Analysis Of Cis-acting Elements In Human MAZ Gene Promoter By EMSA

Objective:MAZ is widely involved in regulation of many genes, which modulate cellular biological and pathological processing, such as matrix metalloproteinases(MMPs) and serum amyloid protein A.These genes are related with rheumatoid arthritis, atherosclerosis,heart disease, cancer and other diseases. Although a pile of papers regarding to regulation of downstream gene expression by MAZ, there are few papers to describe the modulation of expression of MAZ transcript. Considering it is so important transcription factor further probe the regulation of MAZ by its promoter is necessary to reveal the molecular mechanism of inflammation and some other diseases. This paper aims to find the potential cis-elements by analyzing transcription factor binding sites on MAZ gene promoter, and to study these cis-elements by Electrophoretic Mobility Shift Assay(EMSA). We also used mutation probe and competition oligo to identifiy the specific binding protein.Methods:Extraction of nuclear proteins: culture THP-1 cells, extract nuclear proteins and then perform BCA protein assay. Design probes and end-label them with biotin: analysis the potential transcription factor binding sites of 948 bp DNA sequence in MAZ promoter region by PROMO, then design probes and end-label them. EMSA test: labeled probes and nuclear proteins are incubated in gel shift binding buffer, followed by electrophoretic analysis and chemical luminescence. The design of mutant probes and EMSA analysis.Competitive EMSA analysis with a specific transcription factor binding element.Results:There are more than 40 putative transcription factors by PROMO software. Eleven of the 32 probes appeared DNA-protein binding bands. Mutation and competition EMSA results showed that Elk-1 might binding to MAZ proximal promoter, which located in-525~-504 of the transcription start site of MAZ gene. Further identification needs to be done.Conclusion:This study detected that Elk-1 binding cis-element in the promoter of MAZ gene by PROMO software. We initially probe this Elk-1 binding by EMSA. Although we do not have the super-shift evidence to identify the Elk-1 binding to the MAZ promoter. The experiment provided a basis for further study.