Inr Motifs Research Articles

Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the MGMT Gene Expression in Human Glioblastoma Cells.

Background: The therapeutically important DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [Int. J. Mol. Sci. 2021, 22(5), 2492], we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types.

TSS seq based core promoter architecture in blood feeding Tsetse fly (Glossina morsitans morsitans) vector of Trypanosomiasis.

BackgroundTranscription initiation regulation is mediated by sequence-specific interactions between DNA-binding proteins (transcription factors) and cis-elements, where BRE, TATA, INR, DPE and MTE motifs constitute canonical core motifs for basal transcription initiation of genes. Accurate identification of transcription start site (TSS) and their corresponding promoter regions is critical for delineation of these motifs. To this end, the genome scale analysis of core promoter architecture in insects has been confined to Drosophila. The recently sequenced Tsetse fly genome provides a unique opportunity to analyze transcription initiation regulation machinery in blood-feeding insects.ResultsA computational method for identification of TSS in newly sequenced Tsetse fly genome was evaluated, using TSS seq tags sampled from two developmental stages namely; larvae and pupae. There were 3134 tag clusters among which 45.4 % (1424) of the tag clusters mapped to first coding exons or their proximal predicted 5′UTR regions and 1.0 % (31) tag clusters mapping to transposons, within a threshold of 100 tags per cluster. These 1393 non transposon-derived core promoters had propensity for AT nucleotides. The −1/+1 and 1/+1 positions in D. melanogaster, and G. m. morsitans had propensity for CA and AA dinucleotides respectively. The 1393 tag clusters comprised narrow promoters (5 %), broad with peak promoters (23 %) and broad without peak promoters (72 %). Two-way motif co-occurrence analysis showed that the MTE-DPE pair is over-represented in broad core promoters. The frequently occurring triplet motifs in all promoter classes are the INR-MTE-DPE, TATA-MTE-DPE and TATA-INR-DPE. Promoters without the TATA motif had higher frequency of the MTE and INR motifs than those observed in Drosophila, where the DPE motif occur more frequently in promoters without TATA motif. Gene ontology terms associated with developmental processes were overrepresented in the narrow and broad with peak promoters.ConclusionsThe study has identified different motif combinations associated with broad promoters in a blood-feeding insect. In the case of TATA-less core promoters, G.m. morsitans uses the MTE to compensate for the lack of a TATA motif. The increasing availability of TSS seq data allows for revision of existing gene annotation datasets with the potential of identifying new transcriptional units.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1921-6) contains supplementary material, which is available to authorized users.

Heterogeneity of Arabidopsis core promoters revealed by high‐density TSS analysis

Our limited understanding of plant promoters does not allow us to recognize any core promoter elements for the majority of plant promoters. To understand the promoter architecture of Arabidopsis, we used the combined approach of in silico detection of novel core promoter elements and large-scale determination of transcription start sites (TSSs). To this end, we developed a novel methodology for TSS identification, using a combination of the cap-trapper and massively parallel signature sequencing methods. This technique, CT-MPSS, allowed us to identify 158 237 Arabidopsis TSS tags corresponding to 38 311 TSS loci, which provides an opportunity for quantitative analysis of plant promoters. The expression characteristics of these promoters were analyzed with respect to core promoter elements detected by our in silico analyses, revealing that Arabidopsis promoters contain two main types of elements with exclusive characteristics, the TATA type and the GA type. The TATA-type promoters tend to be associated with the Y Patch and the Inr motif, and cause high expression with sharp-peak TSS clusters. By contrast, the GA type produces broad-type TSS clusters. Unlike mammalian promoters, plant promoters are not associated with CpG islands. However, plant-specific GA-type promoters share some characteristics with mammalian CpG-type promoters.

