Promoter In Presence Research Articles

Dynamic regulation of BDNF gene expression by estradiol and lncRNA HOTAIR

Brain derived neurotrophic factor (BDNF) is a major neurotransmitter that controls growth and maintenance of neurons and its misregulation is linked to neurodegeneration and human diseases. Estradiol (E2) is well-known to regulate the process of differentiation and plasticity of hippocampal neurons. Here we examined the mechanisms of BDNF gene regulation under basal conditions and under stimuli such as E2. Our results demonstrated that BDNF expression is induced by E2 in vitro in HT22 cells (hippocampal neuronal cells) and in vivo (in ovariectomized mouse brain under E2-treatment). Using chromatin immunoprecipitation assay, we demonstrated that estrogen receptors (ERα, ERβ) were enriched at the BDNF promoter in presence of E2. Additionally, ER-coregulators (e.g., CBP/p300, MLL3), histone acetylation, H3K4-trimethylation, and RNA polymerase II levels were also elevated at the BDNF promoter in an E2-dependent manner. Additionally, under the basal conditions (in the absence of E2), the long noncoding RNA HOTAIR and its interacting partners PRC2 and LSD1 complexes binds to the promoter of BDNF and represses its expression. HOTAIR knockdown -relieves the repression resulting in elevation of BDNF expression. Further, levels of HOTAIR-interacting partners, EZH2 and LSD1 were reduced at the BDNF promoter upon HOTAIR-knockdown revealing that HOTAIR plays a regulatory role in BDNF gene expression by modulating promoter histone modifications. Additionally, we showed that E2 induced-BDNF expression is mediated by the displacement of silencing factors, EZH2 and LSD1 at BDNF promoter and subsequent recruitment of active transcription machinery. These results reveal the mechanisms of BDNF gene regulation under the basal condition and in presence of a positive regulator such as E2 in neuronal cells.

Designing glucose utilization "highway" for recombinant biosynthesis

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from “glucose repression” are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Enhancing transcription in Escherichia coli and Pseudomonas putida using bacteriophage lambda anti-terminator protein Q.

Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli. Functional expression of heterologous genes can suffer from limitations due to the lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA between a vector-based promoter and the transcription start site. Anti-terminator proteins are a possible solution to overcome this limitation. When bacteriophage lambda infects E. coli, it relies on the host transcription machinery to transcribe and express phage DNA. Lambda anti-terminator protein Q (λQ) regulates the expression of late-genes of phage lambda. E. coli RNA polymerase recognizes the PR' promoter on the lambda genome and forms a complex with λQ, to overcome the terminator tR'. Here we show the use of λQ to efficiently transcribe a capsular polysaccharide cluster, cps3, from Lactobacillus plantarum containing intrinsic terminators in Escherichia coli. In addition, we expand the use of anti-terminator λQ in Pseudomonas putida. The results show ~ fivefold higher expression of a fluorescent reporter located ~ 12.5kbp downstream from the promoter, when the transcription is driven by PR' promoter in presence of λQ compared to a lac promoter. These results suggest that λQ could be used in metabolic engineering to enhance expression of heterologous DNA.

Comparison of the expression and toxicity of AAV2/9 carrying the human A53T α-synuclein gene in presence or absence of WPRE

Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) is thought to enhance transgene expression of target genes delivered by adeno-associated viral (AAV) vectors. This study assessed the protein expression of α-synuclein, phosphorylated α-synuclein at Serine 129, extent of nigrostriatal degeneration as well as subsequent behavioral deficits induced by unilateral intranigral stereotactic injection in male adult C57BL/6J mice of an AAV2/9 expressing A53T human α-synuclein under the control of the synapsin promoter in presence or absence of the WPRE. The presence of WPRE enabled to achieve greater nigrostriatal degeneration and synucleinopathy which was concomitant with worsened forelimb use asymmetry. This work refines a mouse Parkinson's disease model in which anatomo-pathology is related to behavioral deficits.

