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BBA - Gene Structure and Expression (v.1731, #1)

Editorial Board (pp. ii).

Adiponectin is expressed in the brown adipose tissue and surrounding immature tissues in mouse embryos by Noritaka Fujimoto; Noritaka Matsuo; Hideaki Sumiyoshi; Kenji Yamaguchi; Tetunori Saikawa; Hironobu Yoshimatsu; Hidekatsu Yoshioka (pp. 1-12).

Adiponectin is one of the adipocytokines, which are adipose-specific secretory factors. We examined its expression during embryogenesis. Transcripts of adiponectin were detected at a late stage of embryogenesis on embryonic (E) day E16.5. In situ hybridization showed that adiponectin transcripts were localized in brown adipose tissues (BATs) and surrounding immature tissues in mouse embryos. Immunohistochemistry using a specific anti-adiponectin antibody showed that the distribution of adiponectin closely parallels that of its mRNA. Adiponectin was also detected in serum at day E16.5, and its concentration peaked at birth. By contrast, transcripts of both the adiponectin receptors 1 and 2 were already expressed by day E12.5 in many tissues. Thus, their expression profile differed from that of adiponectin itself. Furthermore, experiments using primary cultures of brown adipocytes showed that adiponectin is regulated in brown adipocytes by various modulators, similar to its regulation in white adipose tissues (WATs). These data indicate that adiponectin has important roles in glucose and lipid metabolism during the perinatal period.

Keywords: Adiponectin; Brown adipose tissue; White adipose tissue; Gene expression; Embryogenesis; Adiponectin receptor

Regulation of inhibitor of differentiation gene 3 (Id3) expression by Sp2-motif binding factor in myogenic C2C12 cells: Downregulation of DNA binding activity following skeletal muscle differentiation by Jinmei Wu; Robert W. Lim (pp. 13-22).

Id3 is a member of the Id family of transcriptional regulators that have been implicated in the development of multiple tissues. Altered expression of the Id genes and proteins contribute to carcinogenesis and atherosclerosis. Id3 is highly expressed in proliferating skeletal muscle cells but becomes downregulated upon terminal differentiation. We have identified several DNase I protected footprints within a proximal region of the mouse Id3 promoter that has been shown previously to support high levels of transcriptional activity in proliferating skeletal muscle cells. Two of these sites interacted, respectively, in vitro with Sp2 and Egr-1 proteins present in muscle cell nuclear extracts. Mutation analysis revealed that the Sp2 site accounted for a major part of the Id3 promoter activity in proliferating muscle cells whereas the Egr-1 site was dispensable. Consistent with the previously observed downregulation of the endogenous Id3 gene, protein binding to the Sp2 site was substantially reduced with extracts from differentiated muscle cells. Our results reveal Id3 as a potential target for Sp2 and raise the possibility that acute activation and the chronic and maintained expression of Id3 gene might be regulated by different mechanisms.

Keywords: Myogenesis; Basic helix–loop–helix; Id gene; Id3 promoter; Egr-1; Transcriptional regulation

Analysis of the guinea-pig estrogen-regulated gec1/GABARAPL1 gene promoter and identification of a functional ERE in the first exon by Sandrine Vernier-Magnin; Christophe Nemos; Virginie Mansuy; Fabrice Tolle; Laure Guichard; R�gis Delage-Mourroux; Mich�le Jouvenot; Annick Fraichard (pp. 23-31).

The gec1/GABARAPL1 ( GABA_A -receptor-associated protein like-1) gene has been identified as an early estrogen-regulated gene in guinea-pig cultured endometrial glandular epithelial cells (GEC). Guinea-pig and human gec1/GABARAPL1 proteins share 87% identity with GABARAP, which acts as a protein linker between microtubules and the GABA_A receptor. To investigate the molecular mechanisms regulating gec1/GABARAPL1 gene expression, the 1.5-kbp region upstream of the translation initiation codon of the guinea-pig gec1/GABARAPL1 gene was cloned. A 300-bp fragment encompassing a pyrimidine-rich initiator element (INR) and the transcription start site (+1) was sufficient to initiate transcription. Transfection and gel shift experiments showed that a sequence located at +36/+50 in the first exon permitted induction of expression of this gene by estradiol acting via ERα. This sequence (GGGTCAACGTGACGT) differs only by one base pair from the consensus estrogen response element ERE (GGGTCAACGTGACCT). It can be concluded that the ERE located in the first exon encoding the 5′-untranslated region is sufficient for E₂ activation of gec1/GABARAPL1 transcription.