Regulation of the rose Rh-PIP2;1 promoter by hormones and abiotic stresses in Arabidopsis

Our previous work has indicated that an ethylene-responsive aquaporin gene, Rh-PIP2;1, played an important role in the epidermal cell expansion of rose petals. In this work, we isolated an 896 bp promoter sequence of the Rh-PIP2;1 and found that the promoter was rare in plants, occurring with an Inr motif, but without a TATA box. In transgenic Arabidopsis harboring the Rh-PIP2;1 promoter::GUS construct, the activity of Rh-PIP2;1 promoter was found to be developmental-dependent in almost all of the tested organs, and was particularly active in organs that were rapidly expanding, and in tissues with high water flux capacity. Moreover, the promoter activity was inhibited by ACC, ABA, NaCl, and cold in the roots of 3 or 6-day-old plants, and was increased by GA(3) and mannitol in the rosettes of 9 or 12-day-old plants. Deleting the fragment from -886 to -828 resulted in nearly complete disappearance of the promoter activity in roots, and a substantial decrease in the leaves, hypocotyls and floral organs. Taken together, our results indicated that the Rh-PIP2;1 promoter responded to hormones and abiotic stresses in a developmental- and spatial-dependent manner, and the -886 to -828 region was crucial for the activity of the Rh-PIP2;1 promoter.

Quality of regulatory elements in Drosophila retrogenes

Retrogenes are processed copies of genes that are inserted into new genomic regions and that acquire new regulatory elements from the sequences in their surroundings. Here we use a comparative approach of phylogenetic footprinting and a non-comparative approach of measuring motif over-representation in retrogenes in order to describe putative elements present in cis-regulatory regions of 94 retrogenes recently described in Drosophila. The detailed examination of the motifs found in the core promoter regions of retrogenes reveals an abundance of the DNA replication-related element (DRE), the Initiator (Inr), and a new over-represented motif that we call the GCT motif. Parental genes also show an abundance of DRE and Inr motifs, but these do not seem to have been carried over with retrogenes. In particular, we also examined motifs upstream of retrogenes expressed in adult testis and were able to identify 6 additional over-represented motifs. Comparative analyses provide data on the conservation and origin of some of these motifs and reveal 15 additional conserved motifs in these retrogenes. Some of those conserved motifs are sequences bound by known transcription factors, while others are novel motifs. In this report we provide the first genome-wide data on which specific cis-regulatory regions can be recruited by retrogenes after they are inserted into new coding regions in the genome. Future experiments are needed to determine the function and role of the new elements presented here.

English

During spermatogenesis the X-chromosome is inactivated, but several glycolytic cycle enzymes have been found to have spermatogenic cell specific isoforms encoded by the X-chromosome counterpart genes located on the autosomes. Here, the promoters of the human glycerol kinase gene family were cloned: the somatic gene called GK-Xp, and the two testis-specific isoforms called GK-1 and GK-2, using Alu-GSP primers. The structure and function of the promoters were determined by sequencing and in situ expression studies using GFP as reporter. The transcription initiation sites were mapped by RACE and, these were found at -112 for GK-Xp, -46 for GK-1, and for GK-2 at -57 nucleotide positions, upstream of the translation start codon. The GK-Xp promoter specifically contains a TATA-like motif at -26, and a CCAAT motif at -149, which are not found in either the GK-1 or GK-2 promoters. Interestingly, GK-1 contains an Inr motif, a Sp1 binding site at -11, and a DPE motif at +27 positions, whereas GK-2contains an Inr motif, a CRE at -11, a DPE at +29 and a RARE motif at +43 positions. In addition, they both contain MEP-2 and Ets motifs, in common with other testis-specific metabolic enzymes genes, such as PGK-2 and PDHA-2. Key words: Spermatogenesis, transcription, gene expression.

Two Inr elements are important for mediating the activity of the proximal promoter of the human gonadotropin-releasing hormone receptor gene.

Differential usage of several transcription start sites in the human GnRH receptor gene was evident in human brain and pituitary. To locate the promoter responsible for a cluster of the 3' CAP sites from -635 to -578 (relative to ATG) found in the pituitary, a proximal promoter element was identified at -677/-558 by 5' and 3' deletion mutant analysis. The promoter element drove a 13.1 +/- 0.6-fold increase in reporter gene activity in an orientation-dependent manner in the mouse gonadotrope-derived alphaT3-1 cells. Within the core promoter element, two functional AT-rich Inr motifs, interacting with the same protein factor with different affinities, were identified. By Southwestern blot analysis and competitive gel mobility shift assays, multiple nuclear factors (36-150 kDa) were found to interact specifically with the core promoter element. Interestingly, these nuclear proteins also interacted with a previously identified distal promoter of the human GnRH receptor gene. Taken together, our studies suggested that these two promoters share common protein factors to regulate transcription initiations at two different regions. Additional mechanisms are needed to modulate the efficiencies of individual promoters for developmental and/or tissue-specific regulations.

