Study On The Function Of DCNP1

PART 1 DCNP1 regulates melatonin synthesis via binding to BMAL1 to control the transcription of NATAim:Dendritic cell nuclear protein-1(DCNP1)is dendritic cell nuclear protein-1(DCNP1)is first discovered in the mature or immature dendritic cells(DCs),and is expressed mainly in human brain and skeletal muscle.The function of DCNP1 is still largely unknown.In this research,we attempt to illuminate the the function of DCNP1 and its role in the regulation of the immune system by affecting melatonin synthesis.Methods:Melatonin levels in culture medium of C6 cells transfected with EGFP-DCNP1 or its mutants(1-116 and ARRK)were measured by ELISA.And then analysis the NAT mRNA and protein levels by Q-PCR and western blot.HEK293 cells transfected with EGFP-DCNP1 or its mutants(1-116 and ARRK)were collected for chromatin immunoprecipitation assays to investigate the binding ability to the NAT promoter.Overexpressing of EGFP-DCNP1 or its mutants(1-116 and ARRK)in HEK293 and immunoprecipitation assays were performed to identify the interaction between DCNP1 or its mutants and BMAL1.hnmunofluorescent staining was used to identify the subcellular localization of DCNP1 or its mutants and BMAL1.To identify the effect of DCNP1 on BMAL1 transcription activity,the luciferase reporter gene assay was performed.HEK293 cells were co-transfected with a NAT promoter construct of luciferase reporter or an E-box deleted construct,along with plasmids expressing EGFP or EGFP-DCNP1,followed by the detection of the luciferase activity.HEK293 cells were treated with siRNA of NC or si-BMAL1.Then cells were co-transfected with a NAT promoter construct of luciferase reporter,along with plasmids expressing EGFP,EGFP-DCNP1 or its mutants,and then analysis the luciferase activity.Results:ELISA assay suggested that an overexpression of EGFP-DCNP1 significantly decreased melatonin levels,whereas the mutants(1-116,?RRK)did not.The mRNA and protein levels of NAT were decreased in the C6 cells which were transfected with EGFP-DCNP1,whereas the mutants(1-116,?RRK)can not effect on the level of NAT.In the chromatin immunoprecipitation assay,the NAT promoter could be co-immunoprecipitated by EGFP-DCNPI,but not by its mutants(1-116,ARRK).Immunoprecipitation assays suggested that DCNP1,but not its mutants could interacts with BMAL1.DCNP1 and BMAL1 co-localization in nucleus were observed by fluorescence microscope.The reporter gene assay suggested that DCNP1 repressed NAT reporter activity,but it failed to influence NAT reporter activity without E-box.The full length of DCNP 1 repressed NAT reporter activity in the presence of BMAL1,however,it did not affect NAT reporter activity significantly in the absence of BMAL1.Conclusion:We report that the full length of DCNP1 down-regulates NAT via binding to BMAL1,and regulates the biosynthesis of melatonin.PART ? Study on regulation of neuroflammation by DCNP1Aim:To gain insight into the role and the mechanism of Dendritic cell nuclear protein-1(DCNP1)in LPS-induced neruoinflammation.Methods:BV2 cell were infected with the recombinant lentivirus vector expressing FLAG-DCNP1 or negative control.Then the cells were treated with lipopolysaccharides(LPS)100 ng/ml for 0,4,8 and 16 h.The protein levels of iNOS and COX2 in BV2 cells were detected by western blot.Concentration of IL-6 and TNF-? in the culture media were measured by ELISA assay when the cells were treated with LPS for 16 h.BV2 cells expressing FLAG-DCNP1 or negative control were treated with LPS for 6 h.And then the mRNA levels of inflammatory cytokines genes IL-6,TNF-?,iNOS and COX2 were analyzed by quantitative real-time PCR(qRT-PCR).BV2 cells expressing FLAG-DCNP1 or negative control were treated with LPS for 0 min,15 min,30 min or 1 h,then analysed for the nuclear and cytoplasmic fractions of p65 by immunoblotting.HEK293 cells were co-transfected with a NF-?B responsive promoter construct of luciferase reporter,along with plasmids expressing EGFP,EGFP-DCNP1,observe their effect on the luciferase activity.Overexpression of EGFP or EGFP-DCNP1 in HEK293 and immunoprecipitation assays were performed to identify the interaction between DCNP1 and p65.Results:The results showed relatively low levels of IL-6,iNOS and COX2 production after LPS treatment in FLAG-DCNP1 infected BV2 cells,compared with the control cells.Whereas the concentration of TNF-? had no significant difference between control and FLAG-DCNP1 overexpressed BV2 cells.LPS treatment in FLAG-DCNP1 BV2 cells led to significant decreased mRNA levels of IL-6,TNF-?,iNOS and COX2 compared with the negative control cells.The nuclear and cytoplasmic fractions analysis showed no significant difference in nuclear translocation of p65.The luciferase assay suggested that DCNP1 repressed NF-?B responsive promoter activity.Immunoprecipitation assays suggested that DCNP1 could interacts with p65.Conclusion:Overexpressing DCNPI constitutively inhibited LPS-induced neuroinflammation.This phenomenon may be due to the fact that DCNP1 interactes with p65 and represse NF-?B transcriptional activity.