Characterization Of Cis-elements Research Articles

Characterization of cis-elements in hormonal stress-responsive genes in Oryza sativa

Phytohormones play a key role in plant growth and development. The process of plant’s perception and response to abiotic and biotic stresses is controlled mainly by the phytohormones which act as an endogenous messenger in the regulation of the plant’s status. They can be activated by different signaling pathways in response to environmental stresses. Plants respond to environmental stress through interaction of transcription factors with a handful of cis-regulatory elements (CREs). Some examples of cis elements include abscisic acid-responsive element (ABRE), G-box (CACGTG) element, and W-box. In order to investigate the effects of different hormonal stresses which have a key role in response to biotic and abiotic stresses in rice, microarray data was used. Of the available data, 931 genes revealed significant differences in response to different hormonal stresses such as auxin, cytokinin, abcisic acid, ethylene, salicylic acid, and jasmonic acid. The present results showed that 388 genes were up-regulated, and 543 genes were down-regulated. Most of the genes were up-regulated in response to Indole-3-acetic acid (IAA) hormone. Genes Ontology analysis revealed that they respond to various hormones involved in auxin- responsive genes, auxin-activated signaling pathway and cellular responses to environmental stimuli. G-box had the highest number of cis elements involved in hormonal stress and was regulated by auxin signaling and various stresses. Dehydrin was the only gene up-regulated in response to the six hormones. This gene can be activated in response to abiotic and biotic stresses. As such, dehydrin gene can be used in crop breeding programs to increase tolerance to different environmental stresses in various plant species.

Characterization of cis-elements in the promoter of trz2 encoding Schizosaccharomyces pombe mitochondrial tRNA 3'-end processing enzyme.

The endonuclease tRNase Z is responsible for the 3'-end processing of tRNA precursors, which is one of the essential steps in tRNA maturation. The fission yeast Schizosaccharomyces pombe contains two essential tRNase ZL genes (trz1 and trz2) involved in nuclear and mitochondrial tRNA 3'-end processing, respectively. Our previous studies suggest that trz2 is expressed at a very low level. Here we report characterization of the trz2 promoter. Using lacZ as a reporter, we show that the trz2 promoter contains a HomolD box and a very weak diverged TATA element. The HomolD box is usually found in the promoters of S. pombe ribosomal protein genes. lacZ reporter assays suggest that the HomolD box regulates the expression of both trz2 and the ribosomal protein gene rps2501, which are arranged head-to-head on opposite strands. Overexpression of Rrn7, a candidate HomolD box-binding protein, up-regulates expression of lacZ under the control of the trz2 promoter or the rps2501 promoter. Functional complementation studies suggest that the TATA-like element is essential for trz2 expression, whereas the HomolD box may play a nonessential regulatory role. We also demonstrate that a 57 nt negative regulatory element (NRE) located between the HomolD box and the TATA-like element represses the expression of lacZ under the control of the trz2 promoter. Our results suggest that the low-level trz2 expression may arise from a low level of transcription caused by lack of a strong TATA box and the NRE. Our analysis also suggests that trz2 and rps2501 may be coregulated by the HomolD box.

Molecular mechanism of monoamine oxidase A gene regulation under inflammation and ischemia-like conditions: key roles of the transcription factors GATA2, Sp1 and TBP.

Monoamine oxidase A (MAOA) plays important roles in the pathogenesis of several neurological and cardiovascular disorders. The mechanism of transcriptional regulation of MAOA under basal and pathological conditions, however, remains incompletely understood. Here, we report systematic identification and characterization of cis elements and transcription factors that govern the expression of MAOA gene. Extensive computational analysis of MAOA promoter, followed by 5'-promoter deletion/reporter assays, revealed that the -71/-40bp domain was sufficient for its basal transcription. Gel-shift and chromatin immunoprecipitation assays provided evidence of interactions of the transcription factors GATA-binding protein 2 (GATA2), Sp1 and TATA-binding protein (TBP) with this proximal promoter region. Consistently, over-expression of GATA2, Sp1 and TBP augmented MAOA promoter activity in a coordinated manner. In corroboration, siRNA-mediated down-regulation of GATA2/Sp1/TBP repressed the endogenous MAOA expression as well as transfected MAOA promoter activity. Tumor necrosis factor-α and forskolin activated MAOA transcription that was reversed by Sp1 siRNA; in support, tumor necrosis factor-α- and forskolin-induced activities were enhanced by ectopic over-expression of Sp1. On the other hand, MAOA transcription was diminished upon exposure of neuroblasts or cardiac myoblasts to ischemia-like conditions because of reduced binding of GATA2/Sp1/TBP with MAOA promoter. In conclusion, this study revealed previously unknown roles of GATA2, Sp1 and TBP in modulating MAOA expression under basal as well as pathophysiological conditions such as inflammation and ischemia, thus providing new insights into the molecular basis of aberrant MAOA expression in neuronal/cardiovascular disease states. Dysregulation of monoamine oxidase A (MAOA) have been implicated in several behavioral and neuronal disease states. Here, we identified three crucial transcription factors (GATA2, Sp1 and TBP) that regulate MAOA gene expression in a coordinated manner. Aberrant MAOA expression under pathophysiological conditions including inflammation and ischemia is mediated by altered binding of GATA2/Sp1/TBP with MAOA proximal promoter. Thus, these findings provide new insights into pathogenesis of several common diseases. GATA2, GATA-binding protein 2; Sp1, specificity protein 1; TBP, TATA-binding protein.