Initiator recognition in a primitive eukaryote: IBP39, an initiator-binding protein from Trichomonas vaginalis.

While considerable progress has been made in understanding the mechanisms of transcription in higher eukaryotes, transcription in single-celled, primitive eukaryotes remains poorly understood. Promoters of protein-encoding genes in the parasitic protist Trichomonas vaginalis, which represents one of the deepest-branching eukaryotic lineages, have a bipartite structure with gene-specific regulatory elements and a conserved core promoter encompassing the transcription start site. Core promoters in T. vaginalis appear to consist solely of a highly conserved initiator (Inr) element that is both a structural and a functional homologue of its metazoan counterpart. Using DNA affinity chromatography, we have isolated an Inr-binding protein from T. vaginalis. Cloning of the gene encoding the Inr binding protein identified a novel 39-kDa protein (IBP39). We show that IBP39 binds to both double and single Inr motifs found in T. vaginalis genes and that binding requires the conserved nucleotides necessary for Inr function in vivo. Analyses of the cloned IBP39 gene revealed no homology at the protein sequence level with identified proteins in other organisms or the presence of known DNA-binding domains. The relationship between IBP39 and Inr-binding proteins in metazoa presents interesting evolutionary questions.

Cell Cycle Regulation of the Murine cdc25BPromoter: ESSENTIAL ROLE FOR NUCLEAR FACTOR-Y AND A PROXIMAL REPRESSOR ELEMENT

Expression of the cdc25B gene is up-regulated late during cell cycle progression (S/G(2)). We have cloned the murine cdc25B promoter to identify elements involved in transcriptional regulation. A detailed structure-function analysis led to the identification of several elements that are located upstream of a canonical Inr motif at the site of transcription initiation and are involved in transcriptional activation and regulation. Activation of the promoter is largely mediated by NF-Y and Sp1/3 interacting with one and four proximal binding sites, respectively. In addition, NF-Y plays an essential role in cell cycle regulation in conjunction with a repressor element (cell cycle-regulated repressor) located approximately 30 nucleotides upstream of the putative Inr element and overlapping a consensus TATA motif. The cell cycle-regulated repressor is unrelated to the previously described cell cycle-regulated repressor elements. Taken together, our observations suggest that expression of the cdc25B gene is controlled through a novel mechanism of cell cycle-regulated transcription.

Genomic Organization of the JEM-1 (BLZF1) Gene on Human Chromosome 1q24: Molecular Cloning and Analysis of Its Promoter Region

The Jem-1 (JEM-1, HGMW-approved symbol BLZF1) gene mapping to human chromosome 1q24 codes for a ubiquitously expressed 3-kb mRNA, translated in a 45-kDa nuclear protein. Recent studies have shown a deficient expression of this gene in acute promyelocytic leukemia (APL). However, treatment with retinoids was able to upregulate JEM-1 mRNA in maturing NB4 leukemia cells. Here, we report the characterization of the structural organization of JEM-1. By hybridization screening of a human genomic library derived from blood mononuclear cells, five overlapping genomic DNA clones were isolated. These clones extend over 34 kb of the human genome and comprise the complete JEM-1 gene and a 4-kb 5′flanking region. Determination of the exon–intron structure of Jem-1 revealed seven exons whose junctions with introns exhibited typical splice sequences. A shorter transcript (Jem-1s, 1.3 kb) generated by exon 3 extension and polyadenylation was identified. Its translation generated a 23-kDa protein that exhibited a cytoplasmic localization. 5′RACE–PCR identified a major transcription start site (TSS) located at 403 nt upstream of the ATG. Computer analysis of the 1.8-kb 5′flanking region showed that it lacks a TATA box, Inr motifs or DPE motifs, but it contains a typical CCAAT box located 95 bp upstream of the TSS. Sequencing also revealed potential cis-acting elements for multiple transcription regulators including Sp1, GATA, C/EBP, AP-1, and Pu1. No retinoic acid receptor elements or retinoic X receptor elements were detected. This 1.8-kb DNA sequence showed a strong constitutive promoter activity determined by a luciferase-reporter gene assay in transiently transfected HeLa cells. Retinoids further increased luciferase expression 2.7-fold. We demonstrated that the 1-kb distal sequence contains yet unidentified elements reducing constitutive transcription. Thus, the maximal constitutive promoter activity was assigned to a −432 + 101 region overlapping the TSS. These data support the idea of a constitutive expression of JEM-1, but a negative regulation in APL released by retinoids.

Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element.

Typical metazoan core promoter elements, such as TATA boxes and Inr motifs, have yet to be identified in early-evolving eukaryotes, underscoring the extensive divergence of these organisms. Towards the identification of core promoters in protists, we have studied transcription of protein-encoding genes in one of the earliest-diverging lineages of Eukaryota, that represented by the parasitic protist Trichomonas vaginalis. A highly conserved element, comprised of a motif similar to a metazoan initiator (Inr) element, surrounds the start site of transcription in all examined T. vaginalis genes. In contrast, a metazoan-like TATA element appears to be absent in trichomonad promoters. We demonstrate that the conserved motif found in T. vaginalis protein-encoding genes is an Inr promoter element. This trichomonad Inr is essential for transcription, responsible for accurate start site selection, and interchangeable between genes, demonstrating its role as a core promoter element. The sequence requirements of the trichomonad Inr are similar to metazoan Inrs and can be replaced by a mammalian Inr. These studies show that the Inr is a ubiquitous, core promoter element for protein-encoding genes in an early-evolving eukaryote. Functional and structural similarities between this protist Inr and the metazoan Inr strongly indicate that the Inr promoter element evolved early in eukaryotic evolution.

The Mouse Tumor Necrosis Factor Receptor 2 Gene: Genomic Structure and Characterization of the Two Transcripts

The mouseTNFR2gene has been cloned, sequenced, and characterized as a gene spanning >44 kb of the genome. By alignment of five genomic clones we have established thatTNFR2consists of 10 exons and 9 introns with exons ranging in size from 35 bp to 2.6 kb and introns ranging from 322 bp to >16 kb. All splice acceptor and donor sites conform to the canonical AG/GT rule. The translation initiation and termination sites are located in exon 1 and 10, respectively. AlthoughTNFR2lacks a canonical TATA box, the gene is transcribed from a unique start site located 70 bp upstream of the ATG initiation codon that conforms to the consensus Inr motif. Severalcis-elements for transcription factors were identified in the 5′ flanking region, including NF-1, Sp-1, AP2, γ-IRE, and NF-κB motifs. Functional analysis indicates that the region −705/−412 contains a negativecis-acting element and that the minimal promoter contains motifs that confer LPS inducibility. Two mouseTNFR2mRNAs of 3.2 and 4.1 kb are detected by Northern blot analysis, but until now their origin has not been explained. No evidence of alternative splicing of the coding exons was found. However, hybridization studies and amplification of cDNA ends suggest the use of a noncanonical polyadenylation signal in the untranslated region of exon 10. A comparative analysis of the 3′ untranslated regions of the human and mouseTNFR2genes shows highly divergent 3′ ends. The possibility of an ancestral mouseTNFR2mRNA similar to the short transcript is discussed.

On the Mechanism for Efficient Repression of the Interleukin-6 Promoter by Glucocorticoids: Enhancer, TATA Box, and RNA Start Site (Inr Motif) Occlusion