Mixed anionic-nonionic micelle catalysed oxidation of aliphatic alcohol in aqueous medium

Oxidation of isoamyl alcohol was carried out under pseudo 1st order reaction condition in aqueous medium by chromic acid. In addition to single micelle, mixed anionic-nonionic micelle (SDS-TX 100) was found to be effective catalyst. Promoters in presence of micelle catalyst showed almost million fold rate acceleration. The product was confirmed by IR and NMR study. UV and NMR analysis were carried out to establish the formation of mixed micelle. Formation of active oxidant was confirmed by fluorescence measurement. Interactions between surfactant and substrate were analysed by NMR spectra. In addition to SDS catalysed Bpy promoted reaction combination of mixed micelle with Bpy promoter was found to show higher rate of oxidation. For single micelle catalysed path the observed rate constants follow the order kobs (SDS) > kobs (TX 100) and kobs (Phen) > kobs (Bpy) > kobs (PA) was observed for promoted reactions. In SDS micelle and mixed micelle kobs (Bpy) > kobs (Phen) > kobs (PA) was observed while in TX 100 micelle kobs (Phen) > kobs (Bpy) > kobs (PA) was found.

Expression and role of PAICS, a de novo purine biosynthetic gene in prostate cancer.

BACKGROUND Our goal was to investigate de novo purine biosynthetic gene PAICS expression and evaluate its role in prostate cancer progression. METHODS Next-generation sequencing, qRTPCR and immunoblot analysis revealed an elevated expression of a de novo purine biosynthetic gene, Phosphoribosylaminoimidazole Carboxylase, Phosphoribosylaminoimidazole Succinocarboxamide Synthetase (PAICS) in a progressive manner in prostate cancer. Functional analyses were performed using prostate cancer cell lines- DU145, PC3, LnCaP, and VCaP. The oncogenic properties of PAICS were studied both by transient and stable knockdown strategies, in vivo chicken chorioallantoic membrane (CAM) and murine xenograft models. Effect of BET bromodomain inhibitor JQ1 on the expression level of PAICS was also studied. RESULTS Molecular staging of prostate cancer is important factor in effective diagnosis, prognosis and therapy. In this study, we identified a de novo purine biosynthetic gene; PAICS is overexpressed in PCa and its expression correlated with disease aggressiveness. Through several in vitro and in vivo functional studies, we show that PAICS is necessary for proliferation and invasion in prostate cancer cells. We identified JQ1, a BET bromodomain inhibitor previously implicated in regulating MYC expression and demonstrated role in prostate cancer, abrogates PAICS expression in several prostate cancer cells. Furthermore, we observe loss of MYC occupancy on PAICS promoter in presence of JQ1. CONCLUSIONS Here, we report that evaluation of PAICS in prostate cancer progression and its role in prostate cancer cell proliferation and invasion and suggest it as a valid therapeutic target. We suggest JQ1, a BET-domain inhibitor, as possible therapeutic option in targeting PAICS in prostate cancer. Prostate. © 2016 Wiley Periodicals, Inc.

Expression and Role of PAICS, a De Novo Purine Biosynthetic Gene in Prostate Cancer.

Our goal was to investigate de novo purine biosynthetic gene PAICS expression and evaluate its role in prostate cancer progression. Next-generation sequencing, qRTPCR and immunoblot analysis revealed an elevated expression of a de novo purine biosynthetic gene, Phosphoribosylaminoimidazole Carboxylase, Phosphoribosylaminoimidazole Succinocarboxamide Synthetase (PAICS) in a progressive manner in prostate cancer. Functional analyses were performed using prostate cancer cell lines- DU145, PC3, LnCaP, and VCaP. The oncogenic properties of PAICS were studied both by transient and stable knockdown strategies, in vivo chicken chorioallantoic membrane (CAM) and murine xenograft models. Effect of BET bromodomain inhibitor JQ1 on the expression level of PAICS was also studied. Molecular staging of prostate cancer is important factor in effective diagnosis, prognosis and therapy. In this study, we identified a de novo purine biosynthetic gene; PAICS is overexpressed in PCa and its expression correlated with disease aggressiveness. Through several in vitro and in vivo functional studies, we show that PAICS is necessary for proliferation and invasion in prostate cancer cells. We identified JQ1, a BET bromodomain inhibitor previously implicated in regulating MYC expression and demonstrated role in prostate cancer, abrogates PAICS expression in several prostate cancer cells. Furthermore, we observe loss of MYC occupancy on PAICS promoter in presence of JQ1. Here, we report that evaluation of PAICS in prostate cancer progression and its role in prostate cancer cell proliferation and invasion and suggest it as a valid therapeutic target. We suggest JQ1, a BET-domain inhibitor, as possible therapeutic option in targeting PAICS in prostate cancer. Prostate 77:10-21, 2017. © 2016 Wiley Periodicals, Inc.

