Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR Could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts Occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. (C) 2009 Published by Elsevier Inc.

Promoting developmental transcription.

Animal growth and development depend on the precise control of gene expression at the level of transcription. A central role in the regulation of developmental transcription is attributed to transcription factors that bind DNA enhancer elements, which are often located far from gene transcription start sites. Here, we review recent studies that have uncovered significant regulatory functions in developmental transcription for the TFIID basal transcription factors and for the DNA core promoter elements that are located close to transcription start sites.

Modulation of heat shock transcription factor 1 as a therapeutic target for small molecule intervention in neurodegenerative disease.

Neurodegenerative diseases such as Huntington disease are devastating disorders with no therapeutic approaches to ameliorate the underlying protein misfolding defect inherent to poly-glutamine (polyQ) proteins. Given the mounting evidence that elevated levels of protein chaperones suppress polyQ protein misfolding, the master regulator of protein chaperone gene transcription, HSF1, is an attractive target for small molecule intervention. We describe a humanized yeast-based high-throughput screen to identify small molecule activators of human HSF1. This screen is insensitive to previously characterized activators of the heat shock response that have undesirable proteotoxic activity or that inhibit Hsp90, the central chaperone for cellular signaling and proliferation. A molecule identified in this screen, HSF1A, is structurally distinct from other characterized small molecule human HSF1 activators, activates HSF1 in mammalian and fly cells, elevates protein chaperone expression, ameliorates protein misfolding and cell death in polyQ-expressing neuronal precursor cells and protects against cytotoxicity in a fly model of polyQ-mediated neurodegeneration. In addition, we show that HSF1A interacts with components of the TRiC/CCT complex, suggesting a potentially novel regulatory role for this complex in modulating HSF1 activity. These studies describe a novel approach for the identification of new classes of pharmacological interventions for protein misfolding that underlies devastating neurodegenerative disease.

Characterization of the human and mouse ETV1/ER81 transcription factor genes: Role of the two alternatively spliced isoforms in the human

The Ets transcription factors of the PEA3 group - E1AF/PEA3, ETV1/ER81 and ERM - are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1α) or the absence (ETV1β) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1β) and the full-length isoform (human ETV1α) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1α and human ETV1β expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

Chemotherapy-Induced CXC-Chemokine/CXC-Chemokine Receptor Signaling in Metastatic Prostate Cancer Cells Confers Resistance to Oxaliplatin through Potentiation of Nuclear Factor-κB Transcription and Evasion of Apoptosis

Constitutive activation of nuclear factor (NF)-kappa B is linked with the intrinsic resistance of androgen-independent prostate cancer (AIPC) to cytotoxic chemotherapy. Interleukin-8 (CXCL8) is a transcriptional target of NF-kappa B whose expression is elevated in AIPC. This study sought to determine the significance of CXCL8 signaling in regulating the response of AIPC cells to oxaliplatin, a drug whose activity is reportedly sensitive to NF-kappa B activity. Administration of oxaliplatin to PC3 and DU145 cells increased NF-kappa B activity, promoting antiapoptotic gene transcription. In addition, oxaliplatin increased the transcription and secretion of CXCL8 and the related CXC-chemokine CXCL1 and increased the transcription and expression of CXC-chemokine receptors, especially CXC-chemokine receptor (CXCR) 2, which transduces the biological effects of CXCL8 and CXCL1. Stimulation of AIPC cells with CXCL8 potentiated NF-kappa B activation in AIPC cells, increasing the transcription and expression of NF-kappa B-regulated antiapoptotic genes of the Bcl-2 and IAP families. Coadministration of a CXCR2-selective antagonist, AZ10397767 (Bioorg Med Chem Lett 18:798-803, 2008), attenuated oxaliplatin-induced NF-kappa B activation, increased oxaliplatin cytotoxicity, and potentiated oxaliplatin-induced apoptosis in AIPC cells. Pharmacological inhibition of NF-kappa B or RNA interference-mediated suppression of Bcl-2 and survivin was also shown to sensitize AIPC cells to oxaliplatin. Our results further support NF-kappa B activity as an important determinant of cancer cell sensitivity to oxaliplatin and identify the induction of autocrine CXCR2 signaling as a novel mode of resistance to this drug.

