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

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.

Promoter cloning and characterization of the human programmed cell death protein 4 (pdcd4) gene: evidence for ZBP-89 and Sp-binding motifs as essential Pdcd4 regulators

Pdcd4 (programmed cell death protein 4) is an important novel tumour suppressor inhibiting transformation, translation, invasion and intravasation, and its expression is down-regulated in several cancers. However, little is known about the transcriptional regulation and the promoter of this important tumour suppressor. So far the following is the first comprehensive study to describe the regulation of Pdcd4 transcription by ZBP-89 (zinc-finger-binding protein 89), besides characterizing the gene promoter. We identified the transcriptional start sites of the human pdcd4 promoter, a functional CCAAT-box, and the basal promoter region. Within this basal region, computer-based analysis revealed several potential binding sites for ZBPs, especially for Sp (specificity protein) family members and ZBP-89. We identified four Sp1/Sp3/Sp4-binding elements to be indispensable for basal promoter activity. However, overexpression of Sp1 and Sp3 was not sufficient to enhance Pdcd4 protein expression. Analysis in different solid cancer cell lines showed a significant correlation between pdcd4 and zbp-89 mRNA amounts. In contrast with Sp transcription factors, overexpression of ZBP-89 led to an enhanced expression of Pdcd4 mRNA and protein. Additionally, specific knockdown of ZBP-89 resulted in a decreased pdcd4 gene expression. Reporter gene analysis showed a significant up-regulation of basal promoter activity by co-transfection with ZBP-89, which could be abolished by mithramycin treatment. Predicted binding of ZBP-89 to the basal promoter was confirmed by EMSA (electrophoretic mobility-shift assay) data and supershift analysis for ZBP-89. Taken together, data for the first time implicate ZBP-89 as a regulator of Pdcd4 by binding to the basal promoter either alone or by interacting with Sp family members.

In vivo analyses of constitutive and regulated promoters in halophilic archaea

The two gvpA promoters PcA and PpA of Halobacterium salinarum, and the PmcA promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with β-galactosidase activity, as reporter. For comparison, the Pfdx promoter of the ferredoxin gene of Hbt. salinarum and the PbgaH promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. Pfdx , driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas PbgaH and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to Pfdx , the basal promoter activities of PpA and PmcA were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The PcA promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the PcA -TATA box and the BRE element were the reason for the lack of the basal PcA activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger PpA promoter and investigated in Hfx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the PpA -BRE element was substituted for the PcA -BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring PpA -BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of PmcA -BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of PmcA .

YY1 binding within the human HSD3B2 gene intron 1 is required for maximal basal promoter activity: identification of YY1 as the 3beta1-A factor

The oxidation and isomerization of 3beta-hydroxy-5-ene steroids into keto-4-ene steroids, a pivotal step in the synthesis of all hormonal steroids, is catalyzed by several isoforms of 3beta-hydroxysteroid dehydrogenase. In humans, two highly homologous isoforms exist, type I expressed by the HSD3B1 gene in peripheral tissues, and type II expressed by the HSD3B2 gene in steroidogenic organs. Previously, it was shown that the HSD3B1 gene 3beta1-A element, encompassing 24 nucleotides of intron 1 not perfectly conserved between the two genes and overlapping with a conserved TG box, contributes to maximal basal promoter activity by binding the ubiquitous and unidentified 3beta1-A transcription factor. In this study for the first time we report that similarly, the HSD3B2 gene intron 1 is required for maximal basal promoter activity in reporter gene analyses, as lack of intron 1 results in a 4- to 10-fold reduction in promoter activity. Mutational analysis in gel shift assays revealed that the 3beta1-A factor binds both the HSD3B2 and HSD3B1 gene intron 1 by requiring only seven nucleotides of a conserved segment within the 3beta1-A element. By competition analysis and use of anti-YY1 antibody in both gel shift and Western blot experiments, we identified the 3beta1-A protein as the ubiquitous transcription factor YY1. In addition, we have characterized another similar YY1 binding site differently located with respect to the 3beta1-A element in both genes. Deletion and mutational analysis in transient transfections experiments revealed that contrarily to as previously shown for the HSD3B1 gene, lack of YY1 binding to the type II 3beta1-A element only results in a marginal reduction of basal promoter activity. Instead, YY1 binding to the second site, placed 35 bp downstream from the 3beta1-A element, strongly activates the HSD3B2 gene basal promoter activity, as preventing YY1 binding to this region caused a 50% decrease of basal transcription. Complete abrogation of YY1 binding within type II intron 1 resulted in a gene reporter activity identical to a reporter construct lacking the whole intron 1. These results designate YY1 as the factor responsible for the intron 1-mediated boost of the HSD3B2 gene basal promoter activity. Similarities and dissimilarities between YY1 binding within the HSD3B1 and HSD3B2 gene intron 1 are discussed involving the conserved intron 1 TG box, that suggests different mechanisms are implicated in the YY1-mediated stimulation of these two genes basal promoter activity.

Adrenocorticotropin/3′,5′-Cyclic AMP-Mediated Transcription of the Scavenger akr1-b7 Gene in Adrenocortical Cells Is Dependent on Three Functionally Distinct Steroidogenic Factor-1-Responsive Elements

The akr1-b7 gene encodes a scavenger enzyme expressed in steroidogenic glands under pituitary control. In the zona fasciculata of the adrenal cortex where its expression is controlled by ACTH, AKR1-B7 detoxifies isocaproaldehyde produced during the first step of steroidogenesis. Three steroidogenic factor-1 (SF-1)-responsive elements (SFREs) are contained within the −510/+41 promoter region, which was previously demonstrated to drive gene expression in transgenic mice adrenal cortex. All these sequences bind at least SF-1 in Y1 adrenocortical cell nuclear extracts and can be activated by overexpression of this factor in HeLa cells. However, the three SFREs show distinct properties regarding akr1-b7 promoter activity in Y1 cells. Whereas the proximal −102 SFRE supports basal promoter activity, the −458 bona fide SFRE is essential for both basal promoter activity and cAMP responsiveness, although it is unresponsive to cAMP when isolated from its promoter context. This suggests that SF-1 is not a cAMP-responsive factor per se. The neighboring SFRE at −503 is a palindromic sequence that binds monomeric and heteromeric SF-1 as well as an adrenal-specific complex. Using MA-10 Leydig cells and Y1–10r9 mutant cells, we provide evidence that its activity in adrenocortical cells depends on the binding of the adrenal-specific factor, which is required for basal and cAMP-induced promoter activity. Furthermore, the −503 site has intrinsic cAMP-sensing ability in Y1 cells, which is correlated with increased adrenal-specific complex binding. Collectively, our results suggest that cAMP responsiveness of the akr1-b7 promoter is achieved through cooperation between the adrenal-specific factor bound to the −503 site and SF-1 bound to the −458 site.