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BBA - Gene Structure and Expression (v.1769, #9-10)

Editorial Board (pp. ii).

YB-1 binds to the MMP-13 promoter sequence and represses MMP-13 transactivation via the AP-1 site by Shaija Samuel; Katherine K. Beifuss; Lori R. Bernstein (pp. 525-531).

Matrix metalloproteinases (MMPs) are key enzymes that implement degradation of the extracellular matrix during cellular invasion in development, tissue remodeling, and pathogenic disease states. MMP-13 has pivotal roles in the pathogenesis of invasive cancers and arthritis. Here we report the identification of Y-box binding protein-1 (YB-1) as a new repressor of MMP-13 transactivation. YB-1 binds in vitro in DNA affinity chromatography to the activator protein-1 (AP-1) DNA sequence within the MMP-13 promoter. Chromatin immunoprecipitation assays reveal that YB-1 binds in living cells to the MMP-13 gene promoter to a region of the MMP-13 promoter containing the AP-1 site. YB-1 represses tumor promoter-induced MMP-13 promoter transactivation at the AP-1 site. This is the first report demonstrating YB-1 binding in vitro and in living cells to a mammalian AP-1 target gene, and the first report of YB-1 regulation of the MMP-13 promoter.

Keywords: Activator protein-1 (AP-1); Chromatin immunoprecipitation (ChIP); Matrix metalloproteinase-13 (MMP-13); NAPSTER; Transactivation; Y-box binding protein-1 (YB-1)

Translational regulation of human methionine synthase by upstream open reading frames by Bekir Col; Sebastian Oltean; Ruma Banerjee (pp. 532-540).

Methionine synthase is a key enzyme poised at the intersection of folate and sulfur metabolism and functions to reclaim homocysteine to the methionine cycle. The 5′ leader sequence in human MS is 394 nucleotides long and harbors two open reading frames (uORFs). In this study, regulation of the main open reading frame by the uORFs has been elucidated. Both uORFs downregulate translation as demonstrated by mutation of the upstream AUG codons (uAUG) either singly or simultaneously. The uAUGs are capable of recruiting the 40S ribosomal complex as revealed by their ability to drive reporter expression in constructs in which the luciferase is fused to the uORFs. uORF2, which is predicted to encode a 30 amino acid long polypeptide, has a clustering of rare codons encoding arginine and proline. Mutation of a tandemly repeated rare codon for arginine at positions 3 and 4 in uORF2 to either common codons for the same amino acid or common codons for alanine results in complete alleviation of translation inhibition. This suggests a mechanism for ribosome stalling and demonstrates that the cis-effects on translation by uORF2 is dependent on the nucleotide sequence but is apparently independent of the sequence of the encoded peptide. This study reveals complex regulation of the essential housekeeping gene, methionine synthase, by the uORFs in its leader sequence.

Keywords: Abbreviations; MS; methionine synthase; ORF; open reading frame; UTR; untranslated region; uORF; upstream open reading frame; uAUG1, and 2; initiation codons for uORFs 1 and 2; IRES; internal ribosome entry siteMethionine; Upstream open reading frame; Methionine synthase; Gene regulation

The transcriptional repression by NIPP1 is mediated by Polycomb group proteins by Nivedita Roy; Aleyde Van Eynde; Lijs Beke; Mieke Nuytten; Mathieu Bollen (pp. 541-545).

NIPP1 is a ubiquitously expressed nuclear protein that represses the transcription of targeted genes. Here we show that the transcriptional repression by NIPP1 is alleviated by the RNAi-mediated knockdown of EED and EZH2, two core components of the Polycomb Repressive Complex 2 (PRC2), and by the overexpression of a catalytically dead mutant of the histone methyltransferase EZH2. NIPP1 is present in a complex with EED and EZH2 in vivo and has distinct binding sites for these proteins. These data disclose an essential role for the PRC2 complex in the transcriptional repression by NIPP1.

