Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes

Glucocorticoids and FoxO3 exert similar metabolic effects in skeletal muscle. FoxO3 gene expression was increased by dexamethasone (Dex), a synthetic glucocorticoid, both in vitro and in vivo. In C2C12 myotubes the increased expression is due to, at least in part, the elevated rate of FoxO3 gene transcription. In the mouse FoxO3 gene, we identified three glucocorticoid receptor (GR) binding regions (GBRs): one being upstream of the transcription start site, −17kbGBR; and two in introns, +45kbGBR and +71kbGBR. Together, these three GBRs contain four 15-bp glucocorticoid response elements (GREs). Micrococcal nuclease (MNase) assay revealed that Dex treatment increased the sensitivity to MNase in the GRE of +45kbGBR and +71kbGBR upon 30- and 60-min Dex treatment, respectively. Conversely, Dex treatment did not affect the chromatin structure near the −17kbGBR, in which the GRE is located in the linker region. Dex treatment also increased histone H3 and/or H4 acetylation in genomic regions near all three GBRs. Moreover, using chromatin conformation capture (3C) assay, we showed that Dex treatment increased the interaction between the −17kbGBR and two genomic regions: one located around +500 bp and the other around +73 kb. Finally, the transcriptional coregulator p300 was recruited to all three GBRs upon Dex treatment. The reduction of p300 expression decreased FoxO3 gene expression and Dex-stimulated interaction between distinct genomic regions of FoxO3 gene identified by 3C. Overall, our results demonstrate that glucocorticoids activated FoxO3 gene transcription through multiple GREs by chromatin structural change and DNA looping.

Download Full-text

The β-cell specific transcription factor Nkx6.1 inhibits glucagon gene transcription by interfering with Pax6

Biochemical Journal ◽

10.1042/bj20070053 ◽

2007 ◽

Vol 403 (3) ◽

pp. 593-601 ◽

Cited By ~ 24

Author(s):

Benoit R. Gauthier ◽

Yvan Gosmain ◽

Aline Mamin ◽

Jacques Philippe

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Gene Transcription ◽

Promoter Activity ◽

Islet Cells ◽

Gene Activation ◽

Physical Interaction ◽

Specific Expression

The transcription factor Nkx6.1 is required for the establishment of functional insulin-producing β-cells in the endocrine pancreas. Overexpression of Nkx6.1 has been shown to inhibit glucagon gene expression while favouring insulin gene activation. Down-regulation resulted in the opposite effect, suggesting that absence of Nkx6.1 favours glucagon gene expression. To understand the mechanism by which Nkx6.1 suppresses glucagon gene expression, we studied its effect on the glucagon gene promoter activity in non-islet cells using transient transfections and gel-shift analyses. In glucagonoma cells transfected with an Nkx6.1-encoding vector, the glucagon promoter activity was reduced by 65%. In BHK21 cells, Nkx6.1 inhibited by 93% Pax6-mediated activation of the glucagon promoter, whereas Cdx2/3 and Maf stimulations were unaltered. Although Nkx6.1 could interact with both the G1 and G3 element, only the former displayed specificity for Nkx6.1. Mutagenesis of the three potential AT-rich motifs within the G1 revealed that only the Pax6-binding site preferentially interacted with Nkx6.1. Chromatin immunoprecipitation confirmed interaction of Nkx6.1 with the glucagon promoter and revealed a direct competition for binding between Pax6 and Nkx6.1. A weak physical interaction between Pax6 and Nkx6.1 was detected in vitro and in vivo suggesting that Nkx6.1 predominantly inhibits glucagon gene transcription through G1-binding competition. We suggest that cell-specific expression of the glucagon gene may only proceed when Nkx6.1, in combination with Pdx1 and Pax4, are silenced in early α-cell precursors.

