Mfd translocase is necessary and sufficient for transcription-coupled repair in Escherichia coli.

Nucleotide excision repair in Escherichia coli is stimulated by transcription, specifically in the transcribed strand. Previously, it was shown that this transcription-coupled repair (TCR) is mediated by the Mfd translocase. Recently, it was proposed that in fact the majority of TCR in E. coli is catalyzed by a second pathway ("backtracking-mediated TCR") that is dependent on the UvrD helicase and the guanosine pentaphosphate (ppGpp) alarmone/stringent response regulator. Recently, we reported that as measured by the excision repair-sequencing (XR-seq), UvrD plays no role in TCR genome-wide. Here, we tested the role of ppGpp and UvrD in TCR genome-wide and in the lacZ operon using the XR-seq method, which directly measures repair. We found that the mfd mutation abolishes TCR genome-wide and in the lacZ operon. In contrast, the relA-spoT- mutant deficient in ppGpp synthesis carries out normal TCR. We conclude that UvrD and ppGpp play no role in TCR in E. coli.

A Genome-Wide Search for Ionizing-Radiation-Responsive Elements in Deinococcus radiodurans Reveals a Regulatory Role for the DNA Gyrase Subunit A Gene's 5' Untranslated Region in the Radiation and Desiccation Response.

Tight regulation of gene expression is important for the survival of Deinococcus radiodurans, a model bacterium of extreme stress resistance. Few studies have examined the use of regulatory RNAs as a possible contributing mechanism to ionizing radiation (IR) resistance, despite their proffered efficient and dynamic gene expression regulation under IR stress. This work presents a transcriptome-based approach for the identification of stress-responsive regulatory 5' untranslated region (5'-UTR) elements in D. radiodurans R1 that can be broadly applied to other bacteria. Using this platform and an in vivo fluorescence screen, we uncovered the presence of a radiation-responsive regulatory motif in the 5' UTR of the DNA gyrase subunit A gene. Additional screens under H2O2-induced oxidative stress revealed the specificity of the response of this element to IR stress. Further examination of the sequence revealed a regulatory motif of the radiation and desiccation response (RDR) in the 5' UTR that is necessary for the recovery of D. radiodurans from high doses of IR. Furthermore, we suggest that it is the preservation of predicted RNA structure, in addition to DNA sequence consensus of the motif, that permits this important regulatory ability.IMPORTANCEDeinococcus radiodurans is an extremely stress-resistant bacterium capable of tolerating up to 3,000 times more ionizing radiation than human cells. As an integral part of the stress response mechanism of this organism, we suspect that it maintains stringent control of gene expression. However, understanding of its regulatory pathways remains incomplete to date. Untranslated RNA elements have been demonstrated to play crucial roles in gene regulation throughout bacteria. In this work, we focus on searching for and characterizing responsive RNA elements under radiation stress and propose that multiple levels of gene regulation work simultaneously to enable this organism to efficiently recover from exposure to ionizing radiation. The model we propose serves as a generic template to investigate similar mechanisms of gene regulation under stress that have likely evolved in other bacterial species.

Differential role of an NF-κB transcriptional response element in endothelial versus intimal cell VCAM-1 expression.

RATIONALE: Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown. OBJECTIVE: Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene. METHODS AND RESULTS: Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions. CONCLUSIONS: This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.

The Trichoderma atroviride photolyase-encoding gene is transcriptionally regulated by non-canonical light response elements.

The BLR-1 and BLR-2 proteins of Trichoderma atroviride are the Neurospora crassa homologs of white collar-1 and -2, two transcription factors involved in the regulation of genes by blue light. BLR-1 and BLR-2 are essential for photoinduction of phr-1, a photolyase-encoding gene whose promoter exhibits sequences similar to well-characterized light regulatory elements of Neurospora, including the albino proximal element and the light response element (LRE). However, despite the fact that this gene has been extensively used as a blue light induction marker in Trichoderma, the function of these putative regulatory elements has not been proved. The described LRE core in N. crassa comprises two close but variably spaced GATA boxes to which a WC-1/-2 complex binds transiently upon application of a light stimulus. Using 5' serial deletions of the phr-1 promoter, as well as point mutations of putative LREs, we were able to delimit an ~ 50 bp long region mediating the transcriptional response to blue light. The identified light-responsive region contained five CGATB motifs, three of them displaying opposite polarity to canonical WCC binding sites. Chromatin immunoprecipitation experiments showed that the BLR-2 protein binds along the phr-1 promoter in darkness, whereas the application of a blue light pulse results in decreased BLR-2 binding to the promoter. Our results suggest that BLR-2 and probably BLR-1 are located on the phr-1 promoter in darkness ready to perform their function as transcriptional complex in response to light.

