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BBA - Gene Regulatory Mechanisms (v.1779, #3)
GATA-6 is a novel transcriptional repressor of the human Tenascin-C gene expression in fibroblasts by Angela Ghatnekar; Maria Trojanowska (pp. 145-151).
In this study we show that GATA-6 is a novel repressor of TN-C gene expression. We demonstrated that overexpression of GATA-6 in fibroblasts inhibited basal levels, as well as markedly decreased IL-4- and TGF-β-induced TN-C mRNA and protein levels. A GATA-6 response element was mapped to position −467 to −460 of the TN-C promoter. In addition, we showed that GATA-6 binds this site both in vitro and in vivo.
Keywords: Abbreviations; TN-C; Tenascin-C; CAT; chloramphenicol acetyltransferase; ChIP; Chromatin Immunoprecipitation; IL-4; Interleukin-4; TGF-β; Transforming growth factor-β; ECM; extracellular matrix; COL1A2; α2(I) collagen Tenascin-C; Transcriptional regulation; GATA-6
Multiple ING1 and ING2 genes in Xenopus laevis and evidence for differential association of thyroid hormone receptors and ING proteins to their promoters by Mary J. Wagner; Caren C. Helbing (pp. 152-163).
ING (INhibitor of Growth) tumor suppressor proteins are epigenetic factors involved in numerous cellular processes including apoptosis in species ranging from yeast to humans. We recently isolated ING1 and ING2 transcript variants in Xenopus laevis and showed that these transcripts were differentially regulated by thyroid hormone (TH) during postembryonic development. However, no information exists regarding ING gene structure and how it relates to these differential responses to TH. To further investigate the regulation of ING genes by TH, we isolated ING1 and ING2 gene sequences and demonstrated that there are at least duplicate genes for each. The relationship between transcript variants and their responsiveness to TH were examined through promoter sequence and chromatin immunoprecipitation analyses on tail homogenates. Both TH receptors (TRs) differentially associated with ING1 and ING2 promoter regions with increased recruitment in the presence of TH. This occurred irrespective of gene transcript level response to this hormone. However, differential recruitment of RNA polymerase II corresponded well to transcript levels. ING proteins consistently associated with their own gene promoters except in the region generating the TH-inducible xING1b5 transcript. In this case, a substantial recruitment of TRβ in the absence ING proteins occurred. These data establish the TH-dependent recruitment of transcription factors to ING promoter regions and suggest that differential TR recruitment in response to TH may not be a sufficient indicator for modulating the expression of ING in the tail.
Keywords: Tumor suppressor; Inhibitor of Growth gene; Promoter; Chromatin immunoprecipitation; Thyroid hormone regulation; Frog metamorphosis; Postembryonic development
Signaling pathways mediating the suppression of Arabidopsis thaliana Ku gene expression by abscisic acid by Pei Feng Liu; Wen Chi Chang; Yung Kai Wang; Hwan You Chang; Rong Long Pan (pp. 164-174).
The Ku heterodimer facilitates the regulation of DNA repair, DNA replication, cell cycle, and telomere maintenance. The plant hormone abscisic acid (ABA) is a plant growth inhibitor. This study investigates how Arabidopsis thaliana Ku ( AtKu) genes are regulated by ABA in 3-week-old seedlings. First, β-Glucuronidase assay and real time quantitative PCR analysis results indicate that ABA represses the AtKu gene in a time- and concentration-dependent manner. However, adding of ABA biosynthesis inhibitors, fluride and tungate, did not eliminate AtKu suppression. Moreover, analysis of inhibitor treatments and ABA-responsive mutants suggested that AtKu repression by ABA was mediated through the pathway of extracellular Ca2+, phospholipase D alpha, p38-type mitogen-activated protein kinase (MAPK), MAPK6 and ABA transcription factors, ABI3 and ABI5. Finally, no cross-talk in modulating AtKu gene expression existed between ABA and antagonist hormones (auxins and gibberellic acid).
Keywords: Abbreviations; ABA; abscisic acid; ABRE; abscisic acid responsive element; BAPTA; (1,2-bis(2-aminophenoxy)ethane-; N,N,N’,N’; -tetraacetic acid); bZIP; basic-domain leucine-zipper; DEB; DNA end-binding; 2,4-D; 2,4-dichlorophenoxyacetic acid; EGTA; ethylene glycol bis(2-aminoethylether)-; N,N,N’,N’; -tetraacetic acid; ERK; extracellular-regulated kinase; FLU; fluride; GA; gibberellic acid; GUS; β-glucuronidase; HD-Zip; homeodomain leucine zipper; IAA; indole-3-acetic acid; MAPK; mitogen-activated protein kinase; 4-MUG; 4-methylumbelliferyl β-; d; -glucuronide; NAA; α-naphthalene acetic acid; PLC; phospholipase C; PLD; phospholipase D; TUN; tungate; U-73122; [1-(6-[17 beta-3-methoxyestra-1,3,5- (10) triene-17-yl] amino/hexyl) 1H-pyrroledione]Abscisic acid; DNA end-binding; DNA double-stranded break; Gene regulation; Phytohormone; Signal transduction
Prior interaction of ATP with yeast mRNAs enhances protein synthesis at the initiation step by Juana Ledia Triana; Yda Méndez; Luis Navarro; Ronald Reggio; Ana Celia Ferreras; Cayama Edmundo; Francisco J. Triana-Alonso; Flor Herrera (pp. 175-182).
