Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells

Abstract

Microbiota of the large intestine synthesize considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). There is a specific high-affinity regulated carrier-mediated uptake system for TPP in human colonocytes (product of the SLC44A4 gene). The mechanisms of regulation of SLC44A4 gene expression are currently unknown. In this study, we characterized the SLC44A4 minimal promoter region and identified transcription factors important for basal promoter activity in colonic epithelial cells. The 5'-regulatory region of the SLC44A4 gene (1,022 bp) was cloned and showed promoter activity upon transient transfection into human colonic epithelial NCM460 cells. With the use of a series of 5'- and 3'-deletion luciferase reporter constructs, the minimal genomic region that required basal transcription of the SLC44A4 gene expression was mapped between nucleotides -178 and +88 (using the distal transcriptional start site as +1). Mutational analysis performed on putative cis-regulatory elements established the involvement of ETS/ELF3 [E26 transformation-specific sequence (ETS) proteins], cAMP-responsive element (CRE), and SP1/GC-box sequence motifs in basal SLC44A4 promoter activity. By means of EMSA, binding of ELF3 and CRE-binding protein-1 (CREB-1) transcription factors to the SLC44A4 minimal promoter was shown. Contribution of CREB into SLC44A4 promoter activity was confirmed using NCM460 cells overexpressing CREB. We also found high expression of ELF3 and CREB-1 in colonic (NCM460) compared with noncolonic (ARPE19) cells, suggesting their possible contribution to colon-specific pattern of SLC44A4 expression. This study represents the first characterization of the SLC44A4 promoter and reports the importance of both ELF3 and CREB-1 transcription factors in the maintenance of basal promoter activity in colonic epithelial cells.

Keywords: SLC44A4; colonocytes; promoter; thiamine pyrophosphate; transcription regulation.

Fig. 1.

Activity of the SLC44A4 promoter in NCM460 cells and transient transfection assays with deletion constructs. The 5′- and 3′-deletions were generated for the cloned 5′-regulatory region of the SLC44A4 (−829/+193) as described in materials and methods. Reporter plasmids were transiently transfected into NCM460 cells for luciferase assays. Data are reported as relative firefly luciferase activity normalized to Renilla luciferase activity. Each value represents the means ± SE of at least 3 independent experiments, each performed in triplicate.

Fig. 2.

Schematic diagrams depicting the SLC44A4 gene structure (A) and the genomic sequence of the SLC44A4 minimal promoter region with location of the putative cis-regulatory elements (B). Exon-intron structure of the gene is presented. Location of multiple transcriptional start sitea (TSSs) identified by 5′-rapid amplification of the cDNA ends (5′-RACE) in NCM460 cells and ATG site for transcript variant 1 are indicated by arrows. The minimal promoter sequence (−178 to +88) was subjected to MatInspector analysis. Identified putative cis-elements are shown on a schematic with their core nucleotides underlined. Mutated core nucleotides are shown in bold and italic. The distal TSS mapped on human SLC44A4 mRNA numbered as +1 and shown in bold.

Fig. 3.

Mutational analysis of cis-regulatory elements located in the −178/−78 region of the SLC44A4 promoter and establishment a role of proximal ETS/ELF3 [E26 transformation-specific sequence (ETS) proteins] cis-regulatory element in basal promoter activity. A: nucleotides in the consensus binding sites for transcription factors were mutated by means of site-directed mutagenesis as described in materials and methods. The mutated minimal promoter constructs in pGL3-Basic were transiently expressed in NCM460 cells for luciferase assays. WT, wild-type promoter fragment. See results for additional definitions. Data are reported as relative firefly luciferase activity normalized to Renilla luciferase activity and represent means ± SE of at least 3 independent experiments, each performed in triplicate. B: transient transfection assays with truncated mutants of the minimal promoter region lacking or containing proximal ETS/ELF3 sequence. The experiment was performed as described in A. *P < 0.01.

Fig. 4.

Mutational analysis of cis-regulatory elements located in the −78/+88 region of the SLC44A4 promoter. By means of site-directed mutagenesis mutations were introduced into the consensus binding sites for transcription factors as described in materials and methods. NCM460 cells were transiently transfected with the WT or mutated promoter fragments, followed by luciferase assays. Data are reported as relative firefly luciferase activity normalized to Renilla luciferase activity and represent means ± SE of 3 independent experiments, each performed in triplicate. *P < 0.01.

Fig. 5.

DNA/protein profile from EMSA using SLC44A4 promoter region with the proximal ETS/ELF3 (at −80/−77 bp) sequence. Gel shift assay was performed with the nuclear extracts from NCM460 cells and biotin-labeled 21- and 36-bp regions of the SLC44A4 promoter spanning a sequence between −88 and −68 and between −103 and −68, respectively, as described in materials and methods. A: to establish specificity of the DNA/protein complexes, an assay was conducted with (a 30-fold molar excess) or without identical unlabeled DNA fragments. The major DNA/protein complex is indicated with arrow. B: to study a binding of ELF3 transcription factor, binding reaction was performed in the absence of antibody, in the presence of nonspecific IgG, in the presence of unrelated antibody against SP1, or in the presence of antibody against ELF3. Arrow indicates major specific DNA/protein complex.

Fig. 6.

DNA/protein profile from EMSA using SLC44A4 promoter region with the cAMP-responsive element (CRE) (at −38/−35 bp) sequence. Gel shift assay was performed with the nuclear extracts from NCM460 cells and 2 biotin-labeled regions of the SLC44A4 promoter spanning a sequence between −61 and −22 and between −47 and −6, respectively, as described in materials and methods. A 100-fold molar excess of unlabeled fragment (self) was used to establish specificity of the DNA/protein complexes. Binding reaction was performed in the absence of any antibody or in the presence of antibody against CRE-binding protein-1 (CREB-1), SP1, or combination of both CREB-1 and SP1 (both). Arrows and numbers indicate 2 major DNA/protein complexes.

Fig. 7.

Role of CREB in the SLC44A4 promoter functionality in vivo. NCM460 cells were cotransfected with 2 μg of SLC44A4 minimal promoter construct along with CREB expression plasmid (or vector) and treated with IBMX or H-89, followed by luciferase assay. Data are reported as relative firefly luciferase activity normalized to Renilla luciferase activity and represent means ± SE of 2 independent experiments, each performed in quadruplicate. *P < 0.01 (CREB vs. vector), **P < 0.01 (CREB + IBMX or CREB + H-89 vs. CREB + vehicle).

Fig. 8.

Contribution of CREB-1 and ELF3 in colon-specific expression pattern of SLC44A4 promoter. A: activity of the SLC44A4 promoter in colonic NCM460 and retinal ARPE19 cells. Reporter plasmids were transiently expressed in cells for luciferase assays. Data are reported as relative firefly luciferase activity normalized to Renilla luciferase activity. Each value represents the means ± SE of at least 3 independent experiments, each performed in triplicate. *P < 0.01. B: expression of CREB-1 and ELF3 nuclear factors in colonic NCM460 and retinal ARPE19 cells. Western blot analysis was performed using equal amounts (∼10 μg) of nuclear proteins isolated from NCM460 and ARPE19 cells as described in materials and methods. Blot was probed with the antibody for the protein of interest (anti-CREB-1 or anti-ELF3) and lamin B (loading control). Each Western blot analysis was performed at least twice with similar results. Images from a representative experiments are shown.