Utilization of microRNAs with decreased expression levels in response to X-ray irradiation for fine-tuning radiation-controlled gene regulation.

We previously developed a promoter that was responsive to radiation by randomly combining cis-elements of transcription factors that are activated in response to radiation in prostate cancer cells. The promoter enhanced the expression of the luciferase gene linked downstream by more than 10-fold 12 h after X-ray irradiation at 10 Gy. However, without radiation, it still significantly drove its expression. To suppress expression while retaining its enhancement in response to radiation, we focused our attention on microRNAs (miRNAs). miRNAs are a group of non-coding RNAs approximately 22 nucleotides long that control gene expression by binding to a target sequence residing on the 3'-untranslated region (3'UTR) of a target gene. We identified 8 miRNAs that were downregulated in response to X-ray irradiation, and inserted artificial target sequences composed of randomly combined complementary sequences into 3 representative miRNAs into the 3'UTR of the luciferase gene. The target sequences suppressed the expression, and released the expression, after X-ray irradiation, as expected. When we combined an artificial target sequence with the radiation-responsive promoter, it resulted in a clear-cut gene regulation of expression that was greater than that induced by the promoter alone.

Dose response of soft X-ray-induced bystander cell killing affected by p53 status.

A radiation-induced bystander response, which is generally defined as a cellular response that is induced in nonirradiated cells that received bystander signals from directly irradiated cells within an irradiated cell population. In our earlier X-ray microbeam studies, bystander cell killing in normal human fibroblasts had a parabolic relationship to the irradiation dose. To elucidate the role of p53 in the bystander cell killing, the effects were assessed using human non-small cell lung cancer cells expressing wild-type or temperature-sensitive mutated p53. The surviving fraction of bystander wild-type p53 cells showed a parabolic relationship to the irradiation dose; survival was steeply reduced up to 0.45 Gy, recovered toward to 2 Gy, and remained at control levels up to 5 Gy. In contrast, in the mutated p53 cells at a nonpermissive temperature, the surviving fraction was steeply reduced up to 1 Gy and remained at the reduced level up to 5 Gy. When the mutated p53 cells were incubated at a permissive temperature, the decrease in the surviving fraction at 2 Gy was suppressed. The wild-type p53 cells were not only restrained in releasing bystander signals at 2 Gy, but were also resistant to the signals released by the mutated p53 cells. These results suggest that the X-ray-induced bystander cell killing depends on both the irradiation dose and the p53 status of the targeted cells and the bystander cells.

Transcriptional regulation of bone sialoprotein gene by CO(2) laser irradiation.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Low-power laser irradiation has a stimulating effect on cells and tissues. Although the carbon dioxide (CO(2)) laser is a hard surgical laser, we have attempted to use it at low energy density to achieve biological alterations. To investigate the effects of CO(2) laser irradiation on BSP gene transcription, we used rat osteoblast-like ROS17/2.8 cells. BSP mRNA levels were increased at 12 h after irradiation with the CO(2) laser (2 W, 20 s). Transient transfection assays using various sizes of the rat BSP gene promoter linked to the luciferase reporter gene showed that CO(2) laser irradiation induced luciferase activity of a -116 to +60 BSP promoter construct (pLUC3) at 12 h in the cells. Transcriptional stimulation by CO(2) laser irradiation was abrogated in the pLUC3 construct containing a 2-bp mutation in the fibroblast growth factor 2 response element (FRE). Gel shift analyses showed that CO(2) laser irradiation increased the binding of nuclear protein to FRE. These studies demonstrate that CO(2) laser irradiation increases BSP transcription via FRE in the rat BSP gene promoter.

Thyrotroph embryonic factor regulates light-induced transcription of repair genes in zebrafish embryonic cells.

Numerous responses are triggered by light in the cell. How the light signal is detected and transduced into a cellular response is still an enigma. Each zebrafish cell has the capacity to directly detect light, making this organism particularly suitable for the study of light dependent transcription. To gain insight into the light signalling mechanism we identified genes that are activated by light exposure at an early embryonic stage, when specialised light sensing organs have not yet formed. We screened over 14,900 genes using micro-array GeneChips, and identified 19 light-induced genes that function primarily in light signalling, stress response, and DNA repair. Here we reveal that PAR Response Elements are present in all promoters of the light-induced genes, and demonstrate a pivotal role for the PAR bZip transcription factor Thyrotroph embryonic factor (Tef) in regulating the majority of light-induced genes. We show that tefbeta transcription is directly regulated by light while transcription of tefalpha is under circadian clock control at later stages of development. These data leads us to propose their involvement in light-induced UV tolerance in the zebrafish embryo.

