Screening And Preliminary Functional Study Of Differential Proteins Binding To Sod2 Promoter In Endotoxemic Mice

Superoxide dismutases（SODs） are an ubiquitous family of metalloenzymes. SODs play vital roles in anti-senile,anti-oxidation,anti-inflammation,tumor resistance and autoimmune diseases.According to the distribution difference,three forms of SODs have been identified in eukaryotic cells:the cytoplasmic copper/zinc SOD（Cu/Zn-SOD,SOD1）,the mitochondrial manganese SOD（Mn-SOD,SOD2）, and the extracellular Cu/Zn-SOD（EC-SOD,SOD3）.They function to catalyze the dismutation of superoxide anion and are key antioxidant enzymes that eliminate oxygen radicals.SOD2 is mainly distributed in the mitochondria where energy and oxygen radicals are produced.SOD2 is the first and most important line of antioxidant enzyme against oxidative damages.In contrast to SOD1,which is expressed constitutively in most cases,SOD2 expression is highly regulated.SOD2 expression in eukaryotic cells is up-regulated dramatically by radiation,oxidants, cytokines,proinflammatory mediators.Sod2 gene encoding murine mitochondrial MnSOD protein is a single copy nuclear gene.No upstream TATA or CAAT box elements have been identified in the promoter of mouse Sod2 gene.However,GC-rich regions are present.Such features can be typical of "housekeeping" genes.Sod2 gene expression in eukaryotic cells is up-regulated rapidly by proinflammatory mediators including lipopolysaccharide （LPS）.The inducible expression of Sod2 gene protects the organism against the damage of oxygen free radicals.Research has shown that LPS can activate liver Kupffer cells and neutrophils and make them produce the respiratory burst with substantial release of superoxide anion radicals.Mitochondria exactly are an important place for respiratory burst.SOD2 is mainly distributed in the mitochondria,and functions to catalyze the dismutation of superoxide anion radicals to oxygen and hydrogen peroxide,which is eliminated by catalases,glutathione peroxidases or peroxiredoxins.So LPS-induced upregulation of Sod2 gene plays a vital role against oxygen free radials.Understanding its regulation mechanism is crucial to comprehending its role in cytoprotection.At present,the mechanism about Sod2 gene expression induced by LPS is not fully understood.Visner reported that the expression of Sod2 mRNA in pulmonary epithelia-like cells-L2 cells was up-regulated dramatically upon LPS treatment.This up-regulation was blocked completely by actinomycin D,suggesting that the increase in Sod2 mRNA in response to LPS may at least result from an increase in the rate of transcription of Sod2 gene.But at present,the specific regulatory mechanism about LPS-induced expression of Sod2 gene has not been clarified.Gene expression in eukaryotes is a complicated and strictly regulated process, and it can be regulated and controlled at different levels,such as replication, transcription activation,post-transcription modification,translation and post-translation modification.The transcription initiation is the basic control point of gene expression,which is actually the interaction between the promoter of specific gene and regulatory proteins with spatial and temporal specificity.In fact,many factors but not just a single one,are involved in the transcription initiation of a specific gene,and those factors would interact reciprocally to form a regulatory network.In order to better understand the involved mechanism,lots of strategies have been developed,one of which is to screening novel promoter-binding proteins that may function as transcriptional regulation factors.In order to identify novel proteins involved in transcriptional regulation of Sod2 gene and decipher the mechanism of LPS-induced expression of Sod2,based on DNA-protein interaction principal,we had screened differential proteins binding to the promoter of Sod2 gene in endotoxemic mice by biotin-streptavidin system,magnetic beads isolation technique.We made some researches as follows:1.Effects of LPS on the expression of Sod2 mRNA in mouse liver detected by RT-PCR.The expression of Sod2 mRNA in mouse liver was detected by reverse transcription polymerase chain reaction（RT-PCR） after LPS treatment.The results showed that Sod2 mRNA level in groups injected intraperitoneally with LPS at the dose of 10 mg/kg,20 mg/kg,30mg/kg significantly increasd compared with normal group（F=38.796,P＜0.01）.Compared with control group,the expression of Sod2 mRNA in groups injected intraperitoneally with LPS for 1 h,2 h,4 h,6 h was significantly augmented（F=40.758,P＜0.01）.The results indicated that LPS treatment induced a significant