Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression

RNA interference (RNAi) mediated by DNA-based expression of short hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research.

Activation of the selenopritein SEPS1 gene expression by pro-inflammatory cytokines in Hep G2 cells

SEPS1 (also called selenoprotein S, SelS) plays an important role in the production of inflammatory cytokines and its expression is activated by endoplasmic reticulum (ER) stress. In this report, we have identified two binding sites for the nuclear factor kappa B in the human SEPS1 promoter. SEPS1 gene expression, protein levels and promoter activity were all increased 2–3-fold by TNF-α and IL-1β in HepG2 cells. We have also confirmed that the previously proposed ER stress response element GGATTTCTCCCCCGCCACG in the SEPS1 proximate promoter is fully functional and responsive to ER stress. However, concurrent treatment of HepG2 cells with IL-1β and ER stress produced no additive effect on SEPS1 gene expression. We conclude that SEPS1 is a new target gene of NF-κB. Together with our previous findings that SEPS1 may regulate cytokine production in macrophage cells, we propose a regulatory loop between cytokines and SEPS1 that plays a key role in control of the inflammatory response.

Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors

Retrotransposons have clearly molded the structure of the human genome. The reverse transcriptase coded for by long interspersed nuclear elements (LINEs) accounts for 35% of the human genome, with 8–9 x 105 copies of the most common human LINE element, L1Hs. Retrotransposons cycle through an RNA intermediate with transcription as the rate limiting step. Because various retrotransposons have been demonstrated to be induced by environmental stimuli, we investigated the response of the L1Hs promoter to various agents. L1Hs promoter activity was analyzed by transfecting an L1Hs-expressing cell line with plasmids containing one of two L1Hs promoters fused to the LacZ reporter gene. L1Hs promoter activity was then monitored with a ß-galactosidase assay. Treatment with UV light and heat shock resulted in a small increase in ß-galactosidase activity from one promoter, while treatment with tetradecanoylphorbol 13-acetate resulted in small increases in ß-galactosidase activity from both promoters. No increase in ß-galactosidase activity was observed after exposure to X-rays or hydrogen peroxide.

GAD2 on chromosome 10p12 is a candidate gene for human obesity

The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11–12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of γ-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C>A and +83897 T>A (OR = 0.81, 95% CI [0.681–0.972], p = 0.0049) and an at-risk SNP (−243 A>G) for morbid obesity (OR = 1.3, 95% CI [1.053–1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C>A and +83897 T>A haplotype (χ2 = 7.637, p = 0.02). In the murine insulinoma cell line βTC3, the G at-risk allele of SNP −243 A>G increased six times GAD2 promoter activity (p < 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The −243 A>G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic β cells, we analyzed GAD65 antibody level as a marker of β-cell activity and of insulin secretion. In the control group, −243 A>G, +61450 C>A, and +83897 T>A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T>A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of β-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.

The small molecule, genistein, increases hepcidin expression in human hepatocytes

Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin-6 (IL-6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28-52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole-mount in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction. Genistein's stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3-binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein-treated hepatocytes indicated that genistein up-regulated 68% of the transcripts that were up-regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up-regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. Conclusion: Genistein is the first small-molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.

Effect of arsenic on transcription factor AP-1 and NF-kB DNA binding activity and related gene expression

Both acute (24 h) and chronic (10–20 week) exposure of human fibroblast cells to low dose sodium arsenite (As(III)) significantly affects activating protein-1 (AP-1) and nuclear factor kappa B (NF-κB) DNA binding activity. Short-term treatment with 0.1–5 μM As(III) up-regulates expression of c-Fos and c-Jun and the redox regulators, thioredoxin (Trx) and Redox factor-1 (Ref-1) and activates both AP-1 and NF-κB binding. Chronic exposure to 0.1 or 0.5 μM As(III) decreased c-Jun, c-Fos and Ref-1 protein levels and AP-1 and NF-κB binding activity, but increased Trx expression. Short term exposure to phorbol 12-myristate 13-acetate (TPA), a phorbol ester tumour promoter, or hydrogen peroxide (H₂O₂) also activates AP-1 and NF-κB binding. However, pre-treatment with As(III) prevents this increase. These results suggest that As(III) may alter AP-1 and NF-κB activity, in part, by up-regulating Trx and Ref-1. The different effects of short- versus long-term As(III) treatment on acute-phase response to oxidative stress reflect changes in the expression of Ref-1, c-Fos and c-Jun, but not Trx.

Exercise increases MEF2- and GEF DNA-binding activity in human skeletal muscle

Diabetes is quickly reaching epidemic proportions, with 216 million people worldwide predicted to be diagnosed with the disease by 2010. While it appears that the expression of the insulin responsive glucose transporter isoform 4 (GLUT4) is not reduced in diabetic populations, overexpression of GLUT4 exclusively in muscle enhances insulin action and improves glucose homeostasis. Consequently, understanding the regulation of GLUT4 expression is considered important in identifying potential therapeutic targets for the treatment and management of insulin resistance and related disorders such as type 2 diabetes. Using transgenic mice, we have identified two conserved regions on the GLUT4 gene promoter that are required for normal skeletal muscle GLUT4 expression. The first region contains a binding site for the myocyte enhancer factor 2 (MEF2) transcription factor, between –464 and –473 bp, and it appears that a MEF2A/D heterodimer binds this sequence. However, this site is not sufficient to support full GLUT4 expression, and another region between –712 and –742 bp, termed Domain 1, is also required. A novel transcription factor, named the GLUT4 enhancer factor (GEF), was found to bind to this region. It appears that MEF2 and GEF physically interact in order to induce GLUT4 expression. A single bout of exercise is sufficient to increase both GLUT4 transcription and mRNA abundance. However, the molecular mechanisms underpinning this response remain largely unexplored, particularly in human skeletal muscle. Therefore, the aim of this study was to determine whether a single, acute bout of exercise increases the DNA-binding activity of both MEF2 and GEF in human skeletal muscle.

Functional (GT)n polymorphisms in promoter region of N-methyl-d-aspartate receptor 2A subunit (GRIN2A) gene affect hippocampal and amygdala volumes

The glutamate system including N-methyl-d-aspartate (NMDA) affects synaptic formation, plasticity and maintenance. Recent studies have shown a variable (GT)n polymorphism in the promoter region of the NMDA subunit gene (GRIN2A) and a length-dependent inhibition of transcriptional activity by the (GT)n repeat. In the present study, we examined whether the GRIN2A polymorphism is associated with regional brain volume especially in medial temporal lobe structures, in which the NMDA-dependent synaptic processes have been most extensively studied. Gray matter regions of interest (ROIs) for the bilateral amygdala and hippocampus were outlined manually on the magnetic resonance images of 144 healthy individuals. In addition, voxel-based morphometry (VBM) was conducted to explore the association of genotype with regional gray matter volume from everywhere in the brain in the same sample. The manually measured hippocampal and amygdala volumes were significantly larger in subjects with short allele carriers (n = 89) than in those with homozygous long alleles (n = 55) when individual differences in intracranial volume were accounted for. The VBM showed no significant association between the genotype and regional gray matter volume in any brain region. These findings suggest that the functional GRIN2A (GT)n polymorphism could weakly but significantly impact on human medial temporal lobe volume in a length-dependent manner, providing in vivo evidence of the role of the NMDA receptor in human brain development.