Keywords: EED; EZH2; NIPP1; Polycomb group proteins; PRC2; Transcriptional repression

Transcriptional and epigenetic regulation of the integrin collagen receptor locus ITGA1-PELO-ITGA2 by Yann Cheli; Sachiko Kanaji; Beatrice Jacquelin; Mei Chang; Diane J. Nugent; Thomas J. Kunicki (pp. 546-558).

The integrin collagen receptor locus on human chromosome 5q11.2 includes the integrin genes ITGA1 and ITGA2, and the cell cycle regulation gene PELO, embedded within ITGA1 intron 1. ITGA1 contains a CArG box that is bound by serum response factor (SRF), while PELO contains two Sp1 binding elements. A comparison of mRNA levels in megakaryocytic (MK) and non-megakaryocytic (non-MK) cell lines and an analysis of the transcriptional activity of promoter-LUC reporter gene constructs in transfected cells revealed that ITGA1 is selectively suppressed in the MK lineage. Sodium bisulfite genomic sequencing established that a CpG-rich ITGA1 promoter region (−209/+115) is fully methylated at 19 CpG sites in MK cells that do not express α1β1, but completely demethylated in expressing cells. In vitro methylation of ITGA1 suppresses transcription, while treatment of megakaryocytic cells with 5-aza-2′-deoxycytidine, but not Trichostatin A, resulted in de novo expression of ITGA1. During thrombopoietin-induced in vitro differentiation of primary human cord blood mononuclear cells into megakaryocytes, we observed rapid, progressive CpG methylation of ITGA1, but not PELO or ITGA2. Thus, selective CpG methylation of the ITGA1 promoter is a specific feature of α1β1 regulation that coincides with the initiation of megakaryocyte differentiation.

Keywords: Integrin; Pelota; Collagen; Megakaryocyte; Gene; Differentiation

Identification and functional analysis of Relish homologs in the silkworm, Bombyx mori by Hiromitsu Tanaka; Hiroyuki Matsuki; Seiichi Furukawa; Aki Sagisaka; Eiji Kotani; Hajime Mori; Minoru Yamakawa (pp. 559-568).

Two cDNAs designated BmRelish1 and 2, that encode Relish homologs, were cloned from the silkworm, Bombyx mori. BmRelish1 had an IκB-like domain with 5 ankyrin repeats in addition to Rel homology domain (RHD), nuclear localization signal (NLS), and acidic and hydrophobic amino acids (AHAA) rich regions. On the other hand, BmRelish2 lacked the AHAA and ankyrin repeats (ANK). Knockdown of the BmRelish gene in transgenic silkworms resulted in failure of the activation of antimicrobial peptide genes by Escherichia coli, suggesting that BmRelish plays an important role in antimicrobial peptide gene expression. Functional analysis of BmRelish1 and 2 in mbn-2 cells showed that both Relish homologs do not activate promoters of B. mori antimicrobial peptide genes encoding cecropin B1, attacin, lebocin 3 and lebocin 4. However, a gene construct BmRelish1-d2 lacking the ANK strongly activated promoters of these genes. Another gene construct lacking AHAA and ANK failed to activate these genes, suggesting that BmRelish becomes active by removal of the ANK and that the AHAA-rich region is a transactivation domain. BmRelish2 was shown to repress activation of Cecropin B1 gene expression by BmRelish1-d2, suggesting that BmRelish2 plays a role as a dominant negative factor against the BmRelish1 active form. Necessity of κB sites of Cecropin B1, Attacin and Lebocin 4 genes for the full activation of these genes by BmRelish1-d2 was confirmed. The requirement of the mandatory κB sites for Lebocin 4 gene expression was different between BmRelish1 active form and BmRelA, suggesting differential roles for κB sites in antimicrobial peptide gene activation by different transcription factors. The binding of the RHD portion of BmRelish1 fusion protein to the κB sites of Cecropin B1 and Attacin genes was also confirmed.