Radiation damage to DNA-protein specific complexes: estrogen response element-estrogen receptor complex.

The exposure of a DNA-protein regulatory complex to ionising radiation induces damage to both partner biomolecules and thus can affect its functioning. Our study focuses on a complex formed by the estrogen response element (ERE) DNA and the recombinant human estrogen receptor alpha (ER), which mediates the signalling of female sex hormones, estrogens. The method of native polyacrylamide retardation gel electrophoresis is used to study the stability of the complex under irradiation by low LET radiation ((60)Co gamma rays) and the ability of the separately irradiated partners to form complexes. The relative probabilities of ERE DNA strand breakage and base damages as well as the probabilities of damages to the ER binding domain are calculated using the Monte Carlo method-based model RADACK.

Identification of novel p53 target genes in ionizing radiation response.

The tumor suppressor p53 plays an essential role in cellular adaptation to stress. In response to ionizing radiation, p53 regulates the transcription of genes in a diverse set of pathways including DNA repair, cell cycle arrest, and apoptosis. Previously, we identified by microarray analysis a set of genes that are transcriptionally activated or repressed in response to radiation exposure. In this study, we use computational methods and molecular techniques, including location analysis (ChIP-on-chip assay), to identify ionizing radiation-responsive genes that are directly regulated by p53. Among the 489 ionizing radiation-responsive genes examined, 38 genes were found to be p53 targets. Some of these genes are previously known to be directly regulated by p53 whereas others are novel p53 targets. We further showed that the novel p53 target genes are transcriptionally regulated by p53. The binding of p53 to promoters of target genes correlated with increased transcript levels of these genes in cells with functional p53. However, p53 binding and subsequent transcriptional activation of these target genes were significantly diminished in cells with mutant p53 and in cells from patients with ataxia telangiectasia, which have impaired p53 activation following ionizing radiation exposure. Identification and characterization of ionizing radiation-responsive p53 target genes extend our knowledge of the diverse role that p53 plays in the DNA damage response.

Selective regulation of c-jun gene expression by mitogen-activated protein kinases via the 12-o-tetradecanoylphorbol-13-acetate- responsive element and myocyte enhancer factor 2 binding sites.

To further understand how the mitogen-activated protein kinase (MAPK) signaling pathways regulate AP-1 activity, we have elucidated the physiological role of these cascades in the regulation of c-jun gene expression. c-Jun is a crucial component of AP-1 complexes and has been shown in vitro to be a point of integration of numerous signals that can differentially affect its expression as well as its transcriptional activity. Our strategy was based on the use of (i) genetically modified fibroblasts deficient in components of the MAPK cascades and (ii) pharmacological reagents. The results demonstrate that c-Jun NH(2)-terminal protein kinase (JNK) is essential for a basal level of c-Jun expression and for c-Jun phosphorylation in response to stress. In addition to JNK, p38 MAPK or ERK1/2 and ERK5 are required for mediating UV radiation- or epidermal growth factor (EGF)-induced c-Jun expression, respectively. Further studies indicate that p38 MAPK inhibits the activation of JNK in response to EGF, causing a down-regulation of c-Jun. Overall, these data provide important insights into the mechanisms that ultimately determine the function of c-Jun as a regulator of cell fate.

The as-1 promoter element is an oxidative stress-responsive element and salicylic acid activates it via oxidative species.

The activation sequence-1 (as-1)-like element found in the promoter of some glutathione S-transferase (GST) genes, has been previously described as a salicylic acid (SA)- and auxin-responsive element. In this paper, we tested the hypothesis that the activating effect of SA on the as-1 element is mediated by oxidative species. Supporting this hypothesis, our results show that the antioxidants dimethylthiourea (DMTU) and 3-t-butyl-4-hydroxy-anizole (BHA) inhibit the SA-induced transcription of genes controlled by as-1 elements in tobacco (Nicotiana tabacum) plants [i.e. GNT35 gene coding for a GST and (as-1)(4)/beta-glucuronidase (GUS) reporter transgene]. DMTU and BHA also inhibit SA-activated as-1-binding activity in nuclear extracts. Further support for the hypothesis that the as-1 element is activated by oxidative species comes from our result showing that light potentiates the SA-induced activation of the as-1 element. Furthermore, methyl viologen, a known oxidative stress inducer in plants, also activates the as-1 element. Increasing H(2)O(2) levels by incubation with H(2)O(2) or with the catalase inhibitor 3-amino-1,2,5-triazole does not activate the (as-1)(4)/GUS gene. On the contrary, 3-amino-1,2,5-triazole inhibits the activating effect of SA on the (as-1)(4)/GUS gene. These results suggest that oxidative species other than H(2)O(2) mediate the activation of the as-1 element by SA. Our results also suggest that even though the as-1 binding activity is stimulated by oxidative species, this is not sufficient for the transactivation of genes controlled by this element. The complex interplay between SA and reactive oxygen species in the transcriptional activation of defense genes is discussed.

