Detection and quantification of methylation in DNA using solid-state nanopores.

Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores. Methylation is detected by selectively labeling methylation sites with MBD1 (MBD-1x) proteins, the complex inducing a 3 fold increase in ionic blockage current relative to unmethylated DNA. Furthermore, the discrimination of methylated and unmethylated DNA is demonstrated in the presence of only a single bound protein, thereby giving a resolution of a single methylated CpG dinucleotide. The extent of methylation of a target molecule could also be coarsely quantified using this novel approach. This nanopore-based methylation sensitive assay circumvents the need for bisulfite conversion, fluorescent labeling, and PCR and could therefore prove very useful in studying the role of epigenetics in human disease

17β-Estradiol Modulates Gene Expression in the Female Mouse Cerebral Cortex

<div><p>17β-estradiol (E2) plays critical roles in a number of target tissues including the mammary gland, reproductive tract, bone, and brain. Although it is clear that E2 reduces inflammation and ischemia-induced damage in the cerebral cortex, the molecular mechanisms mediating the effects of E2 in this brain region are lacking. Thus, we examined the cortical transcriptome using a mouse model system. Female adult mice were ovariectomized and implanted with silastic tubing containing oil or E2. After 7 days, the cerebral cortices were dissected and RNA was isolated and analyzed using RNA-sequencing. Analysis of the transcriptomes of control and E2-treated animals revealed that E2 treatment significantly altered the transcript levels of 88 genes. These genes were associated with long term synaptic potentiation, myelination, phosphoprotein phosphatase activity, mitogen activated protein kinase, and phosphatidylinositol 3-kinase signaling. E2 also altered the expression of genes linked to lipid synthesis and metabolism, vasoconstriction and vasodilation, cell-cell communication, and histone modification. These results demonstrate the far-reaching and diverse effects of E2 in the cerebral cortex and provide valuable insight to begin to understand cortical processes that may fluctuate in a dynamic hormonal environment.</p></div

E2 increases pERK protein levels.

<p>(A)Western blot analysis was used to monitor pERK and total ERK levels in the cortices of mice that had been treated with oil or E2 for 7 days. (B) pERK values were normalized to total ERK and are displayed as the normalized fold change ± SEM. The Student’s t-test was used to detect significant differences in oil- and E2- treated animals (*<i>p</i><0.05). The number of animals in each treatment group is indicated at the base of each bar.</p

E2-responsive networks and associated genes.

<p>E2-responsive networks and associated genes.</p

E2-regulated genes with the most decreased expression.

<p>E2-regulated genes with the most decreased expression.</p

Networks of E2-regulated genes.

<p>ClueGO analysis classified the 88 E2-regulated genes into 6 networks. White nodes indicate that a biological process is associated with two networks. Node size indicates the statistical significance of the biological process represented. Thus, larger nodes indicate greater statistical significance.</p

88 E2-responsive genes in the cerebral cortex.

<p>88 E2-responsive genes in the cerebral cortex.</p

E2-regulated genes with the most increased expression.

<p>E2-regulated genes with the most increased expression.</p

Validation of transcripts altered by E2 treatment.

<p>(A) Quantitative real-time PCR was conducted with gene-specific primers. The normalized fold change ± SEM was calculated using the delta-delta Ct method with Rpl7 as a control gene. The Student’s t-test was used to detect significant differences in oil- and E2- treated animals (4 animals/treatment, *<i>p</i><0.05).</p

Nanopore-Based Assay for Detection of Methylation in Double-Stranded DNA Fragments

DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the <i>CpG</i> dyad in DNA fragments and could someday profile the position of methylated <i>CpG</i> sites on DNA fragments