Keywords: Insect immunity; Relish; Silkworm; NF-κB; Antimicrobial peptide gene

HDAC1 bound to the Cyp1a1 promoter blocks histone acetylation associated with Ah receptor-mediated trans-activation by Michael Schnekenburger; Li Peng; Alvaro Puga (pp. 569-578).

Metabolic bioactivation of polycyclic aromatic hydrocarbons, such as the environmental procarcinogen benzo[ a]pyrene, is catalyzed by a cytochrome P450 monooxygenase encoded by the substrate-inducible Cyp1a1 gene. Cyp1a1 induction requires trans-activation by the heterodimeric transcriptional complex formed by the liganded Ah receptor (AHR) and its partner, ARNT. Previously, we showed that constitutively bound HDAC1 dissociates from Cyp1a1 promoter chromatin after ligand-mediated induction, concomitantly with the recruitment of AHR/ARNT complexes and p300. Here, we investigated the hypothesis that HDAC1 binding maintains the Cyp1a1 gene in a silenced state in uninduced cells. We find that Cyp1a1 induction by the AHR/ARNT is associated with modification of specific chromatin marks, including hyperacetylation of histone H3K14 and H4K16, trimethylation of histone H3K4, and phosphorylation of H3S10. HDAC1 and DNMT1 form complexes on the Cyp1a1 promoter of uninduced cells but HDAC1 inhibition alone is not sufficient to induce Cyp1a1 expression, although it allows for the hyperacetylation of H3K14 and H4K16 to levels similar to those found in B[ a]P-induced cells. These results show that by blocking the modification of histone marks, HDAC1 plays a central role in Cyp1a1 expression and that its removal is a necessary but not sufficient condition for Cyp1a1 induction, underscoring the requirement for a concerted series of chromatin-remodeling events to complete the initial steps of gene trans-activation by the Ah receptor.

Keywords: Abbreviations; AHR; aryl hydrocarbon receptor; AhRE; AHR response element; ARNT; aryl hydrocarbon receptor nuclear translocator; Aza; 5-Aza-2′-deoxycytidine; B[; a; ]P; benzo[; a; ]pyrene; BCoR; BCL6 corepressor; bHLH/PAS; basic region/helix–loop–helix/Per–ARNT–Sim; BRG1; brm (brahma)/SWI (singed wings) 2-related gene-1; CBP; CREB (cAMP responsive element binding protein) binding protein; ChIP; chromatin immunoprecipitation; CRM-1; chromosome region maintenance protein-1; DNMT; DNA methyltransferase; DRE; dioxin response element; ERAP140; estrogen receptor-associated protein of 140 kD; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HAH; halogenated aromatic hydrocarbons; HDAC; histone deacetylase; HIF-1β; hypoxia-inducible factor 1β; HSP; heat–shock protein; NaB; sodium butyrate; NCoA-2; nuclear receptor coactivator-2; NCoR; nuclear corepressor; PAH; polycyclic aromatic hydrocarbons; QRT-PCR; quantitative real-time PCR; RIP140; receptor-interacting protein 140; SMRT; silencing mediator for retinoid acid and thyroid hormone receptors; Sp; specific protein; SRC-1; steroid receptor coactivator-1; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; XAP-2; hepatitis B virus X-associated protein-2; XRE; xenobiotic response elementBenzo[; a; ]pyrene; Cyp1a1; Epigenetics; HDAC1; DNMT1; Histone modification

The identification and sequence analysis of a new Reg3gamma and Reg2 in the Syrian golden hamster by Mauro L. Castellarin; Maria Petropavlovskaia; Mark A. Lipsett; Lawrence Rosenberg (pp. 579-585).