The feedback inhibition of interleukin-6 (IL-6) gene expression by glucocorticoids represents a regulatory link between the endocrine and immune systems. The mechanism of the efficient repression of the IL-6 promoter by dexamethasone (Dex) was investigated in HeLa cells transiently transfected with plasmid constructs containing different IL-6 promoter elements linked to the herpesvirus thymidine kinase gene (tk) promoter and the bacterial chloramphenicol acetyltransferase gene (cat) and cotransfected with cDNA vectors constitutively expressing either the active wild-type or inactive mutant human glucocorticoid receptor (GR). The induction by interleukin-1, tumor necrosis factor, phorbol ester, or forskolin of IL-6-tk-cat chimeric constructs containing a single copy of the IL-6 DNA segment from -173 to -151 (MRE I) or from -158 to -145 (MRE II), which derive from within the multiple cytokine- and second-messenger-responsive enhancer (MRE) region, was strongly repressed by Dex in a wild-type GR-dependent fashion irrespective of the inducer used. The induction by pseudorabies virus of an IL-6 construct containing the IL-6 TATA box and the RNA start site ("initiator" or Inr element) but not the MRE region was also repressed by Dex in the presence of wild-type GR. DNase I footprinting showed that the purified DNA-binding fragment of GR bound across the MRE, the TATA box, and the Inr site in the IL-6 promoter; this footprint overlapped that produced by proteins present in nuclear extracts from uninduced or induced HeLa cells. Imperfect palindromic nucleotide sequence motifs moderately related to the consensus GR-responsive element (GRE) motif were present at the Inr, the TATA box, and the MRE II site in the IL-6 promoter; although MRE I and a GR-binding site between -201 and -210 in IL-6 both lacked a discernible inverted repeat motif, their sequences showed considerable similarity with negative GRE sequences in other Dex-repressed genes. Surprisingly, chimeric genes containing MRE II, which lacks a recognizable GACGTCA cyclic AMP- and phorbol ester-responsive motif, were strongly induced by both phorbol ester and forskolin, suggesting that MRE II (ACATTGCACAATCT) may be the prototype of a novel cyclic AMP- and phorbol ester-responsive element. Taken together, these observations suggest that ligand-activated GR represses the IL-6 gene by occlusion not only of the inducible IL-6 MRE enhancer region but also of the basal IL-6 promoter elements.

On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion.

The feedback inhibition of interleukin-6 (IL-6) gene expression by glucocorticoids represents a regulatory link between the endocrine and immune systems. The mechanism of the efficient repression of the IL-6 promoter by dexamethasone (Dex) was investigated in HeLa cells transiently transfected with plasmid constructs containing different IL-6 promoter elements linked to the herpesvirus thymidine kinase gene (tk) promoter and the bacterial chloramphenicol acetyltransferase gene (cat) and cotransfected with cDNA vectors constitutively expressing either the active wild-type or inactive mutant human glucocorticoid receptor (GR). The induction by interleukin-1, tumor necrosis factor, phorbol ester, or forskolin of IL-6-tk-cat chimeric constructs containing a single copy of the IL-6 DNA segment from -173 to -151 (MRE I) or from -158 to -145 (MRE II), which derive from within the multiple cytokine- and second-messenger-responsive enhancer (MRE) region, was strongly repressed by Dex in a wild-type GR-dependent fashion irrespective of the inducer used. The induction by pseudorabies virus of an IL-6 construct containing the IL-6 TATA box and the RNA start site ("initiator" or Inr element) but not the MRE region was also repressed by Dex in the presence of wild-type GR. DNase I footprinting showed that the purified DNA-binding fragment of GR bound across the MRE, the TATA box, and the Inr site in the IL-6 promoter; this footprint overlapped that produced by proteins present in nuclear extracts from uninduced or induced HeLa cells. Imperfect palindromic nucleotide sequence motifs moderately related to the consensus GR-responsive element (GRE) motif were present at the Inr, the TATA box, and the MRE II site in the IL-6 promoter; although MRE I and a GR-binding site between -201 and -210 in IL-6 both lacked a discernible inverted repeat motif, their sequences showed considerable similarity with negative GRE sequences in other Dex-repressed genes. Surprisingly, chimeric genes containing MRE II, which lacks a recognizable GACGTCA cyclic AMP- and phorbol ester-responsive motif, were strongly induced by both phorbol ester and forskolin, suggesting that MRE II (ACATTGCACAATCT) may be the prototype of a novel cyclic AMP- and phorbol ester-responsive element. Taken together, these observations suggest that ligand-activated GR represses the IL-6 gene by occlusion not only of the inducible IL-6 MRE enhancer region but also of the basal IL-6 promoter elements.