SCIRR69 Functions As A Mediator Of Transcription Regulation Of BDNF

Our previous researches have demonstrated that a cDNA(GenBank accession #AY546001)for gene SCIRR69(spinal cord injury regeneration related gene No.69)was temporally up-regulated in injured spinal cord.This gene encodes a protein(521aa),which is primarily located in the ER,homologous to the CREB/ATF family of transcription factors.In the present study,our data show that Chromatin Immunoprecipitation(IP)using antibodies specific to SCIRR69 pulled down a genomic DNA fragment from KCl treated primary neurons.PCR amplification using gene specific primers demonstrated that this fragment was contained in the region for BDNF promoterâ…¡,but not for other BDNF promoters or Bip promoter.Moreover,analysis of the luciferase activity in COS7 cells co-expressing SCIRR69 or its C-terminal truncations(â–³358-521,â–³369-521,â–³379-521) with a luciferase reporter gene under control of the BDNF promoterâ…¡(pGL3-basic BDNF promoter-luc)indicated that the highest enhancement on the reporter gene expression was observed with one of the truncated SCIRR69,â–³379-521.Highly elevated levels of BDNF expression were also observed in KCl treated PC12 cells over-expressing SCIRR69.In contrast,suppression of SCIRR69 expression in the same PC12 cells over expressing SCIRR69 down regulated KCl-induced BDNF expression.We sought next to identify the cis-acting DNA sequences that direct SCIRR69-dependent BDNF transcription.Toward this end,a series of reporter plasmids containing varying lengths of BDNF promoterâ…¡and exonâ…¡were fused to the firefly luciferase reporter gene.A mutation that replaced the 5'-GCACTTCA-3' sequence but left the rest of the BDNF promoterâ…¡intact effectively abolished the reporter gene induction by SCIRR69,suggesting that the 5'-GCACTTCA-3' sequence is the SCIRR69-responsive element(SRE).Furthmore,electrophoretic mobility shift assays(EMSA)were performed using a 56 bp fragment of biotin-labeled DNA corresponding to BDNF promoter sequence that includes the 5'-GCACTTCA-3' element and identified that SCIRR69 is a component of the protein complex that binds to the 5'-GCACTTCA-3' element.After PC12 cells were transfected with plasmid pcDNA-mutants of SCIRR69(FL); SCIRR69(1-378);SCIRR69(1-368);SCIRR69(1-357)and vector pcDNA3.1,the BDNF concentration in the transfected PC 12 cells was obviously up-regulated and the highest was the mutant of SCIRR69(1-378),which verify that mutant SCIRR69(1-378)can most potentially promote the expression of BDNF as a transcription factor.The cortex tissue pieces cultured for 12 hours with the conditional medium of PC12 cells transfected with SCIRR69(1-378)grow the densest and the longest of neuritis among all of groups,even compared with 12.5ng/ml BDNF.To identify whether cleavage of SCIRR69 occurs when treated with 55mM KCl, expressed SCIRR69 with N-terminal flag tag was immunoprecipitated by anti-flag antibodies after stimulation with 55mM KCl for 12h and immunoprecipitated proteins were Western blotted with anti-SCIRR69 antibodies.In addition to an 80kD band,another two bands(52KD,50KD)were detected,suggesting that SCIRR69 can be cleaved and may have the similar mechanism of cleavage to the same family members such as OASIS and ATF6.When construct pCMV-tag-SCIRR69(ARNLL)encoding SCIRR69 with deletion mutation at the S1P site was transfected into COS7 cells,S1P-cleaved SCIRR69 could not be detected after treatment with 55mM KCl for 12h.This indicates that SCIRR69 could be recognized at site RNLL by S1P in response to KCl.Substitution of Glutamine(396)causes S2P-cleaved SCIRR69 disappeared in respond to 55mM KCl stimulation,indicating that SCIRR69 can be cleaved by protease S2P at its Glutamine (396).EMSA demonstrated that cleavage of SCIRR69 by S2P determines its entry into nucleus and the released N terminus fragments of SCIRR69 function as transcription factor.