlnspector software failed to find any canonical TATA box or CAAT-like sequence.However,several potential transcription factor binding motifs were identified in the promoter region between -761 and +1.These include NF-κB,Sp1, MZF1,E2F,and CRE.This result was used as the reference for the design of luciferase reporter vectors. To determine the promoter activity of the proximal region,we prepared the luciferase construct(P-761)containing the region from -761 to +94.Since brain is one of the tissues in which the active SNF2L gene is preferentially expressed,two neuronal cell lines(PC12 cells and C6 cells)and one non-neuronal cell line(HeLa) were used to evaluate the promoter activities.After transient transfection into three different cell types,increased luciferase activity was observed,suggesting that the region between -761 to +94 confers promoter activity.In order to localize the core promoter sequence,six progressive 5' deletion constructs were generated,and were designated as P-650,P-486,P-310,P-185,P-72, and P-58 based on their variable 5' end.These constructs were cotransfected with pRL-TK into HeLa,PC I2 and C6 cells,and luciferase activities were determined.The maximal promoter activity was observed with the construct P-185,and no significant difference in the reporter gene expression levels was observed among three cell types, suggesting that cell specific element may not be present in those sequences.Then,the relative luciferase activities of other constructs were compared to that of pGL3-185 which was set as the 100%.Only background level of luciferase activity was observed with constructs P-72 and P-58.These results indicate that the fragment between -185 and -72 contains cis-elements that are essential to drive high level transcription of human SNF2L gene.When the sequence length increased up to -761,a 50%to 60% reduction in luciferase activities was observed,suggesting the presence of a negative regulatory element between -761 and - 185.To further pinpoint the sequence important for proximal SNF2L promoter activity, three additional deletion constructs(P-152,P-116,P-86)were then generated to truncate different potential transcription factor binding sites between position -185 and -72.Each construct and pRL-TK were cotransfected into HeLa,C6 and PC12 cells,and the relative luciferase activities were compared to that of pGL3-185 construct which was set as the 100%.These deletion experiments suggest that the SNF2L promoter activity detected in diverse cell lines is mostly attributable to the positive regulatory elements located in the regions,-152 to -116 and -116 to -86. PART TWOSp1 regulates basal transcription of the human SNF2L geneThe reporter studies mentioned above had demonstrated a significant reduction in luciferase activity after deletion of nucleotides from -152 to -116.We therefore inspected the sequences of this region and identified a Spl binding site within this region.Spl is a well-characterized transcription factor that binds to the Spl consensus element present in a variety of cellular and viral promoters and is involved in the expression of many genes,including structural proteins,metabolic enzymes,cell cycle regulators,transcription factors,growth factors and signaling receptors.In TATA-less promoters,Sp I often regulates constitutive promoter activity.First,deletion from -152 to -116 removed Spl binding site,and led to a 40% reduction in luciferase activity,suggesting that Sp1 binding site is essencial for the activity of the human SNF2L transcription;Second,to examine whether this putative Spl binding site mediates the transcriptional activity of SNF2L promoter,point mutations were introduced into P-152 construct which changed the sequence of Sp1 consensus core sequence from GGGGGG to TCCTAG and generated Spl mutant construct(Spl-Mut).The luciferase activity driven by mutant Spl construct(Spl-Mut)was reduced to a range as observed in the construct that lacks Spl binding site,indicating that the Sp1 binding site between -145 and -135 of the human SNF2L promoter contributes to the basal activity of the human SNF2L transcription;Third,to test whether endogenous Spl could function as a transcriptional activator of the human SNF2L promoter,we assessed the effects of Spl knockdown via RNA interference.We stably transfected HeLa cells which express high level of Spl protein with SpI-siRNA construct(pS-Spl)and control vector(pSN).Treatment of HeLa cells with siRNA against Spl led to a significant reduction in the Spl protein as demonstrated by Western blot analysis.Then,we transfected HeLa-derived pS-Spl and pSN cells with P-152 construct which contains Spl binding site,In response to decreasing amounts of Spl,the reporter gene activity was decreased by about 50%; Fourth,to further confirm Spl site was essential for the basal promoter activity, we next conducted EMSA to examine the binding of endogenous Spl to this Spl binding site.The retarded mobility of the oligonucleotides was observed in the presence of nuclear extracts.The DNA-protein complex was significantly competed by 10- or 100-fold molar excess of unlabeled wild-type oligonucleotides,but not by the same amount of mutant Spl oligonucleotides.These results indicate that the Spl element in this region specifically binds with endogenous Spl protein.Taken together,our deletion and mutation analysis,RNA interference and gel shift assays demonstrated that the Spl binding site located between -145 and -135 contributes to the basal activity of the human SNF2L promoter.PART THREECREB regulates basal transcription of the human SNF2L geneAs shown in partâ… ,deletion of nucleotides from -116 to -86 led to another drop in luciferase activity.This deletion removed a putative cAMP-responsive element (CRE)consisting of the sequence TGACGTAG at position -99 to -92.A CRE site binds members of the CREB/ATF family of transcription factors,and behaves as a constitutive and/or inducible element in regulating the transcription of target genes.First,to examine whether this CRE site is conserved