TMEM18 is a regulator of adipogenesis and involved in PPARG signalling in vivo

Meeting abstractPolymorphisms in TMEM18 are associated with obesity in children and adults. We have reported previously that TMEM18 is a regulator of human adipogenesis in vitro [1]. The aim of this study was to investigate the role of TMEM18 during adipose tissue (AT) accumulation in vivo. For this, we used the zebrafish as an in vivo model for AT development. In addition, we analysed TMEM18 expression in whole AT samples, isolated adipocytes and cells of the stroma-vascular fraction (SVF) from lean and obese children of our Leipzig Childhood AT cohort [2], and addressed associations with PPARG expression and obesity-related parameters.Using whole mount in situ hybridisation on 9 day old zebrafish larvae, we showed co-expression of tmem18 and pparg in a visceral region where also first adipocytes are detectable by Nile red staining. Morpholino-mediated inhibition of tmem18 expression resulted in a reduction in the number of visceral adipocytes but did not affect zebrafish development per se. There was no effect of tmem18 knockdown on eating behaviour suggesting that the inhibition of adipocyte formation was not mediated by a central effect. Tmem18-mediated inhibition of adipogenesis was accompanied by a significant down-regulation in pparg expression. Using luciferase reporter assays in 3T3-L1 cells, we detected a significant activation of the PPARG promoter in presence of Tmem18 indicating that Tmem18 is an upstream regulator of PPARG signalling. In line with these data, TMEM18 expression correlated with PPARG expression in whole AT and isolated adipocytes but not in SVF cells of children included in our Leipzig AT cohort. Similar to PPARG, TMEM18 expression was down-regulated in adipocytes of obese children compared to lean children and correlated with adipocyte diameter, macrophage infiltration, serum adiponectin levels and HOMA-IR as a measure of insulin resistance.Our findings may indicate a potential role of TMEM18 as a regulator of PPARG signaling during adipogenesis in vivo.

Abstract 3901: Sox9-associated overexpression of IFIT3 leads to pancreatic cancer progression by activation of “pseudoinflammatory” pathways

Abstract The understanding of invasion, angiogenesis and metastasis is essential for the development of new molecular therapy against cancer. Inflammation plays important role in tumour initiation and progression. Here we report the role of the transcription factor Sox9 for regulation of IFIT3 an inflammation-related and tumour-promoting protein in pancreatic cancer. For in vivo and in vitro experiments we utilized the following human pancreatic cancer cell lines: low metastatic FG, high metastatic L3.6pl, and the stable transfected cell line FG-IFIT3. To demonstrate effects on primary tumor growth and metastases in vivo we orthotopically injected the different cell lines in the pancreas of nude mice. To evaluate the VEGF depending angiogenic capacity of the different cell lines ELISA technology was used. By One STrEP technology we were able to identify IFIT3-binding partners. Chromosomal immunoprecipitation (ChIP) using anti-Sox9 antibody, followed by PCR amplifying the IFIT3-promoter was used to identify the interaction of the IFIT3 promoter with the transcription factor Sox9. To investigate Sox9-depending expression of IFIT3 (protein and RNA) we used stable transfected L3.6pl-Sox9-shRNA cells under control of the Tet-CMV promoter in presence or absence of tetracycline, respectively. Analysis of differential gene expression by gene array technology demonstrated that the IFIT3 gene is up-regulated in L3.6pl cells as compared to FG cells. Results of animal experiment and in vitro experiments clearly demonstrated tumor-promoting, pro-metastatic and pro-angiogenic features of IFIT3. RT-PCR has revealed that both treatment with IFna as well as NFkB led to up-regulation of IFIT3-RNA expression. One STrEP experiments identified JNK and STAT1 as binding partners of IFIT3. ChIP has demonstrated binding of the transcription factor Sox9 to the IFIT3 promoter. RT-PCR and immunoblot data demonstrated constitutive up-regulation of Sox9 expression in L3.6pl cells. By Western blotting and RT-PCR we could show that diminishing of Sox9 expression in stable transfected L3.6pl Sox9-shRNA cells leads to a significant down-regulation of IFIT3-epression on the RNA and protein level. The inflammation associated protein IFIT3 is up-regulated in metastatic L3.6pl human pancreatic cancer cells and is in part responsible for the aggressive primary pancreatic tumor growth in vivo. This gene is up-regulated by IFna and NFkB. Interestingly Sox9 binds to the IFIT3P and activates its expression. Since in L3.6pl cells Sox9 is constitutively over-expressed, IFIT3 is up-regulated independent on the presence of the cytokine IFna. Therefore, the pro-inflammatory IFna-signaling pathway is activated even without actual inflammation in absence pro-inflammatory cytokine. The activation of such a “pseudo-inflammatory pathway” seems to be in part responsible for pancreatic cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3901.