De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene

Reverse genetics has facilitated the use of non-segmented negative strand RNA viruses (NNSV) as vectors. Currently, heterologous gene expression necessitates insertion of extra-numeral transcription units (ENTUs), which may alter the NNSV polar transcription gradient and attenuate growth relative to wildtype (Wt). We hypothesized that rescuing recombinant Sendai Virus (rSeV) with a bicistronic gene might circumvent this attenuation but still allow heterologous open reading frame (ORF) expression. Therefore, we used a 9-nucleotide sequence previously described with internal ribosome entry site (IRES) activity, which, when constructed as several repeats, synergistically increased the level of expression of the second cistron [Chappell, S.A., Edelman, G.M., Mauro, V.P., 2000. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U.S.A. 97, 1536-1541]. We inserted the Renilla luciferase (rLuc) ORF, preceded by 1, 3 or 7 IRES copies, downstream of the SeV N ORF in an infectious clone. Corresponding rSeVs were successfully rescued. Interestingly, bicistronic rSeVs grew as fast as or faster than Wt rSeV. Furthermore, SeV gene transcription downstream of the N/rLuc gene was either equivalent to, or slightly enhanced, compared to Wt rSeV. Importantly, all rSeV/rLuc viruses efficiently expressed rLuc. IRES repetition increased rLuc expression at a multiplicity of infection of 0.1, although without evidence of synergistic enhancement. In conclusion, our approach provides a novel way of insertion and expression of foreign genes in NNSVs. (C) 2008 Elsevier B.V. All rights reserved.

Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation

Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIa in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIa is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIa functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.

Salt-inducible kinase 2 regulates mitotic progression and transcription in prostate cancer

UNLABELLED: Salt-inducible kinase 2 (SIK2) is a multifunctional kinase of the AMPK family that plays a role in CREB1-mediated gene transcription and was recently reported to have therapeutic potential in ovarian cancer. The expression of this kinase was investigated in prostate cancer clinical specimens. Interestingly, auto-antibodies against SIK2 were increased in the plasma of patients with aggressive disease. Examination of SIK2 in prostate cancer cells found that it functions both as a positive regulator of cell-cycle progression and a negative regulator of CREB1 activity. Knockdown of SIK2 inhibited cell growth, delayed cell-cycle progression, induced cell death, and enhanced CREB1 activity. Expression of a kinase-dead mutant of SIK2 also inhibited cell growth, induced cell death, and enhanced CREB1 activity. Treatment with a small-molecule SIK2 inhibitor (ARN-3236), currently in preclinical development, also led to enhanced CREB1 activity in a dose- and time-dependent manner. Because CREB1 is a transcription factor and proto-oncogene, it was posited that the effects of SIK2 on cell proliferation and viability might be mediated by changes in gene expression. To test this, gene expression array profiling was performed and while SIK2 knockdown or overexpression of the kinase-dead mutant affected established CREB1 target genes; the overlap with transcripts regulated by forskolin (FSK), the adenylate cyclase/CREB1 pathway activator, was incomplete.

IMPLICATIONS: This study demonstrates that targeting SIK2 genetically or therapeutically will have pleiotropic effects on cell-cycle progression and transcription factor activation, which should be accounted for when characterizing SIK2 inhibitors.

The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440.

The primary enzyme involved in polyphosphate (polyP) synthesis, polyP kinase (ppk), has been deleted in Pseudomonas putida KT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate (PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative (H2O2) stress, when grown on glucose. The accumulation of PHA by Δppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in polyP levels between wild-type and Δppk strains under all growth conditions tested. In the Δppk mutant proteome, polyP kinase (PPK) was undetectable, but up-regulation of the polyp-associated proteins polyP adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide (NAD) kinase (PpnK), a putative polyP adenosine monophosphate (AMP) phosphotransferase (PP_1752), and exopolyphosphatase was observed. Δppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42 h of lag period compared with 24 h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the Δppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting polyP accumulation.