Download Full-text

Yeast TFIID Serves as a Coactivator for Rap1p by Direct Protein-Protein Interaction

Molecular and Cellular Biology ◽

10.1128/mcb.01558-06 ◽

2006 ◽

Vol 27 (1) ◽

pp. 297-311 ◽

Cited By ~ 61

Author(s):

Krassimira A. Garbett ◽

Manish K. Tripathi ◽

Belgin Cencki ◽

Justin H. Layer ◽

P. Anthony Weil

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Gene Transcription ◽

In Vitro Transcription ◽

Tata Binding Protein ◽

In Vivo Studies ◽

Activator Protein 1 ◽

Protein Protein Interaction

ABSTRACT In vivo studies have previously shown that Saccharomyces cerevisiae ribosomal protein (RP) gene expression is controlled by the transcription factor repressor activator protein 1 (Rap1p) in a TFIID-dependent fashion. Here we have tested the hypothesis that yeast TFIID serves as a coactivator for RP gene transcription by directly interacting with Rap1p. We have found that purified recombinant Rap1p specifically interacts with purified TFIID in pull-down assays, and we have mapped the domains of Rap1p and subunits of TFIID responsible. In vitro transcription of a UASRAP1 enhancer-driven reporter gene requires both Rap1p and TFIID and is independent of the Fhl1p-Ifh1p coregulator. UASRAP1 enhancer-driven transactivation in extracts depleted of both Rap1p and TFIID is efficiently rescued by addition of physiological amounts of these two purified factors but not TATA-binding protein. We conclude that Rap1p and TFIID directly interact and that this interaction contributes importantly to RP gene transcription.

Download Full-text

Nonmetastatic Colon Cancer Model C26 Upregulates Glycolysis in Osteocytes in Vitro and Bone in Vivo

Proceedings of IMPRS ◽

10.18060/25742 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

M. Roarke Tollar ◽

Matthew Prideaux ◽

Fabrizio Pin ◽

Lynda F. Bonewald

Keyword(s):

Gene Expression ◽

Culture Media ◽

Bone Mineralization ◽

Metabolic Effects ◽

Whole Body ◽

Rt Pcr ◽

Systemic Complications ◽

Whole Body Metabolism

Background: Developing effective treatments for musculoskeletal complications in cancer patients requires understanding metabolic effects of cancer on bone, and particularly osteocytes, the most abundant bone cell and key regulator of bone remodeling. However, little is known regarding how cancer impacts normal osteocyte energy metabolic pathways, such as glycolysis. Given that changes in metabolism are important regulators of cellular function, it is essential to determine how osteocyte metabolism is disrupted by cancer and how this may impact skeletal and whole-body health. Methods: Mice inoculated with saline (N=5) or C26 cells (N=6) were sacrificed after 2 weeks. Bones were harvested for metabolic profiling by GC-MS, gene expression by RT-PCR and bone morphology by µCT. Differentiated IDG-SW3 osteocyte-like cells were cocultured with C26 cells for 12-24hrs and metabolites and gene expression analyzed by GC-MS and RT-PCR. Results: Trabecular bone mass was significantly decreased in the C26 mice. GC-MS analysis revealed decreased glucose in C26 mice tibiae, but no change in lactate. The bone resorption promoting gene Rankl was upregulated, whereas the inhibitor Opg was unchanged. Bone mineralization regulators Mepe and Phex were decreased. In vitro metabolic studies revealed increased glucose and lactate in IDG-SW3 cell lysate; culture media glucose levels were decreased whereas lactate was increased in the co-cultures with C26 cells. RT-PCR demonstrated increases in the glycolysis promoter Hif1α in addition to glycolysis pathway genes including Glut1, Hk2, Slc16a3 and Pdk1. Rankl was also increased in the IDG-SW3 cells co-cultured with the C26 cells whereas Opg, Phex, and Mepe were downregulated. Conclusion: Glycolysis is upregulated in mouse bone and in vitro IDG-SW3 cells exposed to cancer. Our study provides novel understanding for how cancer affects bone metabolism. Integrating these results with whole body metabolism will aid in the development of novel therapeutic strategies to target musculoskeletal and systemic complications of cancer.

