Regions of variable DNA methylation in human placenta associated with newborn neurobehavior

<p>The placenta regulates the <i>in utero</i> environment and functionally impacts fetal development. Candidate gene studies identified variation in placental DNA methylation is associated with newborn neurologic and behavioral outcomes including movement quality, lethargic behavior, attention, and arousal. We sought to identify novel regions of variable DNA methylation associated with newborn attention, lethargy, quality of movement, and arousal by performing an epigenome-wide association study in 335 infants from a US birth cohort. Methylation status was quantified using the Illumina HumanMethylation450 BeadChip array and associations to newborn outcomes assessed by the NICU Network Neurobehavioral Scales (NNNS) were identified while incorporating established bioinformatics algorithms to control for confounding by cell type composition. Methylation of CpGs within <i>FHIT</i> (cg15970800) and <i>ANKRD11</i> (cg16710656) demonstrated genome-wide significance (<i>P</i> < 1.8 × 10⁻⁷) in specific associations with infant attention. CpGs whose differential methylation was associated with all 4 neurobehavioral outcomes were common to 50 genes involved in biological processes relating to cellular adhesion and nervous system development. Comprehensive methylation profiling identified relationships between methylation of <i>FHIT</i> and <i>ANKRD11</i>, which have been previously linked to neurodevelopment and behavioral outcomes in genetic association studies. Subtle changes in DNA methylation of these genes within the placenta may impact normal variation of a newborn's ability to alter and track visual and auditory stimuli. Gene ontology analysis suggested that those genes with variable methylation related to these outcomes are over-represented in biological pathways involved in brain development and placental physiology, supportive of our hypothesis for a key role of the placenta in neurobehavioral outcomes.</p

Average dietary As intake (μg/day) plotted against the logarithm of drinking water As (μg/L)

<p><b>Copyright information:</b></p><p>Taken from "Dietary Arsenic Exposure in Bangladesh"</p><p></p><p>Environmental Health Perspectives 2007;115(6):889-893.</p><p>Published online 20 Feb 2007</p><p>PMCID:PMC1892146.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI</p> The mean and median regressions were obtained from the model = (α + β) × (log + ɛ), where either the mean or median of ɛ is zero. For the median regression line, α = 40.5 and β = 11.6; for the mean regression line, α = 46.0 and β = 19.5. Shaded areas represent 1 SE around each regression line

Effects of Sulforaphane and 3,3′-Diindolylmethane on Genome-Wide Promoter Methylation in Normal Prostate Epithelial Cells and Prostate Cancer Cells

<div><p>Epigenetic changes, including aberrant DNA methylation, result in altered gene expression and play an important role in carcinogenesis. Phytochemicals such as sulforaphane (SFN) and 3,3′-diindolylmethane (DIM) are promising chemopreventive agents for the treatment of prostate cancer. Both have been shown to induce re-expression of genes, including tumor suppressor genes silenced in cancer cells, via modulation of epigenetic marks including DNA methylation. However, it remained unclear the effects SFN and DIM on DNA methylation at a genomic scale. The goal of this study was to determine the genome-wide effects of SFN and DIM on promoter methylation in normal prostate epithelial cells and prostate cancer cells. Both SFN and DIM treatment decreased DNA methyltransferase expression in normal prostate epithelial cells (PrEC), and androgen-dependent (LnCAP) and androgen-independent (PC3) prostate cancer cells. The effects of SFN and DIM on promoter methylation profiles in normal PrEC, LnCAP and PC3 prostate cancer cells were determined using methyl-DNA immunoprecipitation followed by genome-wide DNA methylation array. We showed widespread changes in promoter methylation patterns, including both increased and decreased methylation, in all three prostate cell lines in response to SFN or DIM treatments. In particular, SFN and DIM altered promoter methylation in distinct sets of genes in PrEC, LnCAP, and PC3 cells, but shared similar gene targets within a single cell line. We further showed that SFN and DIM reversed many of the cancer-associated methylation alterations, including aberrantly methylated genes that are dysregulated or are highly involved in cancer progression. Overall, our data suggested that both SFN and DIM are epigenetic modulators that have broad and complex effects on DNA methylation profiles in both normal and cancerous prostate epithelial cells. Results from our study may provide new insights into the epigenetic mechanisms by which SFN and DIM exert their cancer chemopreventive effects.</p></div

Nightshift work and genome-wide DNA methylation

<div><p>The negative health effects of shift work, including carcinogenesis, may be mediated by changes in DNA methylation, particularly in the circadian genes. Using the Infinium HumanMethylation450 Bead Array (Illumina, San Diego, CA), we compared genome-wide methylation between 65 actively working dayshift workers and 59 actively working nightshift workers in the healthcare industry. A total of 473 800 loci, including 391 loci across the 12 core circadian genes, were analyzed to identify methylation markers associated with shift work status using linear regression models adjusted for gender, age, body mass index, race, smoking status and leukocyte cell profile as measured by flow cytometry. Analyses at the level of gene, CpG island and gene region were also conducted. To account for multiple comparisons, we controlled the false discovery rate (FDR ≤0.05). Significant differences between nightshift and dayshift workers were found at 16 135 of 473 800 loci, across 3769 of 20 164 genes, across 7173 of 22 721 CpG islands and across 5508 of 51 843 gene regions. For each significant loci, gene, CpG island or gene region, average methylation was consistently found to be decreased among nightshift workers compared to dayshift workers. Twenty-one loci located in the circadian genes were also found to be significantly hypomethylated among nightshift workers. The largest differences were observed for three loci located in the gene body of <i>PER3</i>. A total of nine significant loci were found in the <i>CSNK1E</i> gene, most of which were located in a CpG island and near the transcription start site of the gene. Methylation changes in these circadian genes may lead to altered expression of these genes which has been associated with cancer in previous studies. Gene ontology enrichment analysis revealed that among the significantly hypomethylated genes, processes related to host defense and immunity were represented. Our results indicate that the health effects of shift work may be mediated by hypomethylation of a wide variety of genes, including those related to circadian rhythms. While these findings need to be followed-up among a considerably expanded group of shift workers, the data generated by this study supports the need for future targeted research into the potential impacts of shift work on specific carcinogenic mechanisms.</p></div