UV but not gamma irradiation accelerates p53-induced apoptosis of teratocarcinoma cells by repressing MDM2 transcription.

Induction of p53 by DNA damage results in apoptosis of teratocarcinoma cells, whereas MDM2, encoded by a p53-responsive gene, can reverse this phenotype by inhibiting p53 function. Here we report that UV (10 or 20 J/m2), but not gamma irradiation (7 or 10 Gy), caused a massive apoptosis of human teratoma Tera-2 or murine testicular carcinoma F9 cells, both of which contain wild-type p53, but not murine p53 null testicular carcinoma EB-16 cells. Most Tera-2 or F9 cells died overnight after UV but not gamma irradiation. Correlated with this phenotype was a dramatic and continuing accumulation of p53 proteins after UV but not gamma irradiation. This was attributable to UV-responsive repression of MDM2 expression, because both its protein and RNA were not detectable after UV irradiation. This UV-induced repression appeared to be specific to MDM2, because expression of other genes, such as p21, p53, or glyceraldehyde-3-phosphate dehydrogenase, was not reduced. Also, RNase protection analysis showed that a DNA region, excluding the p53 binding site, in the MDM2 promoter mediated transcriptional repression in response to UV. Thus, these results suggest that UV but not gamma irradiation can induce p53 by suppressing MDM2 expression in a p53-independent fashion and subsequently, massive cell death.

Identification of the UV-responsive sequence in the human tissue plasminogen activator gene.

Functional analysis of regulatory elements in the human tissue plasminogen activator (tPA) gene showed that its promoter (-119/+169) is activated by UV irradiation in HeLa cells. We demonstrated here that the AP-2 like CCCCACCC sequence is involved in the UV-mediated activation and Sp1 binds to the sequence.

Identification of UV-B light-responsive regions in the promoter of the tryptophan decarboxylase gene from Catharanthus roseus.

The tryptophan decarboxylase (Tdc) gene encodes a key enzyme in the biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus. TIAs absorb ultraviolet light (UV) and putative functions in plants include a role as UV protectants. In support of this possible function we demonstrate here that UV light induces accumulation of several TIAs as well as expression of the Tdc gene in C. roseus. In addition, in tobacco a Tdc-gusA construct was found to be specifically induced by UV-B light. Lack of induction by UV-A or other wavelengths of light indicate that Tdc expression is regulated by a specific UV-B receptor and corresponding signal transduction pathway. To identify UV-responsive Tdc promoter elements, a loss-of-function analysis was performed, in which deletion derivatives were fused to the gusA reporter gene and analysed in transgenic tobacco plants. Truncation of the Tdc promoter from -1818 (relative to the start of transcription) to -160 reduced expression levels two-fold without affecting the qualitative UV response. Deletion to -37 further reduced expression levels five-fold, but the delta37 promoter also remained UV-responsive. Subsequently, the -160 to -37 region was further studied by gain-of-function experiments, in which the transcriptional activities of tetramerized subfragments fused to truncated promoters were analysed. Combination of the data identified several functional regions in the -160 to +198 promoter. The - 160 to -99 region acts as the main transcriptional enhancer. UV-responsive elements appeared to be redundant in the -160 Tdc promoter and to reside between -99 and -37 and between -37 and + 198.

UV-Laser induced protein/DNA crosslinking reveals sequence variations of DNA elements bound by c-Jun in vivo.

Many proteins involved in the modulation of gene expression exert their function through direct interaction with DNA. The sequence specificity of these interactions provides the basis for many regulatory mechanisms. The sites that are utilized by a transcription factor are usually analyzed using in vitro binding studies. To detect true in vivo binding sites we developed a method, presented here, that allows construction of recognition element DNA (reDNA) libraries which represent in vivo binding sites plus flanking sequences. reDNA libraries can be constructed for any well-characterized transcription factor. Here we used this method for an in vivo study of genomic DNA elements that interact with the transcription factor c-Jun in rat cerebellum.