ACTH receptor (MC2R) promoter variants associated with infantile spasms modulate MC2R expression and responsiveness to ACTH

Adrenocorticotropin hormone (ACTH) has been the standard treatment to infantile spasms (IS). However, the mechanism of ACTH therapy is still unclear. ACTH exerts the function via melanocortin 2 receptor (MC2R). Our previous study showed a common 4-single nucleotide polymorphism (SNP) haplotype TCCT at the MC2R promoter was strongly associated with responsiveness to ACTH therapy, where these 4 SNPs [rs1893219, rs1893220, rs2186944, and a novel SNP (T>C)] were mapped at position -853, -759, -7, and -2 bp based on the transcription start site of the MC2R gene. In this study, we further elucidated functional significances of the TCCT haplotype. To evaluate whether the TCCT haplotype influences MC2R transcription levels, the luciferase reporter vector was used by a transient transfection. Expression of rat MC2R cDNA driven by the TCCT-carrying or TCCC-carrying promoter was detected by the real-time quantitative reverse transcription-PCR. These assays were performed on cell lines cultured in absence or presence of ACTH. In the baseline, the light intensity of the luciferase reporter assay driven by the TCCT promoter was four times higher than that by the TCCC promoter. The intensity was dramatically increased in the pGL3-TCCT after ACTH stimulation, compared to that in the pGL3-TCCC. MC2R expression assay showed a 5-fold increase in the TCCT promoter in presence of ACTH, compared with that in absence of ACTH. The results showed that the haplotype TCCT in MC2R promoter significantly led to increased MC2R expression and strong responses to ACTH, providing evidence of the molecular mechanism of ACTH therapy in IS.

Androgen receptor function is modulated by the tissue‐specific AR45 variant

A naturally occurring variant of the human androgen receptor (AR) named AR45 has been identified. It lacks the entire region encoded by exon 1 of the AR gene and is composed of the AR DNA‐binding domain, hinge region and ligand‐binding domain, preceded by a novel seven amino‐acid long N‐terminal extension. A survey of human tissues revealed that AR45 was expressed mainly in heart and skeletal muscle. In cotransfection experiments, AR45 inhibited AR function, an effect necessitating intact DNA‐ and ligand‐binding properties. Overexpression of AR45 reduced the proliferation rate of the androgen‐dependent LNCaP cells, in line with the repressive effects of AR45 on AR growth‐promoting function. AR45 interacted with the AR N‐terminal domain in two‐hybrid assays, suggesting that AR inhibition was due to the formation of AR–AR45 heterodimers. Under conditions where the transcriptional coactivator TIF2 or the oncogene β‐catenin were overexpressed, AR45 stimulated androgen‐dependent promoters in presence of dihydrotestosterone. AR45 activity was triggered additionally by the adrenal androgen androstenedione in presence of exogenous TIF2. Altogether, the data suggest an important role of AR45 in modulating AR function and add a novel level of complexity to the mode of action of androgens.

