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BBA - Gene Structure and Expression (v.1769, #2)

Editorial Board (pp. ii).

ATF-2 stimulates the human insulin promoter through the conserved CRE2 sequence by Colin W. Hay; Laura A. Ferguson; Kevin Docherty (pp. 79-91).

The insulin promoter contains a number of dissimilar cis-acting regulatory elements that bind a range of tissue specific and ubiquitous transcription factors. Of the regulatory elements within the insulin promoter, the cyclic AMP responsive element (CRE) binds by far the most diverse array of transcription factors. Rodent insulin promoters have a single CRE site, whereas there are four CREs within the human insulin gene, of which CRE2 is the only one conserved between species. The aim of this study was to characterise the human CRE2 site and to investigate the effects of the two principal CRE-associated transcription factors; CREB-1 and ATF-2. Co-transfection of INS-1 pancreatic β-cells with promoter constructs containing the human insulin gene promoter placed upstream of the firefly luciferase reporter gene and expression plasmids for ATF-2 or CREB-1 showed that ATF-2 stimulated transcriptional activity while CREB-1 elicited an inhibitory effect. Mutagenesis of CRE2 diminished the effect of ATF-2 but not that of CREB-1. ATF-2 was shown to bind to the CRE2 site by electrophoretic mobility shift assay and by chromatin immunoprecipitation, while siRNA mediated knockdown of ATF-2 diminished the stimulatory effects of cAMP related signalling on promoter activity. These results suggest that ATF-2 may be a key regulator of the human insulin promoter possibly stimulating activity in response to extracellular signals.

Keywords: Abbreviations; ATF; activating transcription factor; bHLH; basic helix loop helix; bZIP; basic leucine zipper; CRE; cyclic AMP responsive element; CREB; cAMP responsive element binding protein; CREM; CRE-modulator protein; STAT5A; signal transducer and activator of transcription 5ADiabetes mellitus; cAMP responsive element; Gene transcription; Insulin gene

Translational regulation of PGHS-1 mRNA: 5′ untranslated region and first two exons conferring negative regulation by Natalia Bunimov; Jennifer Erin Smith; Dominique Gosselin; Odette Laneuville (pp. 92-105).

Prostaglandin endoperoxide H synthase-1 gene expression is described as inducible in a few contexts such as differentiation of megakaryoblastic MEG-01 cells into platelet-like structures. In the MEG-01 cells model of PGHS-1 gene induction, we previously reported a delay in protein synthesis and identified the translational step of gene expression as being regulated. In the current study, we mapped PGHS-1 mRNA sequences regulating translational efficiency and identified an RNA binding protein. The 5′UTR and first two exons of the PGHS-1 5′ mRNA decreased the synthesis of Luciferase protein by approximately 80% without significant changes in mRNA levels when compared to controls. Both the PGHS-1 5′-UTR and the first two exons were required for activity. Sucrose density gradient fractionations of cytoplasmic extracts from MEG-01 cells infected with reporter constructs, either controls or containing PGHS-1 sequence, presented a similar profile of distribution of reporter transcripts between polysomal and non-polysomal fractions. RNA/protein interaction studies revealed nucleolin binding to the 135 nt PGHS-1 sequence. Mutation of the two NRE elements located in the 5′end of PGHS-1 mRNA sequence partially reduced the negative activity of the 135 nt sequence. Stable secondary structures predicted at the 5′ end of the transcript are potentially involved in translational regulation. We propose that the 5′end of PGHS-1 mRNA represses translation and could delay the synthesis of PGHS-1 enzyme.

Keywords: Luc; Luciferase; MEG-01; megakaryoblastic cells; NRE; nucleolin response element; ORF; open reading frame; PGHS-1; prostaglandin endoperoxide H synthase-1; UTR; untranslated region

Cloning and characterization of a new intestinal inflammation-associated colonic epithelial Ste20-related protein kinase isoform by Y. Yan; H. Nguyen; G. Dalmasso; S.V. Sitaraman; D. Merlin (pp. 106-116).