ATP hydrolysis is important for different stages of the protein synthesis process. A novel effect of this nucleotide was detected using mRNAs isolated from S. cerevisiae after phenol extraction of polysomes. When polysomal mRNA (pmRNA) or poly(A)+ RNA were preincubated with ATP (~3 mM, near physiological concentration), their translational activity in a cell-free system from yeast was stimulated 2–3 fold. This increased translational activity is specific for the poly(A)+ RNA fraction, correlates with facilitated assembly of 80S initiation complexes, and is associated to increased synthesis of high molecular weight polypeptides. TCA precipitation assays of RNA incubated with [14C]ATP suggested an association of the nucleotide with the nucleic acid. The amount of [14C]ATP co-precipitated was dependent on magnesium (optimum at 5–6 mM), was partially inhibited by monovalent ions, and was maximal with poli(A)+ RNA. Existence of RNA-associated kinases or ATPases appear unlikely since neither phosphorylation nor nucleotide hydrolysis were observed during preincubation of pmRNA with ATP. Another evidence of ATP-RNA interaction was an increased absorbance at 260 nm after incubation suggesting unwinding of the RNA secondary structure. Therefore, preincubation with ATP may affect the conformation of mRNAs and thereby facilitate the initiation of protein synthesis. This event could be part of an in vivo energy-dependent mechanism for translational control.
Keywords: Poly(A); +; RNA; ATP; Translation; Initiation complex
ERM transcription factor contains an inhibitory domain which functions in sumoylation-dependent manner by Cindy Degerny; Yvan de Launoit; Jean-Luc Baert (pp. 183-194).
ERM, PEA3 and ETV1 belong to the PEA3 group of ETS transcription factors. They are involved in many developmental processes and are transcriptional regulators in metastasis. The PEA3 group members share an N-terminal transactivation domain (TAD) whose activity is inhibited by a flanking domain named the negative regulatory domain (NRD). The mechanism of this inhibition is still unknown. Here we show that the NRD maps to residues 73 to 298 in ERM and contains three of the five SUMO sites previously identified in the protein. We demonstrate that these three SUMO sites are responsible for NRD's inhibitory function in the Gal4 system. Although the presence of the three sites is required to obtain maximal inhibition, only one SUMO site is sufficient to repress transcription whatever its localization within the NRD. We also show that NRD is a SUMO-dependent repression domain that can act in cis and in trans to downregulate the powerful TAD of the VP16 viral protein. In addition, we find that the SUMO sites outside the NRD also play a role in the negative regulation of full-length ERM activity. We thus postulate that each SUMO site in ERM may function as an inhibitory motif.
Keywords: ETS; ERM; Transcription factor; SUMO; Regulation of transcription
Zinc mesoporphyrin induces rapid and marked degradation of the transcription factor bach1 and up-regulates HO-1 by Weihong Hou; Ying Shan; Jianyu Zheng; Richard W. Lambrecht; Susan E. Donohue; Herbert L. Bonkovsky (pp. 195-203).
Heme oxygenase 1 (HO-1) is the first and rate-controlling enzyme in heme degradation. Bach1 is a mammalian transcriptional repressor of HO-1. To understand how zinc mesoporphyrin (ZnMP) induces the expression of HO-1, we investigated the effects of ZnMP on Bach1 mRNA and protein levels in human hepatoma Huh-7 cells by quantitative RT-PCR and Western blots. We found that ZnMP markedly up-regulated HO-1 mRNA and protein levels, and rapidly and significantly decreased Bach1 protein levels by increasing degradation of Bach1 protein [half life ( t1/2) from 19 h to 45 min], whereas ZnMP did not influence Bach1 mRNA levels. The proteasome inhibitors, epoxomicin and MG132, significantly inhibited degradation of Bach1 by ZnMP in a dose-dependent fashion, indicating that the degradation of Bach1 by ZnMP is proteasome-dependent. Purified Bach1 C-terminal fragment bound heme, but there was no evidence for binding of ZnMP to the heme-binding region of Bach1. In conclusion, ZnMP produces profound post-transcriptional down-regulation of Bach1 protein levels and transcriptional up-regulation of HO-1. Our results indicate that ZnMP up-regulates HO-1 gene expression by markedly increasing Bach1 protein degradation in a proteasome-dependent manner.
Keywords: Zinc mesoporphyrin; Post-transcriptional regulation; Proteasome; Bach1; HO-1; Heme