increase in the Sod2 mRNA level.This experiment provided a premise and basis for the following research and establishment of endotoxemic mouse model.2.Bioinformatics analysis of Sod2 promoter.The transcriptional start site of Sod2 gene was determined and the nucleotide sequence of-2000 bp to 100 bp around the transcriptional start site was obtained by Transcriptional Regulatory Element Database（http://rulai.cshl.edu/cgi-bin/TRED）. The nucleotide sequence was aligned with NCBI / BLAST and Ensembl database. The possible binding sites of transcription factors in Sod2 gene promoter were also predicted by Consite,a promoter analysis web system.The Sod2 promoter sequence of-1554 bp to+48 bp around the transcriptional start site was determined as the object of study.3.Cloning of Sod2 promoter and identification of its transcriptional activity.Genomic DNA was extracted from mouse liver,and the promoter sequence of Sod2（-1554～+48） was amplified by polymerase chain reaction（PCR）.The red fluorescent protein reporter gene vector driven by Sod2 promoter was constructed. After reporter gene plasmids were identified right by sequencing,the recombinant vectors were transiently transfected into murine embryonic fibroblasts（MEF） and mouse hepatoma cell line Hepa 1-6,then the expression of the red fluorescent protein in MEF or Hepa 1-6 cells was observed using Axiovert 200M Fluorescence/Live cell Imaging Microscope at rest or stimulated by NaAsO₂,LPS,by which activity of Sod2 promoter was detected.The results showed that the promoter of Sod2（-1554～+48） presented the transcriptional activity at rest,and the Sod2 expression had been enhanced after the cells were stimulated by inflammatory substance or oxidative stress.The reporter gene vector driven by Sod2 promoter will provide an experimental base and vector for following experiment.4.Screening of differential proteins binding to Sod2 promoter in endotoxemic mice.DNA probes of Sod2 promoter labeled with biotin were amplified by PCR. Nuclear extracts of endotoxemic mouse liver were prepared.DNA pull-down assay was performed by using a magnetic beads conjugated with streptavidin.Proteins binding with the beads were eluted by sample lysis buffer used for 2-D difference gel electrophoresis（2-D DIGE）,and the elution proteins were separated by 2-D DIGE. By DeCyder difference 2-D analysis software,twenty-nine differential protein spots had been identified.Twenty-two proteins were up-regulated,and seven proteins were down-regulated.5.Identification of the differential proteins binding to Sod2 promoter by mass spectrum.The differential proteins were further identified by matrix-assisted laser desorption ionization mass spectrometry（MALDI-TOF/TOF）.Twenty-four proteins were identified.Among them thirteen proteins have been reported to be involved in transcriptional regulation,such as Splicing factor,proline-and glutamine-rich（SFPQ）, Non-POU domain-containing octamer-binding protein（NONO）,Protein S100-AS, Protein S100-A9,Histone H4,Histone 2B,Heterogeneous nuclear ribonucleoprotein （hnRNP） L,hnRNP A2/B,hnRNP H2,Small nuclear ribonucleoprotein F（snRNP F）, RuvB-like 1,Pre-mRNA-processing factor 19 and Pterin-4-alpha-carbinolamine dehydratase 2.6.Bioinformatics analysis of differential proteins.The protein function and subcellular location annotations were from Swiss-Prot and TrEMBL protein database.In cases where Swiss-Prot annotations were not available,NCBI was searched for informations on the function and subcellular location of the novel proteins.Subcellular localization of differential proteins was also predicted by using subcellular localization prediction software WoLF PSORT （http://wolfpsort.org/）.The results showed that eleven proteins specifically localized in nuclei;four proteins were distributed in nuclei or other cell organelles;nine proteins were only located in other subcellular structures but not in nuclei.These results indicated that most of differential proteins were located in nuclei.At physiological or pathological conditions these proteins located in nuclei are more likely to participate in transcriptional regulation of Sod2 gene.String protein interaction database was also used to search primary interaction of the differential proteins.The results showed that two pairs of the proteins interacted reciprocally.The interaction proteins-S100A8 and S100A9,SFPQ and NONO were both up-regulated after the LPS administration,suggesting that these two pairs of proteins were probably involved in LPS-inducible transcriptional regulation of Sod2 gene in the form of protein complexes.7.Location of S100A8,S100A9,SFPQ,NONO in Hepa 1-6 cells deteced by