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BBA - Gene Regulatory Mechanisms (v.1779, #2)
The DRE/DREF transcriptional regulatory system: a master key for cell proliferation by Akio Matsukage; Fumiko Hirose; Mi-Ae Yoo; Masamitsu Yamaguchi (pp. 81-89).
The coordinate expression of many cell proliferation-related genes is required for the cellular shift from the resting state into the proliferating state. One regulatory factor involved in this process, the transcription regulatory factor named DREF (DNA replication-related element-binding factor) was discovered in Drosophila and later found to have orthologues in other species including human. Drosophila DREF is a homo-dimer of a polypeptide of 709 amino acid residues, and shares about 22% identity in its amino acid sequence with the human homolog of 694 amino acid residues. The Drosophila DREF homo-dimer binds specifically to the DRE sequence (5'-TATCGATA) in the promoters of many DNA replication/ cell proliferation-related genes to activate their transcription, and the N-terminal region of DREF carries a domain for specific DRE-binding and homo-dimer formation. Ectopic expression of DREF in eye imaginal discs induces abnormal DNA synthesis, apoptosis and failure to differentiate. Conversely, expression of the dominant negative N-terminal region in larval salivary glands reduces endo-replication. Furthermore, RNA interference-mediated knockdown of DREF in vivo demonstrated its requirement for normal progression through the cell cycle and consequently for growth of imaginal discs and the endoreplicating organs. Both Drosophila and human DREF's interact genetically and physically with regulatory factors related to chromatin structures, suggesting that DREF activates the expression of proliferation-related genes through modification of the 3-D conformation of DNA. A search of the Drosophila genome database identified about 150 genes carrying DRE sequences in their promoter regions, many of which are related to reactions required for cell proliferation such as DNA replication, transcriptional regulation, cell cycle regulation, growth signal transduction and protein metabolism. Thus, DREF appears to be a master key-like factor for cell proliferation. Several differentiation-related transcription factors containing homeodomains down-regulate the function or expression of DREF by distinct mechanisms, suggesting a differentiation-coupled repression of cell proliferation via the DRE/DREF system.
Keywords: DREF; Transcription; Proliferation; DifferentiationAbbreviations; DRE; DNA replication-related element; DREF; DRE-binding factor; PCNA; proliferating cell nuclear antigen; TBP; TATA-box-binding protein; TRF2; TBP-related factor 2; GST; glutathione S-transferase; pol α; DNA polymerase α; bp; base pair; D. melanogaster; Drosophila melanogaster; D. virilis; Drosophila virilis
Isolation and comparative expression analysis of six MBD genes in wheat by Yongchun Li; Fanrong Meng; Jun Yin; Haoying Liu; Zhifei Si; Zhongfu Ni; Qixin Sun; Jiangping Ren; Hongbin Niu (pp. 90-98).
The 5-methylcytosines (m5C) play critical roles in epigenetic control, often being recognized by proteins containing an MBD. In this study, we isolated six wheat cDNAs with open reading frame encoding putative methyl-binding domain proteins, which were designated as TaMBD1– TaMBD6, respectively. BLASTX searches and phylogenetic analysis suggested that the six TaMBD genes belonged to four (I, II, III and VIII) of the eight subclasses of MBD family. Genomic analysis showed that a 1386 bp intron was included in TaMBD1 and a 12-bp intron was found in TaMBD4. The expression profiles of the six TaMBDs were studied via Q-RT-PCR and the results indicated that the TaMBDs were differentially expressed in detected wheat tissues. It was interesting to note that 3 TaMBDs were highly expressed in dry seeds and endosperms. Moreover, the differential expression patterns of TaMBDs were observed in leaves and roots under water-stress. We concluded that multiple wheat MBD genes were present and they might play important roles in wheat growth and development, as well as in the water-stress response.
Keywords: Wheat; MBD genes; Q-RT-PCR; Gene expression; Water-stress
Small RNAs in tomato fruit and leaf development by Asuka Itaya; Ralf Bundschuh; Anthony J. Archual; Je-Gun Joung; Zhangjun Fei; Xinbin Dai; Patrick X. Zhao; Yuhong Tang; Richard S. Nelson; Biao Ding (pp. 99-107).
