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BBA - Gene Structure and Expression (v.1728, #1-2)

Editorial Board (pp. ii).

Transcriptional repression of TFF1 in gastric epithelial cells by CCAAT/enhancer binding protein-β by Narendra V. Sankpal; Marty W. Mayo; Steven M. Powell (pp. 1-10).

TFF1 is a member of a unique family of gastrointestinal peptides. Loss of TFF1 expression has been observed in the majority of human gastric cancers and the biological significance of this loss has been demonstrated in a Tff1 knockout mouse model. However, few TFF1 gene mutations or allelic loss have also been documented. To understand the molecular mechanism repressing the TFF1 gene expression, the 5′-flanking region of the human TFF1 gene was characterized. We found a repressor region (−241 to −84), which is active in MKN45 and IMGE5 cells expressing endogenous TFF1 gene. A consensus binding site for C/EBPβ was identified and EMSA analysis demonstrated specific binding of CEBPβ. Mutation of this C/EBPβ element potentiated the transactivation of TFF1 by 50% and 145% for MKN45 and IMGE5 cells respectively. Furthermore, co-transfection of C/EBPβ isoforms specifically decreased TFF1 promoter activity. These findings suggest that C/EBPβ is involved in the down-regulating of TFF1 gene expression and this mode of repression may account at least in part for the loss of TFF1 gene expression in transformed human and mice gastric epithelial cells.

Keywords: Trefoil Factor-1; Promoter; Transcriptional repression; Gastric cancer; C/EBPβ

Two forms of the large tumor suppressor gene ( Lats1) protein expressed in the vertebrate retina by Novrouz B. Akhmedov; Clyde K. Yamashita; Dai Tran; Natik I. Piri; Gustavo D. Aguirre; Debora B. Farber (pp. 11-17).

The large tumor suppressor gene ( Lats1) encodes a protein kinase that is highly conserved from fly to human, and plays a crucial role in the prevention of tumor formation by controlling mitosis progression. We have found that in addition to the previously isolated 7.5 kb long form of Lats1 ( Lats1_L) mRNA, a less abundant, shorter, 3.4 kb primary transcript ( Lats1_S) also is expressed in the vertebrate retina. Compared to Lats1_L, the sequence of Lats1_S mRNA has a deletion of exons 6, 7, and 8 that corresponds to 792 bp of the open reading frame. Thus, 264 aa of the C-terminal region of the long transcript are missing in the Lats1_S protein. The encoded truncated protein lacks four of eleven conserved kinase domains and the C-terminus. Our results suggest that the 3.4 kb transcript is a splice variant of the 7.5 kb transcript. We have found direct evidence that both the retinal 7.5 and 3.4 kb mRNAs are translated into 170 kDa and 120 kDa proteins, respectively. The expression of both isoforms in vertebrate cells raises the possibility that these Lats1 proteins may act as negative key regulators of the cell cycle, each of them performing a unique role.

Keywords: Large tumor suppressor (Lats/Warts) gene; Serine/threonine protein kinase; Retina

Transcription of brain creatine kinase in U87-MG glioblastoma is modulated by factor AP2 by Dianna Willis; Yanping Zhang; George R. Molloy (pp. 18-33).

Our previous studies established in U87-MG glioblastoma cells that elevated cAMP increased transcription of the endogenous as well as a transiently-transfected brain creatine kinase (CKB) gene, despite the absence of a cAMP response element (CRE) in the CKB proximal promoter. This report employed transfection to show that the transcription of CKB in U87 cells is induced by transcription factor AP2α, which is known to be activated by cAMP. Dominant-negative forms of AP2α not only prevented the AP2α-mediated activation of CKB but also blocked the cAMP-mediated increase in CKB transcription caused by forskolin treatment. The mutation of the four potential AP2 elements within the CKB proximal promoter showed that induction of CKB by AP2 was mediated principally through the AP2 element located at −50 bp in the promoter. Electromobility shift assays revealed a protein in U87 nuclear extracts that bound to a consensus AP2α element as well as to the (−50) AP2 element in CKB. Interestingly, the CKB (−50) AP2 element contains GCCAATGGG which also bound NF-Y, the CCAAT-binding protein, suggesting that interplay between AP2 and NF-Y may modulate CKB transcription. This is the first report of a role for AP2 in the regulation of CKB transcription and of an AP2 element within which an NF-Y site is located.