immunofluorescence.In order to identify intracellular location of SFPQ,NONO,S100A9 and S100A8 proteins,we constructed eukaryotic expression vectors of SFPQ,NONO with HA-tag. The plasmids of pcDNA3-S100A8-HA,pcDNA3-S100A9-HA,pcDNA3-NONO-HA and pcDNA3-SFPQ-HA were respectively transiently transfected into Hepa 1-6 cells, then subcellular location of the proteins were detected by immunofluorescence.The results showed that S100A8,S100A9 were distributed in the cytoplasm and nuclei at rest.After the stimulus of LPS,S100A8 markedly translocated into nuclei,but the distribution of S100A9 in cells had no significant change.The differential distribution of S100A8 and S100A9 indicated they play a different role in gene expression induced by LPS.S100A8 probably participated in the expression regulation of Sod2 gene.SFPQ,NONO were located in the nuclei regardless of LPS-treatment.8.Effects of overexpression of SFPQ,NONO,S100A8,S100A9 on transcriptional expression of Sod2 gene detected by luciferase reporter gene assay.Firstly,We constructed luciferase reporter gene plasmid driven by Sod2 promoter-pGL3-Sod2.Plasmids of pGL3-Sod2 and pSV-β-Gal were transiently transfected into Hepa 1-6 cells,then the relative luciferase activity was detected.The relative luciferase activity in Hepa 1-6 cells transfected with pGL3-Sod2P plasmid signifinantly increased compared with negative control group transfected with pGL3-Basic plasmid（t=19.484,P＜0.01）.After the cells transfected with pGL3-Sod2 were stimulated by LPS,the relative luciferase activity signifinantly increased compared with normal group（t=5.561,P＜0.01）.The results showed that the luciferase reporter gene vector driven by Sod2 promoter presented the transcriptional activity,and the expression of Sod2 had been enhanced by LPS treatment.In order to determine whether NONO,SFPQ,S100A9,S100A8 play roles in transcriptional regulation of Sod2 gene,eukaryotic expression plasmids of pcDNA3 with HA-tag for S100AS,S100A9,NONO,SFPQ were respectively transiently transfected into Hepa 1-6 cells,and the pGL3-Sod2P and pSV-β-Gal were simultaneously cotransfected into cells.Then the relative luciferase activity was detected.The results showed that the relative luciferase activity in Hepa 1-6 cells transfected with different plasmids at rest had no difference compared with control group transfected with pcDNA3-HA plasmids（F=0.876,P＞0.05） which suggested that the overexpression of NONO,SFPQ,S100A9,S100A8 had no effects on the expression of Sod2 gene at rest.After the cells were stimulated by LPS,the relative luciferase activity of cells respectively transfected with pcDNA₃-S100AS-HA or pcDNAa-NONO-HA plus pcDNA3-SFPQ-HA plasmids was significantly increasd compared with control group transfected with pcDNA3-HA plasmid（P＜0.01）, suggestting that the overexpression of S100A8 or cooverexpression of NONO and SFPQ enhanced the expression of Sod2 gene induced by LPS.Taken together the above data,we draw the following conclusions:First,the promoter sequence of Sod2（-1554～+48） presented the transcriptional activity at rest,and the transcriptional expression of Sod2 had been enhanced after the cells were stimulated by inflammatory substance or oxidative stress. Second,twenty-four proteins in the liver nuclear extracts were found probably to be involved in the transcriptional regulation of Sod2 gene in endotoximic mice,such as SFPQ,NONO,S100-A8,S100-A9.Third,S100A8 and S100A9 were distributed in the cytoplasm and nuclei at rest. After the stimulus of LPS,S100A8 markedly translocated into the nuclei.SFPQ and NONO were distributed in the nuclei regardless of LPS-treatment.Forth,the overexpression of S100A8 proteins or cooverexpression of NONO and SFPQ significantly enhanced the LPS-induced Sod2 expression.In general,based on DNA-protein interaction principal,we had screened out the differential proteins binding to Sod2 promoter in endotoxemic mouse liver by biotin-streptavidin technology,which created a novel way to study the transcriptional regulation mechanisms of gene expression from the view of "transcriptional regulation omics".At the same time,this experiment combined biotin-streptavidin technology with DIGE and mass spectrometry technology,and realized the docking of research technique between transcriptional regulation study and the latest differential proteomics study.It provided a new approach to the successful efficient screening and identification of DNA binding proteins.Screening and identifying the differential proteins binding to Sod2 promoter in endotoxemic mice are important for understanding the LPS-inducible expression and regulation mechanism of Sod2 gene.