Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets

Using a unique microarray platform for cytosine methylation profiling, the DNA methylation landscape of the human genome was monitored at more than 21,000 sites, including 79% of the annotated transcriptional start sites (TSS). Analysis of an oligodendroglioma derived cell line LN-18 revealed more than 4000 methylated TSS. The gene-centric analysis indicated a complex pattern of DNA methylation exists along each autosome, with a trend of increasing density approaching the telomeres. Remarkably, 2% of CpG islands (CGI) were densely methylated, and 17% had significant levels of 5 mC, whether or not they corresponded to a TSS. Substantial independent verification, obtained from 95 loci, suggested that this approach is capable of large scale detection of cytosine methylation with an accuracy approaching 90%. In addition, we detected large genomic domains that are also susceptible to DNA methylation reinforced inactivation, such as the HOX cluster on chromosome 7 (CH7). Extrapolation from the data suggests that more than 2000 genomic loci may be susceptible to methylation and associated inactivation, and most have yet to be identified. Finally, we report six new targets of epigenetic inactivation (IRX3, WNT10A, WNT6, RARalpha, BMP7 and ZGPAT). These targets displayed cell line and tumor specific differential methylation when compared with normal brain samples, suggesting they may have utility as biomarkers. Uniquely, hypermethylation of the CGI within an IRX3 exon was correlated with over-expression of IRX3 in tumor tissues and cell lines relative to normal brain samples

Identification of Novel High-Frequency DNA Methylation Changes in Breast Cancer

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (∼200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis

The Efficacy of GeneThresher® Methylation Filtering Technology in the Plant Kingdom

The genomes of many plants are known to be composed of a large fraction of repetitive DNA, while a small portion is dedicated to genes. The bulk of the repetitive DNA constitutes transposable elements and is heavily methylated. GeneThresher technology has been developed to take advantage of these differential methylation patterns by filtering genomic shotgun libraries to exclude methylated sequences (Rabinowicz et al., 1999; Palmer et al., 2003; Martienssen et al., 2004). The result is a gene-enriched genomic shotgun library. Random shotgun sequencing of plant gene space, enabled by GeneThresher technology, is a rapid and cost-effective strategy for comprehensive gene discovery in agriculturally important crop

Gaze holding in healthy subjects

Eccentric gaze in darkness evokes minor centripetal eye drifts in healthy subjects, as cerebellar control sufficiently compensates for the inherent deficiencies of the brainstem gaze-holding network. This behavior is commonly described using a leaky integrator model, which assumes that eye velocity grows linearly with gaze eccentricity. Results from previous studies in patients and healthy subjects suggest caution when this assumption is applied to eye eccentricities larger than 20 degrees. To obtain a detailed characterization of the centripetal gaze-evoked drift, we recorded horizontal eye position in 20 healthy subjects. With their head fixed, they were asked to fixate a flashing dot (50 ms every 2 s)that was quasi-stationary displacing(0.5 deg/s) between ± 40 deg horizontally in otherwise complete darkness. Drift velocity was weak at all angles tested. Linearity was assessed by dividing the range of gaze eccentricity in four bins of 20 deg each, and comparing the slopes of a linear function fitted to the horizontal velocity in each bin. The slopes of single subjects for gaze eccentricities of ± 0-20 deg were, in median,0.41 times the slopes obtained for gaze eccentricities of ± 20-40 deg. By smoothing the individual subjects' eye velocity as a function of gaze eccentricity, we derived a population of position-velocity curves. We show that a tangent function provides a better fit to the mean of these curves when large eccentricities are considered. This implies that the quasi-linear behavior within the typical ocular motor range is the result of a tuning procedure, which is optimized in the most commonly used range of gaze. We hypothesize that the observed non-linearity at eccentric gaze results from a saturation of the input that each neuron in the integrating network receives from the others. As a consequence, gaze-holding performance declines more rapidly at large eccentricities