Androgen receptor function is modulated by the tissue-specific AR45 variant

A naturally occurring variant of the human androgen receptor (AR) named AR45 has been identified. It lacks the entire region encoded by exon 1 of the AR gene and is composed of the AR DNA-binding domain, hinge region and ligand-binding domain, preceded by a novel seven amino-acid long N-terminal extension. A survey of human tissues revealed that AR45 was expressed mainly in heart and skeletal muscle. In cotransfection experiments, AR45 inhibited AR function, an effect necessitating intact DNA- and ligand-binding properties. Overexpression of AR45 reduced the proliferation rate of the androgen-dependent LNCaP cells, in line with the repressive effects of AR45 on AR growth-promoting function. AR45 interacted with the AR N-terminal domain in two-hybrid assays, suggesting that AR inhibition was due to the formation of AR-AR45 heterodimers. Under conditions where the transcriptional coactivator TIF2 or the oncogene beta-catenin were overexpressed, AR45 stimulated androgen-dependent promoters in presence of dihydrotestosterone. AR45 activity was triggered additionally by the adrenal androgen androstenedione in presence of exogenous TIF2. Altogether, the data suggest an important role of AR45 in modulating AR function and add a novel level of complexity to the mode of action of androgens.

Identification of FHOD1-binding proteins and mechanisms of FHOD1-regulated actin dynamics.

Formin homology-2-domain containing protein 1 (FHOD1) regulates gene transcription, actin-cytoskeleton structure, and cell migration. To gain insight into the mechanisms by which FHOD1 mediates these diverse activities, a yeast-two-hybrid screen was performed to identify FHOD1-binding proteins. Three proteins specifically interacted with the carboxy-terminal two-thirds of FHOD1, which includes the FH1, FH2, and diaphanous activating domains (DAD). The newly identified FHOD1-binding proteins are protein kinase C binding protein 1 (PRKCBP1), cyclophilin B, and an isoform of WASP-interacting SH3-domain protein/diaphanous-interacting protein 1 (WISH/DIP1), named WISH-B. The proline-rich FH1 domain of FHOD1 was sufficient to interact with the central portion of PRKCP1 and full-length cyclophilin B. The FH1 domain also interacted with full-length WISH-B, but the extreme amino-terminus was sufficient to associate with WISH-B as well. WISH-B altered the solubility of FHOD1 in vitro and a truncation mutant containing the amino-terminal 227 residues of WISH-B disrupted FHOD1-induced stress fibers. WISH-B did not affect FHOD1-induced gene transcription through the serum response factor (SRF) recognition site on the skeletal alpha actin promoter (SkA). However, stabilization of F-actin prevented FHOD1 dependent activation of this promoter in presence of high, but not low serum concentrations. Thus, the identification of a new FHOD1-binding protein provides insight into the mechanisms by which FHOD1 regulates actin polymerization and transcription.

Characterization of the iron-regulated desA promoter of Streptomyces pilosus as a system for controlled gene expression in actinomycetes

BackgroundThe bioavailability of iron is quite low since it is usually present as insoluble complexes. To solve the bioavailability problem microorganisms have developed highly efficient iron-scavenging systems based on the synthesis of siderophores that have high iron affinity. The systems of iron assimilation in microorganisms are strictly regulated to control the intracellular iron levels since at high concentrations iron is toxic for cells. Streptomyces pilosus synthesizes the siderofore desferrioxamine B. The first step in desferrioxamine biosynthesis is decarboxylation of L-lysine to form cadaverine, a desferrioxamine B precursor. This reaction is catalyzed by the lysine decarboxylase, an enzyme encoded by the desA gene that is repressed by iron.ResultsThe binding of the DmdR (acronym for divalent metal dependent repressor) to the desA promoter in presence of Fe2+ or other divalent ions has been characterized. A 51 bp DNA fragment of the desA promoter containing the 9 bp inverted repeat was sufficient for binding of the DmdR repressor, as observed by the electrophoretic mobility shift assay. The desA mobility shift was prevented by neutralizing DmdR with anti-DmdR antibodies or by chelating the divalent metal in the binding reaction with 2,2'-dipyridyl. Binding to the desA promoter was observed with purified DmdR repressors of Streptomyces coelicolor or Rhodococcus fascians suggesting that there is a common mechanism of iron-regulation in actinomycetes. The complete desA promoter region was coupled using transcriptional fusions to the amy reporter gene (encoding α-amylase) in low copy or multicopy Streptomyces vectors. The iron-regulated desA promoter was induced by addition of the iron chelating agent 2,2'-dipyridyl resulting in a strong expression of the reporter gene.ConclusionsThe iron-regulated desA promoter can be used for inducible expression of genes in Streptomyces species, as shown by de-repression of the promoter when coupled to a reporter gene.