Intestinal epithelial cells respond to inflammatory extracellular stimuli by activating mitogen activated protein kinase (MAPK) signaling, which mediates numerous pathophysiological effects, including intestinal inflammation. Here, we show that a novel isoform of SPS1-related proline alanine-rich kinase (SPAK/STE20) is involved in this inflammatory signaling cascade. We cloned and characterized a SPAK isoform from inflamed colon tissue, and found that this SPAK isoform lacked the characteristic PAPA box and alphaF loop found in SPAK. Based on genomic sequence analysis the lack of PAPA box and alphaF loop in colonic SPAK isoform was the result of specific splicing that affect exon 1 and exon 7 of the SPAK gene. The SPAK isoform was found in inflamed and non-inflamed colon tissues as well as Caco2-BBE cells, but not in other tissues, such as liver, spleen, brain, prostate and kidney. In vitro analyses demonstrated that the SPAK isoform possessed serine/threonine kinase activity, which could be abolished by a substitution of isoleucine for the lysine at position 34 in the ATP-binding site of the catalytic domain. Treatment of Caco2-BBE cells with the pro-inflammatory cytokine, interferon γ, induced expression of the SPAK isoform. Over-expression of the SPAK isoform in Caco2-BBE cells led to nuclear translocation of an N-terminal fragment of the SPAK isoform, as well as activation of p38 MAP kinase signaling cascades and increased intestinal barrier permeability. These findings collectively suggest that pro-inflammatory cytokine signaling may induce expression of this novel SPAK isoform in intestinal epithelia, triggering the signaling cascades that govern intestinal inflammation.

Keywords: Ste20-related protein kinase; Colonic SPAK isoform; p38 cascade; Caco2-BBE; Intestinal inflammation; Interferon γ

Upregulation of Slc39a10 gene expression in response to thyroid hormones in intestine and kidney by Kaler Pawan; Sharma Neeraj; Kumar Sandeep; Radha Kanta Ratho; Prasad Rajendra (pp. 117-123).

A novel zinc transporter has been purified and cloned from rat renal brush border membrane. This transporter was designated as Zip10 encoded by Slc39a10 gene and characterized as zinc importer. Present study documents the impact of thyroid hormones on the expression of Zip10 encoded by Slc39a10 gene in rat model of hypo and hyperthyroidism. Serum T₃ and T₄ levels were reduced significantly in hypothyroid rats whereas these levels were significantly elevated in hyperthyroid rats as compared to euthyroid rats thereby confirming the validity of the model. Kinetic studies revealed a significant increase in the initial and equilibrium uptake of Zn⁺⁺ in both intestinal and renal BBMV of hyperthyroid rats in comparison to hypothyroid and euthyroid rats. By RT-PCR, Slc39a10 mRNA expression was found to be significantly decreased in hypothyroid and increased in hyperthyroid as compared to euthyroid rats. These findings are in conformity with the immunofluorescence studies that revealed markedly higher fluorescence intensity at periphery of both intestinal and renal cells isolated from hyperthyroid rats as compared to hypothyroid and euthyroid rats. Higher expression of Zip10 protein in hyperthroid group was also confirmed by western blot. These findings suggest that expression of zinc transporter protein Zip10 ( Slc39a10) in intestine and kidney is positively regulated by thyroid hormones.

Keywords: Thyroid hormones; Solute linked carrier; (Slc; ) gene family; Zinc transporter; Rat intestine and kidney; Brush border membrane

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure by Nasséra Chalabi; Samir Satih; Laetitia Delort; Yves-Jean Bignon; Dominique J. Bernard-Gallon (pp. 124-130).

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 μM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.

Keywords: Profiling; Hybridization; Breast cancer; Lycopene

Effects of 5′ untranslated region diversity on the posttranscriptional regulation of the human reduced folate carrier by Scott G. Payton; Christina L. Haska; Robin M. Flatley; Yubin Ge; Larry H. Matherly (pp. 131-138).

