Reproducibility and intraindividual variation over days in buccal cell DNA methylation of two asthma genes, interferon γ (IFNγ) and inducible nitric oxide synthase (iNOS)

The biological mechanisms responsible for the onset and exacerbation of asthma symptoms in children may involve the epigenetic regulation of inflammatory genes after environmental exposures. Using buccal cells, we hypothesized that DNA methylation in promoter regions of two asthma genes, inducible nitric oxide synthase (iNOS) and interferon γ (IFNγ), can vary over several days. Repeat buccal samples were collected 4 to 7 days apart from 34 children participating in the Columbia Center for Children's Environmental Health (CCCEH) birth cohort study. Several field duplicates (sequential collection of two samples in the field) and replicates (one sample pyrosequenced twice) also were collected to ensure consistency with collection and laboratory procedures. DNA methylation was assessed by pyrosequencing a PCR of bisulfite-treated DNA. We found that replicate and field duplicate samples were correlated strongly (r = 0.86 to 0.99, P < 0.05), while repeat samples demonstrated low within-subject correlations (r = 0.19 to 0.56, P = 0.06 to 0.30). Our data reveal DNA methylation as a dynamic epigenetic mechanism that can be accessed safely and reproducibly in an inner city pediatric cohort using non-invasive buccal swabs and pyrosequencing technology

Ehrlichia chaffeensis Transcriptome in Mammalian and Arthropod Hosts Reveals Differential Gene Expression and Post Transcriptional Regulation

BACKGROUND: Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. METHODOLOGY/PRINCIPAL FINDINGS: The majority (∼80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30-80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. CONCLUSIONS/SIGNIFICANCE: Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes associated with host-pathogen interactions are differentially expressed and regulated by post transcriptional mechanisms

Hypomethylation of Intragenic LINE-1 Represses Transcription in Cancer Cells through AGO2

In human cancers, the methylation of long interspersed nuclear element -1 (LINE-1 or L1) retrotransposons is reduced. This occurs within the context of genome wide hypomethylation, and although it is common, its role is poorly understood. L1s are widely distributed both inside and outside of genes, intragenic and intergenic, respectively. Interestingly, the insertion of active full-length L1 sequences into host gene introns disrupts gene expression. Here, we evaluated if intragenic L1 hypomethylation influences their host gene expression in cancer. First, we extracted data from L1base (http://l1base.molgen.mpg.de), a database containing putatively active L1 insertions, and compared intragenic and intergenic L1 characters. We found that intragenic L1 sequences have been conserved across evolutionary time with respect to transcriptional activity and CpG dinucleotide sites for mammalian DNA methylation. Then, we compared regulated mRNA levels of cells from two different experiments available from Gene Expression Omnibus (GEO), a database repository of high throughput gene expression data, (http://www.ncbi.nlm.nih.gov/geo) by chi-square. The odds ratio of down-regulated genes between demethylated normal bronchial epithelium and lung cancer was high (p<1E−27; OR = 3.14; 95% CI = 2.54–3.88), suggesting cancer genome wide hypomethylation down-regulating gene expression. Comprehensive analysis between L1 locations and gene expression showed that expression of genes containing L1s had a significantly higher likelihood to be repressed in cancer and hypomethylated normal cells. In contrast, many mRNAs derived from genes containing L1s are elevated in Argonaute 2 (AGO2 or EIF2C2)-depleted cells. Hypomethylated L1s increase L1 mRNA levels. Finally, we found that AGO2 targets intronic L1 pre-mRNA complexes and represses cancer genes. These findings represent one of the mechanisms of cancer genome wide hypomethylation altering gene expression. Hypomethylated intragenic L1s are a nuclear siRNA mediated cis-regulatory element that can repress genes. This epigenetic regulation of retrotransposons likely influences many aspects of genomic biology

Oral tongue cancer gene expression profiling: Identification of novel potential prognosticators by oligonucleotide microarray analysis

<p>Abstract</p> <p>Background</p> <p>The present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling.</p> <p>Methods</p> <p>The gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG_U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, <it>GLUT3</it>, <it>HSAL2</it>, and <it>PACE4</it>, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR.</p> <p>Results</p> <p>Hierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, <it>MMP-1</it> encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of <it>GLUT3</it>, <it>HSAL2</it> and <it>PACE4</it>, respectively. Univariate analyses demonstrated that <it>GLUT3</it> over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). <it>HSAL2</it> was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only <it>GLUT3</it> showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). <it>PACE4</it> mRNA expression failed to show correlation with any of the relevant parameters. </p> <p>Conclusion</p> <p>The characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by real-time RT-PCR offers a powerful strategy for identification of novel targets for prognostication and treatment of oral tongue carcinoma.</p