Keywords: AP2; Astrocyte; Cyclic AMP; Energy metabolism; Transcription; U87 glioblastoma

The mammalian gene pecanex 1 is differentially expressed during spermatogenesis by Adriana Geisinger; Manfred Alsheimer; Andrea Baier; Ricardo Benavente; Rodolfo Wettstein (pp. 34-43).

Using mRNA differential display and cDNA library screening approaches we have identified differential gene expression of pecanex 1 – a mammalian homologue of pecanex gene from Drosophila – in the testes of the rat. Northern blot analyses showed that the transcript is only present in the germ line and not in the somatic cells of the testis, reaching its peak at the pachytene stage of the meiotic prophase. Moreover, nonradioactive in situ hybridization did not detect the expression of the gene in any cell type of the testis other than pachytene spermatocytes. Northern blot assays did not allow the detection of the transcript in nine other tissues. Remarkably, although pecanex exerts a neurogenic role in Drosophila, the transcript was not detectable by Northern blotting in the nervous tissue of adult rats, nor in the brain of neonate and embryonal stages. The protein product of the pecanex 1 gene was detected by immunoblotting in pachytene spermatocytes and round spermatids as well, but not in liver nor brain. From genomic analysis we conclude that, although only one pecanex gene exists in Drosophila, mammalian pecanex 1 belongs to a gene family with three related genes in different chromosomes. We speculate that pecanex 1 could play an important role in the testis, related to spermatogenesis.

Keywords: Abbreviations; DD; differential display; ORF; open reading frame; EST; expressed sequence tag; pcx; Drosophila's pecanex; RT; reverse transcriptase; PCR; polymerase chain reaction; 7-TM; seven transmembrane receptors (refers to receptors with seven transmembrane domains); C-terminal; carboxy-terminal; N-terminal; amino-terminal; o.n.; overnight; GST; gluthation-; S; -transferaseSpermatogenesis; Meiosis; Gene expression; Differential display; Pecanex

Structural characterization and transcriptional regulation of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide in the cotton bollworm, Helicoverpa armigera by Tian-Yi Zhang; Jiu-Song Sun; Wen-Yu Liu; Le Kang; Jin-Liang Shen; Wei-Hua Xu (pp. 44-52).

We have cloned the gene encoding the diapause hormone and the pheromone biosynthesis activating neuropeptide in Helicoverpa armigera (Har-DH-PBAN). The Har-DH-PBAN gene contains six exons and five introns that fall in the same positions as in the Bombyx mori DH-PBAN gene (Bom-DH-PBAN). The transcription initiation site lays 29 bp upstream of the translation initiation site. Southern blot analysis suggests that a single copy of this gene is present per haploid genome. A structural comparison of DH-PBAN promoters between H. armigera and B. mori show similarities in the TATA box and in a potential binding site for a POU family transcription factor, POU-M2. However, testing of these DNA regions for factor binding in vitro and transcription assays in cell culture highlight significant differences in their regulation particularly in reference to the POU-M2 sites. Our results uncover common and different regulatory mechanisms at work in the control of DH-PBAN gene expression in H. armigera and B. mori.

Keywords: Abbreviations; DH-PBAN; diapause hormone-pheromone biosynthesis activating neuropeptide; Har; Helicoverpa armigera; Bom; Bombyx mori; PTTH; prothoracicotropic hormone; SG; suboesophageal ganglion; EMSA; electrophoretic mobility shift assay; HP; Har-DH-PBAN promoter; BP; Bom-DH-PBAN promoterDiapause hormone; Pheromone biosynthesis activating neuropeptide; Gene cloning; Transcription factor; Helicoverpa armigera

T-helper type 2 cell-specific expression of the ST2 gene is regulated by transcription factor GATA-3 by Morisada Hayakawa; Ken Yanagisawa; Shinsuke Aoki; Hiroko Hayakawa; Naoki Takezako; Shin-ichi Tominaga (pp. 53-64).

