|ognizant Communication Corporation|
VOLUME 14, NUMBER 5
Gene Expression, Vol. 14, pp. 251-263
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Gene Expression Pattern and Downregulation of DNA Methyltransferase 1 Using siRNA in Porcine Somatic Cells
Angelica M. Giraldo, Todd D. Vaught, Limin Fu, Alison J. Duncan, Amy M. Vance, Michael Mendicino, and David L. Ayares
Revivicor, Inc., Blacksburg, VA 24060, USA
DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through their influence on gene expression. Manipulation of the DNA methylation marks of differentiated cells will allow a better understanding of the different molecular processes associated with chromatin structure and gene expression. The objective of this study was to identify small interfering RNAs (siRNAs) with the ability to reduce DNA methyltransferase 1 (Dnmt1) mRNA and consequently decrease Dnmt1 protein as well as DNA methylation in porcine cells. Fibroblasts from four porcine fetuses were established and cultured in 5% CO2 in air at 38°C. Optimal transfection conditions were evaluated using a FITC-labeled control siRNA. Four Dnmt1-specific siRNAs were evaluated upon transfection of each cell line. A nonsilencing siRNA was used as a negative control. The expression patterns of Dnmt1 were analyzed by Q-PCR. The combination of 1 mg of siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Downregulation of Dnmt1 varied between siRNAs. Transfection of porcine cells with highly effective siRNAs resulted in a drastic reduction of Dnmt1 mRNA and a slight decrease in protein production. However, this small reduction in the protein concentration induced significant genomic hypomethylation. These data suggest that although Dnmt1 mRNA abundance plays an important role during protein regulation, Dnmt1 enzyme is mainly posttranscriptionally regulated. Subsequent use of these cells for cloning, differentiation, and cancer studies will provide insight as to how methylation of the DNA affects genomic reprogramming.
Key words: DNA methyltransferases; DNA methylation; siRNA; Epigenetics; Gene expression
Address correspondence to Angelica Giraldo, Revivicor, 1700 Kraft Dr., Suite 2400, Blacksburg, VA 24060, USA. Fax: 540 961 7958; E-mail: firstname.lastname@example.org
Comparison of Gene Expression Profiles of Conjunctival Cell Lines With Primary Cultured Conjunctival Epithelial Cells and Human Conjunctival Tissue
Louis Tong,1 Yolanda Diebold,2 Margarita Calonge,2 Jianping Gao,3 Michael E. Stern,3 and Roger W. Beuerman4
1Singapore National Eye Center, Singapore
2Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain
3Allergan, Inc., Irvine, CA, USA
4Singapore Eye Research Institute, Singapore National Eye Center, Singapore
Human conjunctival cell lines are useful tools for modeling ocular surface disease and evaluation of ocular drugs and cosmetics. However, gene expression in these cells may not be comparable to primary cultured cells, raising doubts that they could be used as a substitute. We aimed to ascertain the similarities of global gene expression between commonly used cell lines and primary cells using a microarray approach. The Affymetrix U133A chip (>22,000 genes) was used to investigate conjunctival tissue (CT), primary conjunctival epithelial cells (PCEC), two conjunctival epithelial cell lines (IOBA-NHC and ChWK), and HCEC-T, a human corneal epithelial cell line (control). Using principal component analysis, the PCEC profile was clustered more closely to conjunctival tissue than either of the two cell lines. Certain extracellular matrix genes were differentially upregulated in CT compared to PCEC, suggesting presence of fibroblasts in addition to epithelial cells in CT. Overall, 67.3% (95% CI: 66.7-67.9) of transcripts in IOBA-NHC were within 1.5-fold of the corresponding transcripts in PCEC, but only 62.2% (95% CI: 61.5-62.9) in the case of ChWK. In HCEC-T, the proportion was only 58.8% (95% CI 58.1-59.4), suggesting less resemblance to PCEC than the conjunctival epithelial cell lines. The IOBA-NHC profile was more similar to PCEC than ChWK, for all genes and genes concerned with membrane association, communication, development, and regulation of metabolism, especially protein and nucleic acid metabolism. The correlation of normalized gene expression levels was high between either the IOBA-NHC or ChWK and PCEC for genes concerned with cell defense, viral life cycle, antigen presentation, antioxidation, or ubiquitin ligation. In order to evaluate the functional significance of the altered gene expression in IOBA-NHC cells, we evaluated a few proteins important for epithelial differentiation or defense, corresponding to the transcripts for S100A9, TGM2, and TLR4. Protein levels of S100A9 and TGM2 were indeed raised, and TLR4 decreased, in IOBA-NHC compared to PCEC. Gene expression in conjunctival cell lines differs from primary cells, but the profile varies according to functional gene categories. Depending on the methodology of proposed studies, if there is limited availability of PCEC, NHC-IOBA may be more suitable than ChWK, but even then, epithelial differentiation and innate immunity functions in NHC-IOBA may differ from primary cells.