Tomato fruit and leaf development offers excellent systems to study the evolution of gene regulation underlying development of different organs. We have identified over 350 and 700 small RNAs from tomato fruit and leaf, respectively. Except for conserved microRNAs, more than 90% of the small RNAs are unique to tomato. We confirmed expression of some conserved as well as novel putative microRNAs by Northern hybridization. Our results help form a basis for comparative studies on how small RNA-mediated gene expression has contributed to the evolution of common and distinct developmental pathways of fruits and leaves. We have established a website (http://ted.bti.cornell.edu/digital/sRNA/) for public access to all of our small RNA sequences, their expression patterns in respective tissues, and their matching genes or predicted target genes in a searchable manner.
Keywords: Tomato; Small RNA; miRNA; siRNA; Fruit development; Leaf development
Protein synthesis inhibitors enhance the expression of mRNAs for early inducible inflammatory genes via mRNA stabilization by Soh Yamazaki; Koichiro Takeshige (pp. 108-114).
Expression of inflammatory genes is regulated at multiple steps, including transcriptional activation and mRNA stabilization. During an investigation into the requirement of de novo protein synthesis for the induction of inflammatory genes, it was revealed that protein synthesis inhibitors unexpectedly potentiated the induction of mRNAs for primary response genes, while the inhibitors suppressed the induction of secondary inducible genes as previously described. Stimulus-induced nuclear translocation and promoter recruitment of NF-κB, which is responsible for the transcriptional activation of many inflammatory genes, were largely unaffected by the inhibitors. Instead, these inhibitors prolonged the half-lives of all of the primary inducible mRNAs tested. Thus, these findings emphasize the important contribution of regulated mRNA longevity to gene expression induced by pro-inflammatory stimulation.
Keywords: Abbreviations; NF-κB; nuclear factor-κB; LPS; lipopolysaccharide; CHX; cycloheximide; qPCR; quantitative real-time PCR; ChIP; chromatin immunoprecipitationGene expression; Inflammation; Transcription; NF-κB; Post-transcriptional regulation; mRNA stability
Pax3 regulates Wnt1 expression via a conserved binding site in the 5′ proximal promoter by Benjamin T. Fenby; Vassiliki Fotaki; John O. Mason (pp. 115-121).
The development of the neural crest is orchestrated by a complex interplay between intercellular signalling molecules and transcription factors. Here, we demonstrate a direct interaction between two such factors, the paired-type transcription factor Pax3 and the secretory glycoprotein Wnt1. We found that the Wnt1 promoter can be regulated by Pax3 in a dose-dependent manner. Sequence analysis predicted a conserved binding site for Pax3 within the Wnt1 promoter region. Deletion or mutation of this sequence abolished the promoter response to Pax3. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated that Pax3 interacts with the Wnt1 promoter in vivo. These data indicate that Pax3 directly regulates the expression of Wnt1 in the developing embryo.
Keywords: Neural crest; Transcription; Growth factor; Pax gene; Wnt
Molecular cloning and functional analysis of an ERF gene from cotton ( Gossypium hirsutum) by Zhi-Xin Qiao; Bo Huang; Jin-Yuan Liu (pp. 122-127).
Ethylene response factors (ERFs) play important roles in regulating plant biotic and abiotic stress tolerance. In this paper, a new ethylene response factor gene GhERF1 was isolated from cotton. The deduced amino acid sequence of GhERF1 contained an AP2/ERF domain, which shared high similarity with other reported AP2/ERF domains and was most closely related to the B3 subgroup of the ERF subfamily. The particle bombardment assay showed that GhERF1 functioned as an in vivo transcription activator in tobacco cells and was localized in the nuclei of onion epidermis cells. In addition, semi-quantitative RT-PCR revealed that GhERF1 accumulated highly and rapidly when plants were treated with exogenous ethylene, abscisic acid (ABA), high salinity, cold and drought. These results suggested that GhERF1 might be functionally important in acclimation of cotton to stress.