ST2 is a member of the interleukin-1 receptor family and is expressed in type-2 T helper (Th2) cells. Here, we have studied the molecular mechanism responsible for the transcriptional regulation of the ST2 gene in Th2 cells using a mouse thymoma cell line, EL-4. The ST2 gene has distal and proximal promoters. ST2 mRNA was produced from the distal promoter in EL-4 cells stimulated with both phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (Bt₂cAMP). The region of approximately 100 bp upstream of transcription start site, containing two GATA consensus sites, was indispensable for the activation of the distal promoter in reporter gene analysis. An electrophoretic mobility shift assay showed that transcription factor GATA-3 bound one of the GATA consensus sites (from −84 to −79) with nuclear extracts from PMA plus Bt₂cAMP-stimulated EL-4 cells. The overexpression of GATA-3 enhanced the activity of the distal promoter. On the other hand, mutations of the GATA consensus site canceled out the enhancement by GATA-3. These data suggest that GATA-3 is an important transcription factor for the expression of the ST2 gene in Th2 cells.

Keywords: Abbreviations; IL; interleukin; Th; T helper; IFN-γ; interferon-γ; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; PMA; phorbol 12-myristate 13-acetate; Bt; ₂; cAMP; dibutyryl cAMP; FBS; fetal bovine serum; PCR; polymerase chain reaction; RT-PCR; reverse transcription-polymerase chain reaction; EMSA; electrophoretic mobility shift assayST2; Interleukin-1 receptor family; GATA-3; Type-2 T helper cell

Positive and negative regulatory elements in the late lactation protein-A gene promoter from the tammar wallaby ( Macropus eugenii) by Josephine F. Trott; Timothy E. Adams; Michael Wilson; Kevin R. Nicholas (pp. 65-76).

Little is known about the regulation of the marsupial-specific late lactation protein-A (LLP-A) gene, first expressed at mid-lactation in the mammary gland of the tammar wallaby. A genomic clone of LLP-A was sequenced and shown to include seven exons. The LLP-A promoter region of 1969 bp ligated to a secreted alkaline phosphatase (SEAP) gene reporter was co-transfected into CHO-K1 cells with prolactin (PRL) receptor cDNA. Transfected cells cultured with insulin, cortisol and PRL did not secrete SEAP into media. Similarly, this construct was not expressed in the mammary gland of eight lines of transgenic mice. In contrast, when the LLP-A promoter region was reduced to 850 bp, the expression of the SEAP reporter in CHO-K1 cells was constitutive and PRL-independent, despite the presence of two low affinity Stat5 binding sites. The 1969 bp promoter was analyzed using nine serial deletions ligated to the SEAP gene. The expression of these constructs was PRL-independent. Five putative inhibitory elements were identified between −1969 and −1796, −1404 and −1184, −1184 and −992, −992 and −757, and −591 and −425, and a putative enhancer or core transcription element between −425 and−239. These studies indicate that the complex temporal regulation of the LLP-A gene involves elements in its 5′-regulatory region.

Keywords: Abbreviations; BSA; bovine serum albumin; CAT; chloramphenicol acetyl transferase; CHO-K1; Chinese hamster ovary; EMSA; electrophoretic mobility shift assay; LLP-A; late lactation protein-A; oBLG; ovine β-lactoglobulin; PRL; prolactin; PRLR; prolactin receptor; SEAP; secreted alkaline phosphataseMarsupial; Inhibitor element; Milk protein; Gene regulation; Exon structure; Transgenic

Expression and transcriptional regulation of Munc18 isoforms in mast cells by Rupesh Nigam; Jorge Sepulveda; Michael Tuvim; Youlia Petrova; Roberto Adachi; Burton F. Dickey; Anurag Agrawal (pp. 77-83).

Mast cells are specialized immune cells with a central pathophysiological role in allergic reactions and important roles in pathogen defense. Their main effector response is the exocytic release of preformed inflammatory mediators from secretory granules. Munc18 proteins are essential for exocytic function, so we analyzed the expression of Munc18 transcripts in RBL-2H3 mast cells and mouse bone marrow derived mast cells (BMMC). All three isoforms of Munc18 are expressed in both cell types, but Munc18-2 transcripts are most abundant. The proximal 181 bp region of the Munc18-2 gene promoter is conserved between mice and humans, and shows maximal promoter activity among a series of truncation mutants. Binding sites for Ets, E-box and CREB transcription factors that are known to be important for mast cell development are highly conserved and functionally active. Thus, mast cells upregulate an essential component of their exocytic machinery as they develop morphologic and functional features of the regulated secretory phenotype.