The human RFC (hRFC) gene is regulated by five major 5′ non-coding exons, characterized by alternate transcription start sites and splice forms. The result is up to 14 hRFC transcripts for which different 5′ untranslated regions (UTRs) are fused to a common coding sequence. By in vitro translation assays with hRFC constructs corresponding to the major transcript forms, most of the forms were translated poorly. Upon expression of the 5′UTR-hRFC constructs in hRFC-null HeLa cells, a range of steady state hRFC proteins and transcripts were detected that reflected relative transcript stabilities and, to a lesser extent, translation efficiencies. Transcripts including 5′ UTRs derived from non-coding exon A encoded a modified hRFC protein translated from an upstream initiation site. When this modified hRFC protein was expressed in hRFC-null K562 cells, there were only minor differences in surface targeting, stability, or transport function from wild type hRFC. Our results demonstrate an important role for posttranscriptional determinants of cellular hRFC levels and activity.

Keywords: Reduced folate carrier; Translation; Untranslated region; Transcript; Membrane transport; Methotrexate; Folate

Promoter analysis of the Catharanthus roseus geraniol 10-hydroxylase gene involved in terpenoid indole alkaloid biosynthesis by Nitima Suttipanta; Sitakanta Pattanaik; Samir Gunjan; Claire H. Xie; John Littleton; Ling Yuan (pp. 139-148).

Geraniol 10-hydroxylase (G10H) is an important enzyme in the biosynthetic pathway of monoterpenoid alkaloids found in diverse plant species. The Catharanthus roseus G10H controls the first committed step in biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. The major transcription start site of the promoter was mapped to an adenine 31 bp downstream of the TATA-box. For functional characterization, transcriptional fusions between the G10H promoter fragments with 5′ or 3′ deletions and the GUS reporter gene were generated and their expressions were analyzed in a tobacco protoplast transient expression assay. Deletion of the promoter down to −318 bp had little effect on GUS activity. However, further deletion of the promoter to position −103 resulted in approximately 5-fold reduction of GUS activity. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between −191 and −147, −266 and −188, and −318 and −266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings GUS expression was tissue-specific, restricted to leaf and actively growing cells around the root tip, and not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitor and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade.

Keywords: Promoter; Geraniol 10 hydroxylase gene; Terpenoid indole alkaloid; Catharanthus roseus; Transgenic plants

A 4.3 kb Smad7 promoter is able to specify gene expression during mouse development by Xubao Liu; Qian Chen; Chenzhong Kuang; Meiyu Zhang; Yiwen Ruan; Zao C. Xu; Zhenzhen Wang; Yan Chen (pp. 149-152).

Members of transforming growth factor-β (TGF-β) superfamily play important roles in diverse biological functions including early development. These extracellular factors exert their effects by interacting with membrane receptors followed by signal transduction by a group of Smad proteins. Smad7 is an inhibitory Smad protein that specifically antagonizes TGF-β and activin signaling. To characterize the developmental role of Smad7, a transgenic mouse model was generated using a 4.3 kb mouse Smad7 promoter driving β-galactosidase expression. In these mice, the Smad7 promoter defined a restrictive expression pattern of β-galactosidase in a tightly regulated temporal and spatial manner. The β-galactosidase gene was transiently expressed in the cardiovascular structures including heart cushion tissues and the endothelium of major arteries at E11.5 to E12.5. Through E12.5 to E17.5, β-galactosidase expression was prominently detected in the epithelium of developing cochlea and nasolacrimal duct. In addition, it was temporally expressed in trigeminal ganglion, the skeletal muscles surrounding major joints, primordium of the jaws, as well as genital tubercle. These studies indicated that the 4.3 kb Smad7 promoter contains sufficient regulatory elements to define controlled gene expression during mouse development.

Keywords: TGF-β; Activin; Smad7; Heart; Aorta; Cochlea; Nasolacrimal duct; Development; Transgenic

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BBA - Gene Structure and Expression (v.1769, #2)