Identification of a cis‐element that mediates multiple pathways of the endoplasmic reticulum stress response in rice

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen leads to ER stress. Intracellular signalling pathways are activated to alleviate the stress. The ER stress sensor IRE1 induces the active form of key transcription factors, such as XBP1 in mammals and bZIP50 in Oryza sativa (rice), by mediating the unconventional splicing of their mRNAs. Although the characterization of cis-elements that are recognized by these transcription factors is essential for understanding ER stress responses, such cis-elements remain unidentified in plants. Here, a cis-element named pUPRE-II was identified from promoters of bZIP50-dependent genes using chromatin immunoprecipitation assays and electrophoretic mobility shift assays. The sequence of pUPRE-II (e.g., 5'-GATGACGCGTAC-3' in the OsSAR1 promoter) was found to be flexible and not identical with that of mUPRE, a cis-element that preferentially interacts with mammalian XBP1. Unexpectedly, the transcription factor bZIP60, another ER stress sensor in rice, and a counterpart of mammalian ATF6, also showed strong binding affinity for pUPRE-II without assistance from co-factors. Reporter assays indicated that pUPRE-II significantly contributes to gene expression mediated by bZIP50 or bZIP60 in rice. Although both bZIP50 and bZIP60 bound to pUPRE-II, these transcription factors showed distinct requirements for transcriptional activation. This study provides a missing link between ER stress sensors and stress-responsive genes in rice. Furthermore, the characteristics of pUPRE-II highlight the uniqueness of ER stress-responsive transcription in plants.

Induction of the Human Oxidized Base-specific DNA Glycosylase NEIL1 by Reactive Oxygen Species

NEIL1, a mammalian DNA glycosylase and ortholog of Escherichia coli Nei/Fpg, is involved in the repair of oxidatively damaged bases in mammalian cells. Exposure of HCT116 human colon carcinoma cells to reactive oxygen species, generated by glucose oxidase (GO), enhanced the levels of NEIL1 mRNA and polypeptide by 2-4-fold by 6 h after GO treatment. A similar oxidative stress-induced increase in human NEIL1 (hNEIL1) promoter-dependent luciferase expression in HCT116 cells indicates that reactive oxygen species activates NEIL1 transcription. The transcriptional start site of hNEIL1 was mapped, and the upstream promoter sequence was characterized via luciferase reporter assay. Two identical CRE/AP-1-binding sites were identified in the promoter that binds transcription factors c-Jun and CREB/ATF2. This binding was significantly enhanced in extracts of cells treated with GO. Furthermore, a simultaneous increase in the level of phosphorylated c-Jun suggests its involvement in up-regulating the NEIL1 promoter. Oxidative stress-induced activation of NEIL1 appears to be involved in the feedback regulation of cellular repair activity needed to handle an increase in the level of oxidative base damage.

Characterization of cis elements that regulate the expression of glnA in Synechococcus sp. strain PCC 7942.

The upstream noncoding region of the Synechococcus sp. strain PCC 7942 (hereafter referred to as Synechococcus 7942) glnA gene was fused to the cat gene in order to study the expression of glnA both in Synechococcus 7942 and in Escherichia coli. The lack of cat expression in E. coli indicated that the glnA promoter was not recognized by E. coli RNA polymerase. The fused construct was integrated into the Synechococcus 7942 chromosome at a neutral site. Expression of the cat reporter gene was regulated under various nitrogen conditions in a way similar to that of the glnA gene. A deletion introduced at the binding site of the NtcA regulatory protein abolished derepression of the glnA promoter during growth in nitrate and under nitrogen starvation. Deletion of the sequence between the transcription and translation start sites of glnA prevented the repression observed during growth in ammonium. These results indicate that the glnA promoter is subject to complex regulation that involves sequences upstream and downstream from the transcription start site.