SFN and DIM had distinct methylation gene targets in normal prostate epithelial cells and prostate cancer cells but high level of gene targets overlap within a single cell line.

<p>(A) Venn diagrams showing the number of genes that were differentially methylated in PrEC, LnCAP, and PC3 cells upon treatments with SFN or DIM compared to vehicle control. (B) Venn diagrams showing the number of differentially methylated gene targets of SFN and DIM within each cell line.</p

Functional annotation of genes with altered promoter methylation in LnCAP cells that were reversed with SFN and/or DIM treatments.

<p>(A) Number of differentially methylated genes in LnCAP cells relative to PrEC cells that were reversed with SFN or DIM treatments. Black bar represents genes that had decreased methylation in LnCAP cells that were increased with SFN/DIM treatments. White bar represents genes that had increased methylation in LnCAP cells that were decreased with SFN/DIM treatments. (B) Two gene examples, CCR4 and TGFBR1, with dysregulated methylation profiles in LnCAP cells that were reversed with SFN and/or DIM treatments. Colored bars represent the genomic position of DNA methylation probes that had decreased methylation (blue) or increase methylation (red) when comparing the probe-specific log2 fold-change in LnCAP cells versus PrEC, or DIM versus DMSO vehicle control in LnCAP cells. TSS (blue circle) represents transcription start site of each gene. (C) Functional annotation of differentially methylated gene targets in LnCAP cells that were reversed with SFN or DIM treatments. (D) Venn diagram showing the number of gene targets with promoter methylation that were dysregulated in LnCAP cells and reversed with SFN or DIM.</p

Biological functions of genes with promoter methylation affected by SFN or DIM treatments in normal prostate epithelial cells and prostate cancer cells.

<p>Functional annotation of differentially methylated gene targets of SFN and DIM in PrEC, LnCAP, and PC3 cells were examined and the number of genes involved in select biological processes and functions were shown. Data are expressed as the number of genes in each significantly enriched GO category.</p

Comparison of the overlap of DNA methylation probes with epigenetic marks showed the outcome of DIM-mediated alteration in DNA methylation may be influenced by local histone modifications.

<p>(A) The genomic region coordinates of DNA methylation probes that had decreased methylation (black bar) or increased methylation (white bar) in DIM-treated LnCAP cells were uploaded into EpiExplorer to determine potential overlap with various histone modifications from the ENCODE database. A randomized control set was generated by reshuffling the genomic positions of each dataset (light and dark gray bars for decreased methylation and increased methylation, respectively). Data summarize the percent overlap of DNA methylation probes and their respective randomized control to histone methylation mark H3K4me3, and histone acetylation marks H3K9ac. (B) ChIP assays for H3K4me3 at the TGFBR1 and CYR61 promoters in LnCAP cells after vehicle (DMSO) or 15 µM DIM treatments for 48 h. ChIP-qPCR data were expressed as % input. Results represent the mean % input fold-change ± SEM compared to DMSO vehicle control of two independent experiments. *p-value <0.05.</p

Genome-wide promoter methylation effects of SFN or DIM on normal prostate epithelial cells and prostate cancer cells.

<p>(A) Effects of SFN and DIM on the methylation profile in PrEC, LnCAP, and PC3 cells compared to vehicle control. In each of the three cell lines, significant methylated probes in SFN or DIM-treated groups were compared to their respective vehicle control to determine probe-specific log2 fold-change. The distribution of probes with significant log2 fold-change is shown. (B) Average methylation level in individual genes in SFN and DIM-treated PrEC, LnCAP, and PC3 cells compared to vehicle control. Log2 fold-change per gene was determined by averaging the log2 fold-change of all differentially methylated probes assigned to each gene, and represented as box and whisker plots. Number above the bar denotes the number of genes in each group. Whiskers represent maximum and minimum values, and “+” represents mean value.</p

Increased DNA methyltransferase gene expression in prostate cancer cell lines was decreased with SFN or DIM treatments.

<p>(A) Gene expression of DNMT1, DNMT3A, and DNMT3B in untreated PrEC, LnCAP, and PC3 cells (n = 3–5 per group). Data represent mean normalized fold-change ± SEM compared to PrEC. (B–D) Effects of SFN and DIM on DNMT gene expression in PrEC cells (B), LnCAP cells (C), and PC3 cells (D). Cells were treated with vehicle control (DMSO), 15 µM SFN, or 15 µM DIM (n = 6 per group in B–D). DNMT1, DNMT3A, and DNMT3B gene expression were analyzed 48 h post-treatment. Data represent mean normalized fold-change ± SEM compared to DMSO. *p-value <0.05.</p