The Molecular Consequences of CK2-mediated Phosphorylation of the TGA2 Transcription Factor within Systemic Acquired Resistance of Arabidopsis thaliana

During infection, the model plant Arabidopsis thaliana is capable of activating long lasting defence responses both in tissue directly affected by the pathogen and in more distal tissue. Systemic acquired resistance (SAR) is a type of systemic defence response deployed against biotrophic pathogens resulting in altered plant gene expression and production of antimicrobial compounds. One such gene involved in plant defence is called pathogenesis-related 1 (PR1) and is under the control of several protein regulators. TGA II-clade transcription factors (namely TGA2) repress PR1 activity prior to infection by forming large oligomeric complexes effectively blocking gene transcription. After pathogen detection, these complexes are dispersed by a mechanism unknown until now and free TGA molecules interact with the non-expressor of pathogenesis-related gene 1 (NPR1) protein forming an activating complex enabling PR1 transcription. This study elucidates the TGA2 dissociation mechanism by introducing protein kinase CK2 into this process. This enzyme efficiently phosphorylates TGA2 resulting in two crucial events. Firstly, the DNA-binding ability of this transcription factor is completely abolished explaining how the large TGA2 complexes are quickly evicted from the PR1 promoter. Secondly, a portion of TGA2 molecules dissociate from the complexes after phosphorylation which likely makes them available for the formation of the TGA2-NPR1 activating complex. We also show that phosphorylation of a multiserine motif found within TGA2’s N terminus is responsible for the change of affinity to DNA, while modification of a single threonine in the leucine zipper domain seems to be responsible for deoligomerization. Despite the substantial changes caused by phosphorylation, TGA2 is still capable of interacting with NPR1 and these proteins together form a complex on DNA promoting PR1 transcription. Therefore, we propose a change in the current model of how PR1 is regulated by adding CK2 which targets TGA2 displacing it’s complexes from the promoter and providing solitary TGA2 molecules for assembly of the activating complex. Amino acid sequences of regions targeted by CK2 in Arabidopsis TGA2 are similar to those found in TGA2 homologs in rice and tobacco. Therefore, the molecular mechanism that we have identified may be conserved among various plants, including important crop species, adding to the significance of our findings.

The euchromatic and heterochromatic landscapes are shaped by antagonizing effects of transcription on H2A.Z deposition

A role for variant histone H2A.Z in gene expression is now well established but little is known about the mechanisms by which it operates. Using a combination of ChIP-chip, knockdown and expression profiling experiments, we show that upon gene induction, human H2A.Z associates with gene promoters and helps in recruiting the transcriptional machinery. Surprisingly, we also found that H2A.Z is randomly incorporated in the genome at low levels and that active transcription antagonizes this incorporation in transcribed regions. After cessation of transcription, random H2A.Z quickly reappears on genes, demonstrating that this incorporation utilizes an active mechanism. Within facultative heterochromatin, we observe a hyper accumulation of the variant histone, which might be due to the lack of transcription in these regions. These results show how chromatin structure and transcription can antagonize each other, therefore shaping chromatin and controlling gene expression.

Rôle de l’enzyme PAS kinase dans la régulation du facteur de transcription PDX-1 dans la cellule bêta pancréatique.