Keywords: Ethylene response factor (ERF); Stress; Nuclear localization; Transactivation; Gossypium hirsutum
Regulatory elements in the KlHEM1 promoter by Laura Núñez; Ana Rodríguez-Torres; María Esperanza Cerdán (pp. 128-133).
In Kluyveromyces lactis the gene encoding 5-aminolevulinate synthase, KlHEM1, is regulated at the transcriptional level by carbon source and oxygen availability. The KlHEM1 promoter, fused to the reporter lacZ gene, has been analysed by deletion and direct mutagenesis techniques in order to find regulatory elements functionally relevant in this transcriptional regulation. Two regulatory regions which contain the consensuses for KlGcr1p and KlMig1p binding are functional in the promoter. Regulation by carbon source is oppositely dependent on KlGcr1p and KlMig1p, as also confirmed by the use of mutants in these regulatory factors. A pyrimidine-rich element is necessary for full expression of KlHEM1 both in aerobic and hypoxic conditions. Deletion analyses show that a regulatory sequence included in the region −656 to −558 is also necessary to reach high KlHEM1 expression in hypoxia. Hypoxic expression and repression caused in this condition by externally added deuteroporphyrin IX is independent of the consensuses for KlHap1p and KlBuf1p binding. Effects caused by disruption of the genes coding for the regulatory factors KlHap1p, KlRox1p and KlMot3p on KlHEM1 aerobic transcription do not fully explain the differences observed between normoxic and hypoxic expression.
Keywords: Abbreviations; ALAS; 5-aminolevulinate synthase; dpIX; deuteroporphyrin IX; Kl; followed by the name of a gene indicates that this gene is from; Kluyveromyces lactis; Sc; followed by the name of a gene indicates that this gene is from; Saccharomyces cerevisiae; Hap2c; the protein complex Hap2–Hap3–Hap4–Hap55-aminolevulinate synthase; Heme; Carbon source; Kluyveromyces lactis
Promoter analysis of the mouse Peg3 gene by Jun Song; Yingchun Lu; An Giang; Shen Pang; Robert Chiu (pp. 134-138).
Mouse Peg3 is a paternally expressed gene. Study of methylation of the Peg3 gene in P19 embryonal carcinoma cells suggested that monoallelic methylation of CpG dinucleotides is not only present in the promoter region, but also in the first exon and the first intron. Promoter activity analysis demonstrated that the minimal promoter of the Peg3 gene is located in the region between −827 and +712 and the critical region for promoter activity is between +423 and +712. We further identified the roles of the cis-elements, conserved sequence element (CSE) and YY1-binding sites, in the regulation of Peg3 expression and found that CSE is involved in the inhibition of Peg3 expression, while YY1-binding sites serve as activating cis-elements to antagonize CSE-mediated inhibition.
Keywords: Peg3; YY1; Promoter; Transcription; Imprinting gene; DNA methylation
Characterization of a neuregulin-1 gene promoter: Positive regulation of type I isoforms by NF-κB by Timo Frensing; Christian Kaltschmidt; Thomas Schmitt-John (pp. 139-144).
The neuregulin-1 ( Nrg1) gene encodes for a group of growth factors with multiple functions during mammalian development. Overexpression of Nrg1 is found in many different cancer types and correlates with cancer progression and an aggressive phenotype. In this study we identified the promoter of Nrg1 type I isoforms. Reporter gene assays revealed that 850 bp upstream from the translation initiation codon are necessary for high transcriptional activity in murine Neuro-2A neuroblastoma cells. The core promoter is highly conserved among mammals, has multiple transcription start sites and is located in a CpG island. The conserved promoter region contains GC-and GT-box elements and overexpression of Sp1 increased promoter activity, while ZBP-89 decreased the activity. Overexpression of the NF-κB subunit p65 (RelA) led to a strong activation of the promoter mediated through a single NF-κB binding site. Reflecting that the transcriptional activity of NF-κB and Sp1 are increased upon Nrg-stimulation in breast cancer cells this study suggests a potential mechanism of a positive feedback loop/autoregulation of neuregulin.
Keywords: Sp1; ZBP-89; Heregulin; Breast cancer; Autoregulation; Transcriptional regulation