Keywords: 17β-estradiol (E; ₂; ); Early estrogen-regulated gene; ERE (estrogen response element); INR (initiator element); Estrogen receptors (ERα and ERβ); gec1/GABARAPL1

Role of nucleolin in posttranscriptional control of MMP-9 expression by Michael F�hling; Andreas Steege; Andrea Perlewitz; Benno Nafz; Ralf Mrowka; Pontus B. Persson; Bernd J. Thiele (pp. 32-40).

Matrix-metalloproteinases (MMPs), which are able to degrade extra cellular matrix (ECM) components, are crucial in ECM-remodeling, under physiological (e.g., embryogenesis, wound healing, angiogenesis) or pathophysiological conditions (e.g., arthritis, cancer progression and metastasis, fibrosis). Treating HT1080 cells, a human fibrosarcoma cell line, with the iron chelator 2,2-Dipyridyl, which mimics certain aspects of hypoxia, leads to a 3-fold elevated Matrix-metalloproteinase-9 (MMP-9) protein level. This elevation occurs within 3 h, without any change of mRNA-concentration. The rapid increase in MMP-9 expression is caused by an enhancement of translational efficiency characterized by a recruitment of translationally inactive MMP-9 mRNP-complexes into the rough endoplasmatic reticulum (rER). Reporter gene assays, which depend on the untranslated regions (UTR) of MMP-9 mRNA, reveal that the posttranscriptional regulation is mainly attributed to the 3′UTR. RNA/protein interaction studies indicate that the elevated binding of nucleolin (∼64 kDa form) to the 3′UTR may be of major importance for the increased efficiency of MMP-9 translation. The results show that MMP-9 expression can be regulated posttranscriptionally, affecting the efficiency of translation and localization of the mRNA.

Keywords: Matrix metalloproteinase 9 (MMP-9); Nucleolin; Posttranscriptional regulation; Untranslated region (UTR); 2,2-Dipyridyl; Hypometabolism

A novel SREBP-1 splice variant: Tissue abundance and transactivation potency by Thomas Klaus Felder; Kerstin Klein; Wolfgang Patsch; Hannes Oberkofler (pp. 41-47).

Sterol regulatory element binding proteins (SREBPs) belong to the family of basic helix–loop–helix–leucine zipper transcription factors. The SREBP-1 gene encodes two different isoforms, SREBP-1a and -1c, that are expressed at varying levels in different tissues and cultured cells and exhibit common and distinct functions. We identified an additional SREBP-1 isoform, termed SREBP-1ac, and determined its mRNA abundance in different human tissues and cell lines. SREBP-1ac mRNA was detectable in all tissues studied, although at lower levels than the major SREBP-1a and -1c isoforms. Transcription of the novel SREBP isoform was not induced by insulin or cholesterol depletion. SREBP-1ac did not transactivate the human LDLR and UCP2 promoters but robustly attenuated the transactivation capacity of SREBP-1a, -1c and -2 in cotransfection experiments.

Keywords: Abbreviations; SREBP; sterol regulatory element binding protein; LDLR; low density lipoprotein receptor; bHLH-LZ; basic helix–loop–helix–leucine zipper; UCP2; uncoupling protein 2SREBP-1; Alternative splicing; Transcriptional regulation

Retinoic acid represses a cassette of candidate pluripotency chromosome 12p genes during induced loss of human embryonal carcinoma tumorigenicity by Caryl J. Giuliano; Joanna S. Kerley-Hamilton; Tom Bee; Sarah J. Freemantle; Ranjan Manickaratnam; Ethan Dmitrovsky; Michael J. Spinella (pp. 48-56).

