Transcription factors（TFs）play critical roles in the development of the nervous system,but the transcriptional regulatory mechanisms of these genes are poorly understood.Here we analyzed 5-kb of the 5＇ flanking genomic DNA sequences of 41 TF genes involved in neuronal development.The results showed that the TF genes tend to have higher GC contents in the proximal region and most of the TF genes have at least one proximal GC-rich（GC content60%）promoter with a CpG island.The promoter distribution analysis showed that the GC-poor promoters were sporadically distributed within the 5-kb flanking genomic sequence（FGS）;however,more than half（37 of 70）of the GC-rich promoters were located in the proximal region between nucleotides—1 and—500.Luciferase assays showed that partial GC-rich promoters increased gene expression in SH-SY5Y cells and that CpG methylation repressed the promoter activity.This study suggests a potential general mechanism for regulation of TF expression.
LIM kinase 1 （LIMK1）, a cytosolic serine/threonine kinase, regulates actin filament dynamics and reorganization and is involved in neuronal development and brain function. Abnormal expression of LIMK1 is associated with several neurological disorders. In this study, we performed a conservation analysis using Vector NTI （8.0） software. The dualluciferase reporter assay and real-time quantitative RT-PCR were used to assess the protein and mRNA levels of the reporter gene, respectively. We found that a region ranging from nt ＋884 to ＋966 in the human LIMK1 3＇ untranslated region （UTR） was highly conserved in the mouse Limkl 3＇ UTR and formed a structure containing several loops and stems. Luciferase assay showed that the relative luciferase activity of the mutated construct with the conserved region deleted, pGL4-hLIMK1-3U-M, in SH-SY5Y and HEK-293 cells was only -60% of that of the wild-type construct pGL4-hLIMK1-3U, indicating that the conserved region is critical for the reporter gene expression. Real-time quantitative RT-PCR analysis demonstrated that the relative Luc2 mRNA levels in SH-SY5Y and HEK293 cells transfected with pGL4-hLIMK1-3U-M decreased to50% of that in cells transfected with pGL4-hLIMK1- 3U, suggesting an important role of the conserved region in maintaining Luc2 mRNA stability. Our study suggests that the conserved region in the LIMK1 3＇ UTR is involved in regulating LIMK1 expression at the post-transcriptional level, which may help reveal the mechanism underlying the regulation of LIMK1 expression in the central nervous system and explore the relationship between the 3＇-UTR mutant and neuroloqical disorders.
Guang-Fei DengShu-Jing LiuXun-Sha SunWei-Wen SunQi-Hua ZhaoWei-Ping LiaoYong-Hong YiYue-Sheng Long