among different species,we have compared the genomic sequences between -117 and +1 in human,mouse and rat. We found that this region is highly conserved with more than 90%homology among different species;and particularly,the relative location and sequence of the CRE is exactly identical in these species,further suggesting that this CRE is functionally important for the transcriptional control of the SNF2L gene;Second,deletion from -116 to -86 removed CRE site,and led to a significant reduction in luciferase activity,suggesting that CRE site is essencial for the activity of the human SNF2L transcription;Third,to determine whether this CRE is critical for constitutive promoter activity of human SNF2L gene,CRE-Mut was constructed by site-directed mutagenesis, which contains a mutant CRE by changing the sequence TGACGTAG(the wild type CRE)in P-116 to CTCTGTAG.The luciferase activities driven by the native and mutated constructs were determined in HeLa,C6 and PC12 cells.Compared to the activities of P-116 in three cell lines,the mutation of CRE site(CRE-Mut)reduced promoter activity by 80%,70%and 60%in each respective cell line.The promoter activity of this mutant construct was not significantly different from that of P-86 construct which lacks CRE.These results show that this site is critical for basal SNF2L promoter activity in the cell lines tested;Fourth,to confirm that CREB were involved in the regulation of SNF2L gene expression,we assessed the effects of CREB knockdown via RNA interference.We stably transfected HeLa cells which express high level of CREB protein with CREB-miRNA construct and control vector(miRNA-neg).Treatment of HeLa cells with miRNA against CREB led to a significant reduction in the CREB protein as demonstrated by Western blot analysis.To directly evaluate the effect of CREB status on SNF2L promoter,we transfected HeLa-derived CREB-miRNA and miRNA-neg cells with P-116 construct which contains CRE site.Luciferase assays revealed that knockdown of endogenous CREB resulted in a nearly 70%reduction of the promoter activity;Fifth,to test the ability of the CRE site to interact with endogenous CREB family protein,nuclear extracts from HeLa cells were prepared and EMSA were performed. The specificity of this complex for wild type probe was shown by competition assay, in which the binding was competed away by a 10- or 100-fold excess of unlabeled wild type probe,but not by either a 10- or 100-fold excess of unlabeled mutant probe. These results indicate the CRE site in this region specifically binds with CREB transcription factor;Sixth,to test the effects of agents that activate protein kinases on SNF2L gene expression,foskolin and dbcAMP were used.Transient expression of a luciferase fusion gene under the control of the SNF2L promoter region is increased by foskolin or dbcAMP in PC12 cells and HeLa cells,but not in C6 cells.Unexpectedly,treatment with foskolin or dbcAMP was not found to enhance endogenous SNF2L mRNA levels in PC12 cells and NuTul9 cells.Taken together,our deletion and mutation analysis demonstrated that the CRE site in the proximal region of human SNF2L promoter is responsible for the constitutive transcription.Our RNA interference and gel shift assays demonstrated that CREB is at least one protein from CREB/ATF family that stimulates SNF2L promoter activity.These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation but no regulation by cAMP in neural cells.PART FOURAnalysis of negative regulatory elements upstream of human SNF2L basal promoterDeletion from -761 to -185 caused a significant reduction in luciferase activity, suggesting the presence of negative regulatory elements located between -761 to -185 of human SNF2L gene.Using Matlnspector software,we identified several putative negative transcription factor binding sites within this region,including YYI,POZ/ZF, Blimp-1,E4BP4,NRSE and En-1.First,deletion from -486 to -185 led to a nearly 20%reduction in luciferase activity.Inspection of this region revealed that a YYI site was located within this region.Then YY1-Mut construct was generated with P-486 construct,and luciferase activity was determined.Compared to P-486,no significant effect was observed with mutated construct,indicating that the YY1 site in this region is not critical for the negative regulation of human SNF2L gene expression;Second,to identify the negative elements between -486 to -185,the deletion construct P-396 was generated.The luciferase activity of P-396 construct is slightly lower than that of P-486.However,compared to P-185,a significant reduction was observed with P-396,suggesting that the negative elements are more likely to be located between -396 and-185; Third,Software analysis revealed that two candidate negative elements,NRSE (neural-restrictive silencer element)and En-1(Homeobox protein engrailed)binding sites,were located between -396 to -185.NRSE-Mut and En-l-Mut constructs were then generated with P-396,and luciferase activities of these constructs were determined.Unexpectedly,mutation of En-1 site did not result in significant change when compared to P-396 construct.Point mutation in the core sequence of NRSE caused a significant reduction in luciferase activity.These results indicated that the two elements tested cound not play a key role in negative regulation mechanism of human SNF2L gene;Fourth,there were three candidate negative elements located between -761 and -486,including POZ/ZF,Blimp-1 and E4BP4.Point mutations were induced to change the core sequence of these binding sites,and the luciferase activities were detected.Unexpectedly,mutation of these binding sites led to a reduction,instead of increase,in luciferase activities,indicating that these three elements are not responsible for the human SNF2L gene transcription individually.Although the exact negative elements were not identified,5' deletion analysis revealed that the presence of some negative functional elements between -761 to -185 of human SNF2L gene,and further study will be needed to characterize these elements.