Abstract
Microbiota of the large intestine synthesizes considerable amount of vitamin B1 (thiamine) in the form of thiamine pyrophosphate (TPP). We have recently demonstrated the existence of an efficient and specific carrier-mediated uptake process for TPP in human colonocytes, identified the TPP transporter (TPPT) involved (product of the SLC44A4 gene), and shown that expression of TPPT along the gastrointestinal (GI) tract is restricted to the colon. Our aim in this study was to determine the molecular basis of the colon-specific expression of TPPT focusing on a possible epigenetic mechanism. Our results showed that the CpG island predicted in the SLC44A4 promoter is non-methylated in the human colonic epithelial NCM460 cells, but is hyper-methylated in the human duodenal epithelial HuTu80 cells (as well as in the human retinal pigment epithelial ARPE19 cells). In the mouse (where TPPT expression in the GI tract is also restricted to the colon), the CpG island predicted in the Slc44a4 promoter is non-methylated in both the jejunum and colon, thus arguing against possible contribution of DNA methylation in the colon-specific expression of TPPT. A role for histone modifications in the tissue-specific pattern of Slc44a4 expression, however, was suggested by the findings that in mouse colon, histone H3 in the 5’-regulatory region of Slc44a4 is tri-methylated at lysine 4 and acetylated at lysine 9, whereas the tri-methylation at lysine 27 modification was negligible. In contrast, in the mouse jejunum, histone H3 is hyper-trimethylated at lysine 27 (repressor mark). Similarly, possible involvement of miRNA(s) in the tissue-specific expression of TPPT was also suggested by the findings that the 3’-UTR of SLC44A4 is targeted by specific miRNAs/RNA binding proteins in non-colonic, but not in colonic, epithelial cells. These studies show, for the first time, epigenetic mechanisms (histone modifications) play a role in determining the tissue-specific pattern of expression of TPPT in the GI tract.
Highlights
The water-soluble vitamin thiamine is essential for cellular metabolism mainly through of its role as a co-factor for multiple enzymes that catalyze oxidative energy metabolism, and of reducing cellular oxidative stress [1,2,3,4,5]
Our results showed that the high colon-specific expression of TPP transporter (TPPT) and the absence/negligible expression of this transporter in other parts of GI tract are associated with certain histone H3 modifications in its promoter region, while the gene regulation through promoter methylation appears to play no role
Our aim in this study was to establish the molecular basis of the colon-specific expression of the TPPT in the intestinal tract
Summary
The water-soluble vitamin thiamine (vitamin B1) is essential for cellular metabolism mainly through of its role as a co-factor (in the form of thiamin pyrophosphate/diphosphate) for multiple enzymes that catalyze oxidative energy metabolism, and of reducing cellular oxidative stress [1,2,3,4,5]. We have established the molecular identity of the colonic TPP transporter (TPPT) as the product of the SLC44A4 gene [14], and have further cloned and characterized its 5’-regulatory region and showed an important role for the ETS/ELF3 (E74-Like Factor 3 (Ets Domain Transcription Factor, Epithelial-Specific)) and CREB-1 (cAMP-responsive element-binding protein-1) transcriptional factors in regulating the basal activity of the SLC44A4 promoter [15]. We determined the DNA methylation and histone H3 modification profiles in the promoter regions of the human and mouse TPP transporter genes, and estimated the possible contribution of miRNAs towards the regulation of TPPT expression in the intestine. Our results showed that the high colon-specific expression of TPPT and the absence/negligible expression of this transporter in other parts of GI tract are associated with certain histone H3 modifications in its promoter region, while the gene regulation through promoter methylation appears to play no role. The results provide clear evidence that TPPT expression is not regulated by miRNAs in the colon, but has a potential to be down-regulated by miRNAs/RNA binding proteins in a cell-specific manner
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