Key words: Human; Cell culture; Gene expression; Microarray; Ocular surface disease
Address correspondence to Dr. Louis Tong, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751. Tel: +65-62277255; Fax: +65-63224599; E-mail: Louis.email@example.com
Expression of Genes Involved in Nitrogen Assimilation and the C/N Balance Sensing in Prochlorococcus sp. Strain SS120
Antonio López-Lozano, Guadalupe Gómez-Baena, María Del Carmen Muñoz-Marín, Oriol Alberto Rangel, Jesús Diez, and Jose Manuel García-Fernández
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, 14071-Córdoba, Spain
The expression of five genes involved in nitrogen assimilation in cyanobacteria, namely glnA, glsF, icd, ntcA, and glnB, encoding three key enzymes from that pathway (glutamine synthetase, glutamate synthase, isocitrate dehydrogenase) and two regulatory proteins (NtcA and PII), was studied in this work. Their changes under different conditions were analyzed by quantitative real-time RT-PCR. Nutrient limitation induced clear modifications on the expression of most studied genes: lack of nitrogen provoked an initial increase, followed by a marked decrease; in the cases of phosphorus and iron starvation, a general, stronger expression decrease was observed, particularly striking in the case of iron. Darkness and addition of the photosynthethic inhibitors DCMU and DBMIB also had a strong effect on gene expression. Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, provoked a sharp increase in icd expression. These results, together with previous studies, suggest that 2-oxoglutarate could be the molecule utilized by Prochlorococcus to sense the C/N balance. Besides, our results confirm the different regulation of nitrogen assimilation in Prochlorococcus with regard to other cyanobacteria.
Key words: Carbon and nitrogen assimilation; Cyanobacteria; Gene expression; Metabolic regulation
Address correspondence to Jose Manuel García-Fernández, Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Edificio Severo Ochoa, planta 1, Campus de Rabanales, 14071-Córdoba, Spain. Tel/fax: +34 957 211075; E-mail: firstname.lastname@example.org
Conditional Ablation of Mediator Subunit MED1 (MED1/PPARBP) Gene in Mouse Liver Attenuates Glucocorticoid Receptor Agonist Dexamethasone-Induced Hepatic Steatosis
Yuzhi Jia, Navin Viswakarma, Tao Fu, Songtao Yu, M. Sambasiva Rao, Jayme Borensztajn, and Janardan K. Reddy
Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Glucocorticoid receptor (GR) agonist dexamethasone (Dex) induces hepatic steatosis and enhances constitutive androstane receptor (CAR) expression in the liver. CAR is known to worsen hepatic injury in nonalcoholic hepatic steatosis. Because transcription coactivator MED1/PPARBP gene is required for GR- and CAR-mediated transcriptional activation, we hypothesized that disruption of MED1/PPARBP gene in liver cells would result in the attenuation of Dex-induced hepatic steatosis. Here we show that liver-specific disruption of MED1 gene (MED1DLiv) improves Dex-induced steatotic phenotype in the liver. In wild-type mice Dex induced severe hepatic steatosis and caused reduction in medium- and short-chain acyl-CoA dehydrogenases that are responsible for mitochondrial b-oxidation. In contrast, Dex did not induce hepatic steatosis in mice conditionally null for hepatic MED1, as it failed to inhibit fatty acid oxidation enzymes in the liver. MED1DLiv livers had lower levels of GRregulated CAR mRNA compared to wild-type mouse livers. Microarray gene expression profiling showed that absence of MED1 affects the expression of the GR-regulated genes responsible for energy metabolism in the liver. These results establish that absence of MED1 in the liver diminishes Dex-induced hepatic steatosis by altering the GR- and CAR-dependent gene functions.
Key words: Mediator complex subunit 1 (MED1); PPARBP; Hepatic steatosis; Dexamethasone; Glucocorticoid receptor; Constitutive androstane receptor
Address correspondence to Jayme Borensztajn, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611, USA. Fax: 1 312 5038249; E-mail: email@example.com or Janardan K. Reddy, Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611, USA. Fax: 1 312 5038249; E-mail: firstname.lastname@example.org