Le diabète de type 2 (DT2) se caractérise par une production insuffisante d'insuline par le pancréas ainsi qu'une résistance des tissus périphériques à l'action de l'insuline. Dans les cellules bêta pancréatiques, le glucose stimule la production de l'insuline en induisant la transcription de son gène et la traduction ainsi que la sécrétion de sa protéine. Paradoxalement, une exposition prolongée et simultanée de ces cellules à de hautes concentrations de glucose en présence d'acides gras conduit à la détérioration de la fonction bêta pancréatique et au développement du DT2. Toutefois, les mécanismes moléculaires responsables de ces effets du glucose ne sont que partiellement connus. L'objectif du travail décrit dans cette thèse est d'identifier les mécanismes responsables de la régulation de la transcription du gène de l'insuline. PDX-1 (de l’anglais pour pancreatic and duodenal homeobox 1) est un facteur de transcription majeur et essentiel tant pour le développement du pancréas que pour le maintien de sa fonction à l'état adulte. En réponse au glucose, PDX-1 se lie au promoteur du gène de l'insuline et induit sa transcription. Ceci est inhibé par l'acide gras palmitate. Dans la première partie des travaux effectués dans le cadre de cette thèse, nous avons identifié deux mécanismes de régulation de la transcription du gène de l'insuline: le premier via ERK1/2 (de l'anglais pour extracellular-signal-regulated protein kinases 1 and 2) et le second par l’enzyme PASK (pour per-arnt-sim kinase). Nous avons également mis en évidence l'existence d'un troisième mécanisme impliquant l'inhibition de l'expression du facteur de transcription MafA par le palmitate. Nos travaux indiquent que la contribution de la signalisation via PASK est majeure. L'expression de PASK est augmentée par le glucose et inhibée par le palmitate. Sa surexpression dans les cellules MIN6 et les îlots isolés de rats, mime les effets du glucose sur l'expression du gène de l'insuline ainsi que sur l'expression de PDX-1 et prévient les effets délétères du palmitate. Dans la deuxième partie de la thèse, nous avons identifié un nouveau mécanisme par lequel PASK augmente la stabilité protéique de PDX-1, soit via la phosphorylation et l'inactivation de la protéine kinase GSK3 bêta (de l'anglais pour glycogen synthase kinase 3 beta). Le glucose induit la translocation de PDX-1 du cytoplasme vers le noyau, ce qui est essentiel à sa liaison au promoteur de ses gènes cibles. L'exclusion nucléaire de PDX-1 a été observée dans plusieurs modèles ex vivo et in vivo de dysfonction de la cellule bêta pancréatique. Dans le dernier volet de cette thèse, nous avons démontré l'importance de l'utilisation de cellules primaires (îlots isolés et dispersés) pour étudier la translocation nucléaire de PDX-1 endogène étant donné que ce mode de régulation est absent dans les lignées insulino-sécrétrices MIN6 et HIT-T15. Ces études nous ont permis d'identifier et de mieux comprendre les mécanismes régulant la transcription du gène de l'insuline via le facteur de transcription PDX-1. Les cibles moléculaires ainsi identifiées pourraient contribuer au développement de nouvelles approches thérapeutiques pour le traitement du diabète de type 2. Mots-clés : Diabète, îlots de Langerhans, cellule bêta pancréatique, gène de l'insuline, PDX-1, PASK, GSK3 bêta, ERK1/2, PKB, glucose, palmitate.

Transcription factor NF-kappaB is transported to the nucleus via cytoplasmic dynein/dynactin motor complex in hippocampal neurons

Background Long-term changes in synaptic plasticity require gene transcription, indicating that signals generated at the synapse must be transported to the nucleus. Synaptic activation of hippocampal neurons is known to trigger retrograde transport of transcription factor NF-κB. Transcription factors of the NF-κB family are widely expressed in the nervous system and regulate expression of several genes involved in neuroplasticity, cell survival, learning and memory. Principal Findings In this study, we examine the role of the dynein/dynactin motor complex in the cellular mechanism targeting and transporting activated NF-κB to the nucleus in response to synaptic stimulation. We demonstrate that overexpression of dynamitin, which is known to dissociate dynein from microtubules, and treatment with microtubule-disrupting drugs inhibits nuclear accumulation of NF-κB p65 and reduces NF-κB-dependent transcription activity. In this line, we show that p65 is associated with components of the dynein/dynactin complex in vivo and in vitro and that the nuclear localization sequence (NLS) within NF-κB p65 is essential for this binding. Conclusion This study shows the molecular mechanism for the retrograde transport of activated NF-κB from distant synaptic sites towards the nucleus.

Ascl1 coordinately regulates gene expression and the chromatin landscape during neurogenesis

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.