Download Full-text

199 IN VITRO-DEVELOPED BOVINE BLASTOCYSTS ARE MARKED WITH ABERRANT HYPER- AND HYPO-METHYLATED GENOMIC REGIONS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab199 ◽

2015 ◽

Vol 27 (1) ◽

pp. 190

Author(s):

D. Salilew-Wondim ◽

M. Hoelker ◽

U. Besenfelder ◽

V. Havlicek ◽

F. Rings ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Expression Patterns ◽

Proximal Promoter ◽

Altered Gene Expression ◽

Genome Wide ◽

Altered Gene ◽

Genomic Regions

Most often, in vitro produced embryos display poor quality and altered gene expression patterns compared to their in vivo counterparts. Aberrant DNA methylation occurring during in vitro embryo development is believed to be one of the multifaceted factors which may cause altered gene expression and poor embryo quality. Here, we investigated the genome-wide DNA methylation patterns of in vitro derived embryos using the recently developed Bovine EmbryoGENE Methylation Platform (BEGMP) array (Shojaei Saadi et al. BMC Genomics 2014 15, 451. doi: 10.1186/1471-2164-15-451) to unravel the aberrantly methylated genomic region in in vitro developed embryos. For this, in vitro and in vivo produced blastocysts were produced and used for genome-wide DNA methylation analysis. In vitro blastocysts were produced from oocytes retrieved from ovaries collected from the local abattoir and matured, fertilized, and cultured in vitro using SOF media. The in vivo blastocysts were produced by superovulation and AI of Simmental heifers followed by uterine flushing. Genomic DNA (gDNA) was then isolated from four replicates (each 10 blastocysts) of in vivo and in vitro derived blastocysts using Allprep DNA/RNA micro kit (Qiagen, Valencia, CA, USA) and the gDNA was then fragmented using the MseI enzyme. Following this, MseLig21 and MseLig were ligated to the MseI-digested genomic fragments in the presence of Ligase enzyme. Methyl-sensitive enzymes, HpaII, AciI, and Hinp1I, were used to cleave unmethlayted genomic regions within the MseI-MseI region of the fragmented DNA. The gDNA was subjected to two rounds of ligation-mediated polymerase chain reaction (LM-PCR) amplification. After removal of the adapters, the amplified gDNA samples from in vivo or in vitro groups were labelled either Cy-3 or Cy-5 dyes in dye-swap design using ULS Fluorescent gDNA labelling kit (Kreatech Biotechnology BV, Amsterdam, The Netherlands). Hybridization was performed for 40 h at 65°C. Slides were scanned using Agilent's High-Resolution C Scanner (Agilent Technologies Inc., Santa Clara, CA, USA) and features were extracted with Agilent's Feature Extraction software (Agilent Technologies Inc.). The results have shown that from a total of 414 566 probes harboured by the BEGMP array, 248 453 and 253 147 probes were detected in in vitro and in vivo derived blastocysts, respectively. Data analysis using the linear modelling for microarray (LIMMA) package and R software (The R Project for Statistical Computing, Vienna, Austria) revealed a total of 3434 differentially methylated regions (DMRs; Fold change ≥1.5, P-value <0.05), of which 42 and 58% were hyper- and hypo-methylated, respectively, in in vitro derived blastocysts compared to their in vivo counterparts. The DMRs were found to be localised in the intronic, exonic, promoter, proximal promoter, and distal promoter, and some of the probes did not have nearby genes. In addition, 10.8% of the DMRs were found to be stretched in short, long, or intermediate CpG islands. Thus, this study demonstrated genome-wide dysregulation in the epigenome landscape of in vitro-derived embryos by the time they reach to the blastocysts stage.

Download Full-text

A DNA methylation reader complex that enhances gene transcription

Science ◽

10.1126/science.aar7854 ◽

2018 ◽

Vol 362 (6419) ◽

pp. 1182-1186 ◽

Cited By ~ 53

Author(s):

C. Jake Harris ◽

Marion Scheibe ◽

Somsakul Pop Wongpalee ◽

Wanlu Liu ◽

Evan M. Cornett ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Arabidopsis Thaliana ◽

Gene Transcription ◽

Methylated Dna ◽

Transposon Insertion ◽

Activate Gene

DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.

