Detection Of Ap-1 Binding To Cyclind1 And Caspase3 Gene Promoter In A549 Cells With Chromatin Immunoprecipitation

Background and AimsLung cancer is the most common malignant tumor worldwide and its incidence and mortality have increased significantly in past decades,which has been severely threatening human health.The mechianism of lung cancer is that carcinogens can induce DNA damage on respiratory tract epithelial cells which change the expression of some correlational genes,and change cellular genetic information,then cells begin to transfer and become carcinogenesis.Ap-1(activating protein 1) is a kind of sequence specific transcription activation factor,being made up of Jun,Fos or AFT family members,participating in homodimers or heterodimers,which combined to the same spot of DNA-AP-1 combing spot.AP-1,especially those of Jun family,could regulate cell survival and death by regulating expression of cell cycle regulating factor,such as p53,p21,p19 and p16.Chromatin Immunoprecipitation(CHIP) is a widely used method to identify specific proteins associated with a region of the genome,or in reverse,to identify regions of the genome associated with specific proteins.These proteins can be isoforms of histones modified at a particular amino acid or other.Unlike other methods such as EMSA,DNA microarrays,and report gene assays which analyze direct interactions between protein and DNA in vitro,ChIP can detect that a specific protein binds to the specific sequences of a gene in living cells.Analysis by Immunohistochemical study shows that AP-1,CyclinD1 and caspase3 are overexpression in non-small cell lung cancer.So Speculate that AP-1 may be one of the transcription factors of CyclinD1 and caspase3,and it may be play an important role in non-small cell lung cancer's formation and development However,lack of direct evidence.In this study,AP-1 binding to CyclinD1 and caspase3 gene promoter region were examined by Chromatin immunoprecipitation(CHIP) assay using c-jun specific antibody,The immunoprecipitated DNA fragments were amplified through PCR, using the specific CyclinD1 and caspase3 primers.To detect and verify AP-1 binding to CyclinD1 and caspase3 gene promoter region in A549 cell lines in vivo.Materials and methods1 A549 cell was used in this study.2 Chromatin immunoprecipitation(CHIP)2.1 In Vivo Crosslinking and LysisAdd 270μl of 37%formaldehyde to 10ml of growth media to crosslink and gently swirl dish to mix,Incubate at room temperature for 10 minutes.Add 1ml of 10×Glycine to each dish to quench unreacted formaldehyde,Swirl to mix and incubate at room temperature for 5 minutes,Add 10ml of cold 1×PBS to wash cells. Scrape cells from each dish into a microfuge tube,Spin at 2800rpm at 4℃for 3 minutes to pellet cells.Remove supematant,Resuspend cell pellet in 1ml of SDS Lysis Buffer,Aliquot between 330μl per microfuge tube.2.2 Sonication to Shear DNAOptimal conditions required for shearing crosslinked DNA to 200～1000 base pairs in length need to be determined. 2.3 Agarose gel analysis of the sheared DNA.2.4 "Preclear" the chromatinAdd 60μl of Protein G Agarose for each IP,Incubate for 1 hour at 4℃with rotation,Pellet agarose by brief centrifugation(6500rpm 1 minute).Collect the supematant by aliquoting 1ml into fresh microfuge tubes.2.5 Immunoprecipitation(IP) of Crosslinked Protein/DNAAdd the immunoprecipitating antibody to the supernatant fraction:add 4.0μg of antibody(the positive control,anti-RNA Polymerase,the negative control, Normal Mouse IgG and c-jun antibody) per tube.Incubate overnight at 4℃with rotation,Add 60μl of Protein G Agarose for 1 hour at 4℃with rotation,Pellet Protein G Agarose by brief centrifugation(6500rpm 1 minute) and remove the supernatant fraction,Wash the Protein G Agarose-antibody/chromatin complex.2.6 Elution of Protein/DNA ComplexesAdd 100μl of Elution Buffer to each tube containing the antibody/agarose complex.Mix by flicking tube gently,Incubate at room temperature for 15 minutes Pellet agarose by brief centrifugation(6500rpm 1 minute) and collect supernatant into new microfuge tubes.Repeat again and combine eluates(total volume=200μl).2.7 Reverse Crosslinks of Protein/DNA Complexes to Free DNATo all tubes(IPs and Inputs) add 8μl 5 M NaCl and incubate at 65℃overnight to reverse the DNA/Protein crosslinks.Then add 1μl of RNase A and incubate for 30 min at 37℃.Add 4μl 0.5M EDTA,8μl 1M Tris-HCl and 1μl Proteinase K and incubate at 45℃for 2 hours.2.8 DNA Purification Using Spin Columns3 PCR AnalysisResultsThe CyclinD1 gene specific fragment(218bp) were detected in the DNA fragment immunoprecipitated by c-jun antibody,but the caspase3 gene specific fragment(150bp) were not detected in the DNA fragment immunoprecipitated by c-jun antibody.Conclusions1 AP-1 binds to CyclinD1 gene promoter region in A549 cells,and regulates its expression.Direct detective that AP-1 may be one of the transcription factors of CyelinD1.and it may be play an important role in non-small cell lung cancer's Formation and development.2 AP-1 does not bind to caspase3 gene promoter region in A549 cells,and does not regulate its expression.