The regenerating ( Reg) genes are associated with tissue repair and have been directly implicated in pancreatic β-cell regeneration. A hamster Reg3, Islet neogenesis associated protein (INGAP), has been shown to possess anti-diabetic properties in rodent models. Although several Reg3 proteins have been identified in other species, INGAP is the only Reg3 found in hamsters. To identify new Reg3 genes in the hamster pancreas we employed homology reverse transcription polymerase chain reaction (RT-PCR) using degenerate Reg3 primers, followed by rapid amplification of cDNA ends (RACE). We report here the discovery of a new hamster Reg3 gene of 765 nucleotides (nt) that encodes a 174-amino acid (aa) protein. This protein sequence was identified as a novel hamster Reg3γ with 78% and 75% identity to the rat Reg3γ and mouse Reg3γ protein, respectively. We also fully sequenced the previously reported partial sequence of the hamster Reg1 gene coding region using RACE to yield a 756-nt transcript that encodes a deduced 173 aa protein. This protein was identified as hamster Reg2, rather than Reg1 as was initially reported, with an 81% identity to mouse Reg2. The spatial gene expression patterns of the hamster Reg genes, analyzed by RT-PCR, were similarly distributed with low level expression being found globally throughout the body. Mice and hamsters are the only species known to carry either of the functional INGAP or Reg2 genes. It remains to be determined whether these genes bestow mice and hamsters with special regenerative abilities in the pancreas.

Keywords: Reg; INGAP; Tissue regeneration; Islet neogenesis; Gene discovery

The Popdc gene family in the rat: Molecular cloning, characterization and expression analysis in the heart and cultured cardiomyocytes by Doris Parnes; Vered Jacoby; Avital Sharabi; Hadassa Schlesinger; Thomas Brand; Gania Kessler-Icekson (pp. 586-592).

Three Popeye domain-containing ( Popdc 1–3) family-members are known in vertebrates. Their exact function is as yet unknown although involvement in cell adhesion has been suggested. We report herein sequencing of the rat Popdc 1–3 cDNAs that show high homology to other vertebrate orthologs and are expressed primarily in the heart and skeletal muscles. Popdc2 splice variants were identified, with Popdc2C showing a distinctive age-dependent decline. In isolated cardiomyocytes, Popdc genes were negatively regulated by serum, an effect that was reversed by EGFR-kinase inhibition, suggesting an EGFR-dependent modulation of Popdc gene expression.

Keywords: Popeye domain containing (Popdc); Rat; Heart; Postnatal-regulation; Cardiomyocytes; Serum; EGF receptor

Functional characterization of the promoter of human kinetochore protein HEC1: Novel link between regulation of the cell cycle protein and CREB family transcription factors by Liansheng Cheng; Liangwei Li; Xinxian Qiao; Jing Liu; Xuebiao Yao (pp. 593-602).

HEC1 (highly expressed in cancer), which localizes to kinetochore in cell mitosis, plays an essential role in chromosome segregation for M phase progression. To clarify the mechanism of its transcriptional regulation, we searched out and isolated its 5′-flanking region. Mapping of this region identified that it is a TATA-less promoter and contains several putative binding sites for different transcription factors. The results from HeLa cells transfected with pGL3 luciferase reporter vectors containing progressive deletion of the HEC1 5′-flanking region demonstrated that two elements containing binding sites for cAMP responsive element binding (CREB) protein and activating transcription factor 4 (ATF4 or CREB2) are critical for transcriptional activity. Mutation of the two elements, not downstream E2F box, resulted in a significant reduction of the promoter activity. Gel shift and supershift assays also demonstrated specific binding of transcription factors to their putative binding sites. Furthermore, overexpression of either CREB or ATF4 enhanced the activation of the HEC1 promoter and overexpression of both of them had an additive effect on the activation of the HEC1 transcription. Conversely, overexpression of dominant negative mutants of either CREB or ATF4 resulted in downregulation of HEC1 mRNA significantly. Our study provided a new insight into a potential mechanism of how transcription factors of CREB family are involved in the regulation of kinetochore protein HEC1 in cancer-related cells.

Keywords: HEC1; Kinetochore; Promoter; CREB; ATF4

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BBA - Gene Structure and Expression (v.1769, #9-10)

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BBA - Gene Structure and Expression (v.1769, #9-10)