Keywords: Sec1; Munc18; BMMC; Gata; Mast cell

Dehydroepiandrosterone negatively regulates the p38 mitogen-activated protein kinase pathway by a novel mitogen-activated protein kinase phosphatase by Kenji Ashida; Kiminobu Goto; Yue Zhao; Taijiro Okabe; Toshihiko Yanase; Ryoichi Takayanagi; Masatoshi Nomura; Hajime Nawata (pp. 84-94).

Dehydroepiandrosterone-sulfate, the sulfated form of dehydroepiandrosterone, is the most abundant steroid in young adults, but gradually declines with aging. In humans, the clinical application of dehydroepiandrosterone targeting some collagen diseases, such as systemic lupus erythematosus, as an adjunctive treatment has been applied in clinical trial. Here, we report that dehydroepiandrosterone may negatively regulate the mitogen-activated protein kinase pathway in humans via a novel dual specificity protein phosphatase, DDSP (dehydroepiandrosterone-enhanced dual specificity protein phosphatase). DDSP is highly homologous to LCPTP/HePTP, a tissue-specific protein tyrosine phosphatase (PTP) which negatively regulates both ERK and p38-mitogen-activated protein kinase, and is transcribed from the PTPN7 locus by alternative splicing. Although previous reports have shown that the mRNA expression of the LCPTP/HePTP gene was inducible by extracellular signals such as T-cell antigen receptor stimulation, reverse transcribed (RT)-PCR experiments using specific sets of primers suggested that the expression of LCPTP/HePTP was constitutive while the actual inducible sequence was that of DDSP. Furthermore DDSP was widely distributed among different types of human tissues and specifically interacted with p38-mitogen-activated protein kinase. This inducible negative regulation of the p38-mitogen-activated protein kinase-dependent pathway may help to clarify the broad range of dehydroepiandrosterone actions, thereby aiding the development of new preventive or adjunctive applications for human diseases.

Keywords: Abbreviations; RT-PCR; reverse transcribed PCR; MAPK; mitogen-activated protein kinase; PTP; protein tyrosine phosphatase; DSP; protein dual specificity phosphatase; LCPTP; leukocyte-specific protein tyrosine phosphatase; HePTP; hematopoietic tissue-specific protein tyrosine phosphatase; DHEA; dehydroepiandrosterone; DDSP; dehydroepiandrosterone-induced protein dual specificity phosphatase; ERK; extracellular stimulus-regulated kinase; JNK; c-; Jun; N-terminus kinase; TcR; T-cell antigen receptor; GFP; green fluorescence protein; PMA; phorbol-12-myristate-13-acetate; aa; amino acid; SSH; suppression subtraction hybridizationDehydroepiandrosterone; Dual specificity protein phosphatase; Protein tyrosine phosphatase; p38-mitogen-activated protein kinase; Mitogen-activated protein kinase; Mitogen-activated protein kinase phosphatase

A PPAR response element regulates transcription of the gene for human adipose differentiation-related protein by Paul Targett-Adams; Marion J. McElwee; Ewa Ehrenborg; Mattias C. Gustafsson; Colin N. Palmer; John McLauchlan (pp. 95-104).

Lipid droplets are cytoplasmic organelles which serve as storage sites for neutral lipids. Adipose differentiation-related protein (ADRP) is intrinsically associated with the surface of lipid droplets and is believed to play a major role in the maintenance of lipid stores in non-adipocytes. ADRP abundance is intimately linked to the amount of lipid found within cells and agents which increase the levels of intracellular lipid, such as certain agonists of the peroxisome proliferator-activated receptors (PPARs), also are capable of modulating ADRP gene transcription. However, little is known about the molecular mechanisms and promoter control elements, which regulate the transcription of the human gene. Using a reporter system to investigate ADRP transcription, we have identified a PPAR response element (PPRE) with the sequence 5′-AGGTGA A AGGGCG-3′ within its promoter region. Mutational analysis revealed that the ADRP PPRE specifically mediated the upregulation of transcription in response to activation by agonists of PPAR subtypes α and δ in both rat and human hepatocyte-derived cell lines. These findings offer insight into the mechanisms which serve to regulate ADRP transcription and intracellular lipid storage.

Keywords: Abbreviations; PPAR; peroxisome proliferator-activated receptor; PPRE; peroxisome proliferator-activated receptor response element; ADRP; adipose differentiation-related protein; RACE; rapid amplification of cDNA ends; PCR; polymerase chain reactionPPRE; PPAR; ADRP; Promoter; Regulation

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BBA - Gene Structure and Expression (v.1728, #1-2)