Cloning and Characterization of the Human Trefoil Factor 3 Gene Promoter

Abstract

Human trefoil factor 3 (hTFF3) is a small-molecule peptide with potential medicinal value. Its main pharmacological function is to alleviate gastrointestinal mucosal injuries caused by various factors and promote the repair of damaged mucosa. However, how its transcription is regulated is not yet known. The aim of this study was to clone the hTFF3 gene promoter region, identify the core promoter and any transcription factors that bind to the promoter, and begin to clarify the regulation of its expression. The 5′ flanking sequence of the hTFF3 gene was cloned from human whole blood genomic DNA by PCR. Truncated promoter fragments with different were cloned and inserted into the pGL3-Basic vector to determine the position of the core hTFF3 promoter. Transcription element maintaining basic transcriptional activity was assessed by mutation techniques. Protein-DNA interactions were analyzed by chromatin immunoprecipitation (ChIP). RNA interference and gene over-expression were performed to assay the effect of transcription factor on the hTFF3 expression. The results showed that approximately 1,826 bp of the fragment upstream of hTFF3 was successfully amplified, and its core promoter region was determined to be from −300 bp to −280 bp through analysis of truncated mutants. Mutation analysis confirmed that the sequence required to maintain basic transcriptional activity was accurately positioned from −300 bp to −296 bp. Bioinformatic analysis indicated that this area contained a Sp1 binding site. Sp1 binding to the hTFF3 promoter was confirmed by ChIP experiments. Sp1 over-expression and interference experiments showed that Sp1 enhanced the transcriptional activity of the hTFF3 promoter and increased hTFF3 expression. This study demonstrated that Sp1 plays an important role in maintaining the transcription of hTFF3.

Hypomethylation associated enhanced transcription of trefoil factor-3 mediates tamoxifen-stimulated oncogenicity of ER+ endometrial carcinoma cells

“…Amongst its many targets, SP1 can also work together with ERα to regulate the expression of many E2-responsive genes, as reported in a study utilising BC cells [ 44 ]. A previous study of the human TFF3 promoter in colon cancer cells had also found that the transcription of TFF3 was precisely controlled by an SP1 binding site, corroborating the results obtained in our experiments [ 45 ]. Hence, there appears to be a combinatorial control of TFF3 expression by chronic tamoxifen exposure in EC cells; the hypomethylation of the TFF3 promoter to potentially increase availability of TF binding sites as well as the specific increase in phospho-c-JUN activity and SP1 expression that interact with the binding sites on TFF3 promoter and stimulate transcription of TFF3.…”

Epigenetic repression of Krüppel-like factor 4 through Dnmt1 contributes to EMT in renal fibrosis

“…ChIP assay was carried out as previously described ( 20 ). ChIP assays were performed using the ChIP-IT kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions.…”

ITF promotes migration of intestinal epithelial cells through crosstalk between the ERK and JAK/STAT3 pathways

“…The intestinal trefoil factor (ITF) is a low-molecular weight polypeptide reported to protect and repair the gastrointestinal mucosa through the maintenance of intestinal epithelial cell integrity and restoration of normal intestinal permeability 2 . ITF has a unique domain in which six cysteine residues in a sequence of 38 or 39 amino acid residues form three disulfide bonds.…”