Testicular germ cell tumors (TGCTs) are the most common carcinomas of young men aged 15–35. The molecular events involved in TGCT genesis are poorly understood. TGCTs have near universal amplification of the short arm of chromosome 12, however positional cloning efforts have not identified causative genes on 12p involved in formation or progression of TGCTs. Human embryonal carcinoma (EC) are the stem cells of TGCTs and are pluripotent. EC cells terminally differentiate toward a neuronal lineage with all- trans retinoic acid (RA) treatment resulting in a concomitant G1 cell cycle arrest and loss of tumorigenicity. Our efforts to define the molecular mechanisms of RA-mediated tumor cell differentiation at a critical “commitment to differentiate�? window has identified a cassette of genes on 12p that are repressed with RA precisely as EC cells lose tumorigenic potential. These are Nanog, CD9, EDR1 (PHC1), SCNN1A, GDF3, Glut3 and Stella. The master pluripotency regulator Oct4 is located on chromosome 6 and is also repressed by RA. Notably, knockdown of Oct4 with siRNA results in repression of basal Nanog, EDR1, GDF3 and Stella gene expression. Nanog has recently been identified to play a role in maintenance of the pluripotency of mouse embryonic stem cells and CD9, EDR1, GDF3, and Stella have each been implicated as stem cell markers. Since RA suppresses the tumorigenicity of EC cells, these genes may have a critical role in the etiology of TGCTs, suggesting a link between enforced pluripotency and transformation.

Keywords: Embryonal carcinoma; Germ cell tumor; Pluripotency; Cancer stem cell; Tumorigenicity; Oct4

A new alternative splice variant of BRCA1 containing an additional in-frame exon by Jessyka Fortin; Anne-Marie Moisan; Martine Dumont; Gilles Leblanc; Yvan Labrie; Francine Durocher; Paul Bessette; Peter Bridge; Jocelyne Chiquette; Rachel Laframboise; Jean L�pine; Bernard Lesp�rance; Roxanne Pichette; Marie Plante; Louise Provencher; Patricia Voyer; Jacques Simard (pp. 57-65).

The breast/ovarian cancer susceptibility gene BRCA1 interact with multiple protein complexes involved in cellular mechanisms, such as DNA repair, transcription, homologous recombination and cell cycle regulation. Extensive analyses over the past decade led to the detection of several BRCA1 alternative splice variants. Here, we identify the first BRCA1 alternative splice variant containing an additional in-frame exon. This previously unknown exon 13A-containing transcript is generated by the insertion of 66 nucleotides between exons 13 and 14, due to alternative splicing in intron 13 (IVS13-2786-2720). Furthermore, exon 13A-containing transcript was detectable in total RNA samples from 12 normal tissues and several breast and other cancer cell lines. As revealed by real-time PCR analysis, this transcript corresponds to 20 to 25% of the total BRCA1 mRNA expression levels in leukocytes, brain and cerebellum tissues, whereas its relative level of expression is less than 5% in other tested tissues and cancer cell lines. This novel alternative transcript adds 22 amino acids after residue 1452, thus modifying the primary structure of the trans-activation domain 1 (AD1) and the protein–protein interacting domain of BRCA1 with BRCA2, AR and MSH2. No sequence variant has been detected by direct genomic sequencing of exon 13A in individuals originating from high-risk breast/ovarian cancer families.

Keywords: BRCA1; Breast cancer predisposition; Alternative splicing

Promoter analysis of the human α1,3/4-fucosyltransferase gene ( FUT III) by Anna Dąbrowska; Dagmara Baczyńska; Katarzyna Widerak; Anna Laskowska; Maciej Ugorski (pp. 66-73).

α1,3/4-Fucosyltransferase (FUT3) is involved in the synthesis of sialyl Le^a tetrasaccharide, a tumor-associated carbohydrate antigen. Fucosyltransferases are thought to be important regulatory enzymes in the synthesis of fucosylated structures. However, there are conflicting data on the role of FUT3 in the synthesis of this carbohydrate structure and more studies on the regulation of FUT III gene expression are needed. Therefore, as first step, the promoter of FUT III gene was cloned and characterized. Sequencing data showed the absence of TATA, CAAT, and GC boxes, but many binding sites for transcription factors, previously described in colon cancer cells, were identified. Analysis of enhancer and silencing elements of deletion mutants revealed the presence of basal promoter elements of the FUT III gene in the region −636 to −674 bp from the translation initiation site, and positive and negative regulatory elements within the −674 bp to −854 bp and −854 to −1220 regions, respectively. 5′-RACE analysis showed the presence of two transcripts with 5′-ends localized within the exon A. The 5′-end of the longer transcript extended −229 nucleotides from the translation start codon and contained a sequence corresponding to an Inr element, localizing the putative transcription initiation site within this sequence. The strong correlation between the promoter activity of the FUT III gene and the high expression of sialyl Le^a observed in different colon carcinoma cell lines seem to confirm the important regulatory role of FUT3 in the synthesis of sialyl Le^a.

Keywords: FUT III; promoter; Cloning; sLe; ^a; Colon cancer

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BBA - Gene Structure and Expression (v.1731, #1)