| FAM76B(Family with sequence similarity 76B),also known as MGC33371,its gene is located on human chromosome 11q21.The full length of FAM76B cDNA is-1020bp,encoding 339 amino acids,FAM76B protein is localized in the nucleus.Our previous studies confirmed that FAM76B could interact with PGRN by yeast two-hybrid experiments,but the specific biological function of FAM76B is still unclear.PGRN(Progranulin,granule protein precursor),the most important protein in the function associated with FAM76B,also known as GEP(Granulin-epithelinprecursor),proepithelin,PCDGF(PC cell derived growth factor).PGRN is the member of growing factor family,which is widely distributed and has variety of functions in the organization.PGRN involved in many physiological and pathological processes such as embryonic development,cartilage generation,neurological development,wound healing,inflammation and immune response,frontotemporal lobar degeneration(Frontotemporal Lobar Degeneration,FTLD),tumorigenesis and metastasis,but its biological function is not very clear.Therefore,the studying of FAM76B has important significance in elucidating the molecular mechanism of biological function for PGRN.The contents of this research include the following three aspects:1.The cloning of FAM76B promoters with different lengths and the testing of their activities.2.1kb in the upstream of FAM76B 5’-UTR region was cloned by seeking the information of FAN76B gene from NCBI.After testing its biological activity,three truncated promoters with the length of 1376bp,811bp and 395bp,respectively were obtained by the analysis of the promoter with full length using bioinformatics software.The four promoters with different length above were cloned into a reporter gene vector pGL3-Basic-mCherry and pGL3-Basic-Luciferase respectively,which were then transfected into HEK293 cells,respectively.Then,the transcriptional activity of the promoters was determined by the analysis of the expression of reporter gene mCherry or luciferase.2.The prediction of transcription factors binding to the promoter region of FAM76B and their functional analysis.The transcription factors having both an ability to bind to the promoter region of FAM76B promoter and a related function with FAM76B were selected by the bioinformatics analysis of the region of FAM76B promoter based on the variation of the activity of FAM76B promoter with different length.Then these transcription factors were cloned and ligated with the eukaryotic expression vector pAd5-E1-CMV shuttle,respectively.The expression vectors carrying the transcription factors were co-transfected into HEK293 cells with the FAM76B promoters with different length respectively,which could further verify the function of these factors by the analysis of the expression of reporter gene mCherry in FAM76B promoter expression vectors.3.Identification of transcription factors expression change between FAM76B knockout HEK293 cells and wild type HEK293 cells.The change of the transcription factors expression change between FAM76B knockout HEK293 cells and wild type HEK293 cells was detected by using real-time PCR.The results were obtained as follows through the research above1.Four FAM76B promoters with different lengths were successfully obtained and then cloned into an expression vector containing a reporter gene.The expression vectors obtained above then were transfected into HEK293 cells,and reporter gene expression was detected.The results showed that all four FAM76B promoters had transcriptional activity.The shorter promoter has highest activity.2.14 transcription factors related to the function of the FAM76B promoter were selected by the prediction and bioinformatic analysis of FAM76B promoter region,the names of 14 transcription factors were E2F1,AP-2alpha,USF1,C-MYB,SP1,c-Ets-1,API,.STAT5A,ATF1,RAR-alphal,SRF,PU.1,AREB6 and ATF1.Then full-length cDNAs of 14 cDNAs encoding transcription factors were cloned.The transcription factors were ligated with the eukaryotic expression vector,respectively.Then those eukaryotic expression vector were co-transfected into HEK293 cells with FAM76B promoters with different lengths,followed by the detection of reporter gene expression.The results showed that transcription factors AP1,c-Ets-1,SRF,PU.l,ATF1,STAT5A and USF1 could promote the transcriptional activity of FAM76B promoter;transcription factors E2F1 and C-MYB could inhibit the transcriptional activity of FAM76B.3.Using RT-PCR to detect the changes of the expression of functional cells,the transcription factors in wild type HEK293 cells and FAM76B knockout HEK293 function of transcription factors were further validated.The results suggested that the expression of transcription factors API,c-Ets-1,PU.1,ATF1,STAT5A,USF1 and E2F1 was upregulation in FAM76B knockout HEK293 cell lines,and the expression of transcription factors SRF and C-MYB was downregulation in FAM76B knockout HEK293 cell lines.In summary,FAM76B promoter activity was found to be decreased with increasing length of promoter within a defined region of FAM76B promoter in this study by the cloning of FAM76B promoter region and the functional analysis of FAM76B promoter with the different truncations.The related transcription factors that possibly regulate the activity of FAM76B were cloned and validated by the bioinformatics analysis of the regions of FAM76B.The studies above lay the foundation for elucidating the molecular mechanisms of the regulation of FAM76B expression and further studying on molecular mechanisms of the biological function involving of FAM76B and PGRN. |