Download Full-text

Higher glucose availability augments the metabolic responses of the C2C12 myotubes to exercise-like electrical pulse stimulation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00133.2021 ◽

2021 ◽

Author(s):

Juulia H. Lautaoja ◽

Thomas O'Connell ◽

Sakari Mäntyselkä ◽

Juuli Peräkylä ◽

Heikki Kainulainen ◽

...

Keyword(s):

High Glucose ◽

Electrical Pulse ◽

Metabolic Effects ◽

C2c12 Myotubes ◽

Branched Chain ◽

Free Media ◽

C2c12 Myotube ◽

Glucose Availability

The application of exercise-like electrical pulse simulation (EL-EPS) has become a widely used exercise mimetic in vitro. EL-EPS produces similar physiological responses as in vivo exercise, while less is known about the detailed metabolic effects. Routinely the C2C12 myotubes are cultured in high glucose medium (4.5 g/l), which may alter EL-EPS responses. In this study, we evaluate the metabolic effects of EL-EPS under the high and low glucose (1.0 g/l) conditions to understand how substrate availability affects the myotube response to EL-EPS.The C2C12 myotube, media and cell-free media metabolites were analyzed using untargeted nuclear magnetic resonance (NMR)-based metabolomics. Further, translational and metabolic changes and possible exerkine effects were analyzed. EL-EPS enhanced substrate utilization as well as production and secretion of lactate, acetate, 3-hydroxybutyrate and branched chain fatty acids (BCFAs). The increase in BCFAs correlated with branched chain amino acids (BCAAs) and BCFAs were strongly decreased when myotubes were cultured without BCAAs suggesting the action of acyl-CoA thioesterases on BCAA catabolites. Notably, not all EL-EPS responses were augmented by high glucose because EL-EPS increased phosphorylated c-Jun N-terminal kinase and interleukin-6 secretion independent of glucose availability. Administration of acetate and EL-EPS conditioned media on HepG2 hepatocytes had no adverse effects on lipolysis or triacylglycerol content.Our results demonstrate that unlike in cell-free media, the C2C12 myotube and media metabolites were affected by EL-EPS, particularly under high glucose condition suggesting that media composition should be considered in future EL-EPS studies. Further, acetate and BCFAs were identified as putative exerkines warranting more research.

Download Full-text

Identification of genes early responsive to gamma-irradiation in isolated rat hepatocytes in vitro and rat liver in vivo by cDNA array gene expression analysis

Zeitschrift für Gastroenterologie ◽

10.1055/s-2005-920060 ◽

2005 ◽

Vol 43 (05) ◽

Author(s):

DS Batusic ◽

H Christiansen ◽

B Saile ◽

RM Hermann ◽

J Dudas ◽

...

Keyword(s):

Gene Expression ◽

Gamma Irradiation ◽

Rat Liver ◽

Gene Expression Analysis ◽

Cdna Array ◽

Rat Hepatocytes ◽

Isolated Rat Hepatocytes ◽

Isolated Rat

Download Full-text

Effect of X-irradiation on gene expression of rat liver chemokines: in vivo and in vitro studies

Zeitschrift für Gastroenterologie ◽

10.1055/s-2008-1037499 ◽

2008 ◽

Vol 46 (01) ◽

Cited By ~ 1

Author(s):

F Moriconi ◽

H Christiansen ◽

N Sheikh ◽

J Dudas ◽

...

Keyword(s):

Gene Expression ◽

Rat Liver ◽

In Vitro Studies ◽

X Irradiation

Download Full-text

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

Diseases of Aquatic Organisms ◽

10.3354/dao03475 ◽

2020 ◽

Vol 139 ◽

pp. 153-160

Author(s):

S Peeralil ◽

TC Joseph ◽

V Murugadas ◽

PG Akhilnath ◽

VN Sreejith ◽

...

Keyword(s):

Gene Expression ◽

Virulence Genes ◽

Vibrio Harveyi ◽

Penaeus Monodon ◽

Challenge Test ◽

Virulence Gene ◽

Economic Losses ◽

Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

Download Full-text