The Transcription Activity Regulation Of Lipid Droplet-Associated Proteins LSDP5 By C/EBPα

Lipid droplets are intracellular storehouses of triglyceride, lipid droplets in animals also are known as the fat drops, called oil bodies in plants and micro-fat body in yeast. Mature lipid droplets contain a core of neutral lipid surrounded by a phospholipid monolayer and coated by many specific proteins named lipid droplet-associated proteins. Lipid droplet-associated proteins are involved in lipid metabolism, generation and transport and degradation. As an important material of lipid droplets, lipid droplet-associated proteins play a important role in function. There are a lot kinds of lipid droplet-associated proteins, in which proteins of PAT family are mainly in animals. LSDP5 is a newly discovered member of the PAT family.We predicted the C/EBPa binding elements on the sequences of LSDP5 promoters in pig. C/EBPa maybe interact with the promoters of LSDP5 to regulate the expression of LSDP5. C/EBPa is one of CCAAT/enhancer binding proteins family. They bind to sequences of DNA and regulate targeted genes transcription, therefore it's targeted genes can be regulated. In this study, we cloned the promoter sequences of swine LSDP5 and analysis the promoter activity by the dual luciferase reporter gene assay system and validated the binding of transcription factor to LSDP5 promoter by Chromatin immunoprecipitation. We determine the the effect of C/EBPa on the promoter and obtained the following results:1.The primers of LSDP5 promoters were designed according to published sequence of pig in NCBI. We obtained an 1900bp DNA sequences. Sequences of 1579bp LSDP5 promoters were loaded into pGL3-Basic vector. We detected the activity of promoters with the dual luciferase reporter gene assay system in NIH/3T3 cells line. The test results showed that the obtained sequences have significant activity of promoters. Promoters of LSDP5 were truncated in accordance with the different length. We found that there were the highest activity detected on the promoters from-642 to-176 bp position.2. The activity of this promoter regulated by different transcription factors was detected with the dual luciferase reporter gene detection system in NIH/3T3 cell lines. The results of this experiment showed that the activity of this promoter was significantly up regulated by C/EBPa compared with the control vector pcDNA3.1. Different length of LSDP5 promoters were co-transfected with C/EBPa in NIH cell lines. The activity of the promoters was detected with the dual luciferase reporter gene detection system. The results indicated activity of pGL3-LSDP5 (-642/+60bp) promoter was maximum compared with the control vector pcDNA3.1.3. The LSDP5 mRNA expression level of the IBRS2 cells in which C/EBPa was over expresssed was detected by real-time PCR. The data showed the LSDP5 mRNA level was increased.4. The activity of the predicted C/EBPa binding sites mutant promoter was significantly decreased in NIH/3T3 cell lines when co-transfected C/EBPa compared with the wild-type promoter in order to find C/EBPa binding sites. That suggested C/EBPa could bind to the promoter of LSDP5 through predicted binding sites to regulate activity of LSDP5 promoter.5. The direct interaction between C/EBPa and the promoter of LSDP5 was detected in IBRS2 cells by the ChIP experiment. The result confirmed that C/EBPa can bind to the region around -294bp sites of LSDP5 promoter to regulate LSDP5 gene expression.