Epigenomic profiling of men exposed to early-life stress reveals DNA methylation differences in association with current mental state

Peer reviewe

A statistical method for excluding non-variable CpG sites in high-throughput DNA methylation profiling

<p>Abstract</p> <p>Background</p> <p>High-throughput DNA methylation arrays are likely to accelerate the pace of methylation biomarker discovery for a wide variety of diseases. A potential problem with a standard set of probes measuring the methylation status of CpG sites across the whole genome is that many sites may not show inter-individual methylation variation among the biosamples for the disease outcome being studied. Inclusion of these so-called "non-variable sites" will increase the risk of false discoveries and reduce statistical power to detect biologically relevant methylation markers.</p> <p>Results</p> <p>We propose a method to estimate the proportion of non-variable CpG sites and eliminate those sites from further analyses. Our method is illustrated using data obtained by hybridizing DNA extracted from the peripheral blood mononuclear cells of 311 samples to an array assaying 1505 CpG sites. Results showed that a large proportion of the CpG sites did not show inter-individual variation in methylation.</p> <p>Conclusions</p> <p>Our method resulted in a substantial improvement in association signals between methylation sites and outcome variables while controlling the false discovery rate at the same level.</p

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities

Active behaviour during early development shapes glucocorticoid reactivity

TGlucocorticoids are the final effectors of the stress axis, with numerous targets in the central nervous system and the periphery. They are essential for adaptation, yet currently it is unclear how early life events program the glucocorticoid response to stress. Here we provide evidence that involuntary swimming at early developmental stages can reconfigure the cortisol response to homotypic and heterotypic stress in larval zebrafish (Danio rerio), also reducing startle reactivity and increasing spontaneous activity as well as energy efficiency during active behaviour. Collectively, these data identify a role of the genetically malleable zebrafish for linking early life stress with glucocorticoid function in later life

Preprocessing differential methylation hybridization microarray data

<p>Abstract</p> <p>Background</p> <p>DNA methylation plays a very important role in the silencing of tumor suppressor genes in various tumor types. In order to gain a genome-wide understanding of how changes in methylation affect tumor growth, the differential methylation hybridization (DMH) protocol has been developed and large amounts of DMH microarray data have been generated. However, it is still unclear how to preprocess this type of microarray data and how different background correction and normalization methods used for two-color gene expression arrays perform for the methylation microarray data. In this paper, we demonstrate our discovery of a set of internal control probes that have log ratios (M) theoretically equal to zero according to this DMH protocol. With the aid of this set of control probes, we propose two LOESS (or LOWESS, locally weighted scatter-plot smoothing) normalization methods that are novel and unique for DMH microarray data. Combining with other normalization methods (global LOESS and no normalization), we compare four normalization methods. In addition, we compare five different background correction methods.</p> <p>Results</p> <p>We study 20 different preprocessing methods, which are the combination of five background correction methods and four normalization methods. In order to compare these 20 methods, we evaluate their performance of identifying known methylated and un-methylated housekeeping genes based on two statistics. Comparison details are illustrated using breast cancer cell line and ovarian cancer patient methylation microarray data. Our comparison results show that different background correction methods perform similarly; however, four normalization methods perform very differently. In particular, all three different LOESS normalization methods perform better than the one without any normalization.</p> <p>Conclusions</p> <p>It is necessary to do within-array normalization, and the two LOESS normalization methods based on specific DMH internal control probes produce more stable and relatively better results than the global LOESS normalization method.</p

Comparative isoschizomer profiling of cytosine methylation:the HELP assay

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome. ©2006 by Cold Spring Harbor Laboratory Press

Generation of a genomic tiling array of the human Major Histocompatibility Complex (MHC) and its application for DNA methylation analysis

Background: The major histocompatibility complex (MHC) is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome.Methods: To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP), methylated DNA immunoprecipitation (MeDIP), array comparative genomic hybridization (aCGH) and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls.Results: Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs). Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation.Conclusion: A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics

Genome Wide Analysis of Acute Myeloid Leukemia Reveal Leukemia Specific Methylome and Subtype Specific Hypomethylation of Repeats

Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R2 = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array

An Integrative Genomic and Epigenomic Approach for the Study of Transcriptional Regulation

The molecular heterogeneity of acute leukemias and other tumors constitutes a major obstacle towards understanding disease pathogenesis and developing new targeted-therapies. Aberrant gene regulation is a hallmark of cancer and plays a central role in determining tumor phenotype. We predicted that integration of different genome-wide epigenetic regulatory marks along with gene expression levels would provide greater power in capturing biological differences between leukemia subtypes. Gene expression, cytosine methylation and histone H3 lysine 9 (H3K9) acetylation were measured using high-density oligonucleotide microarrays in primary human acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) specimens. We found that DNA methylation and H3K9 acetylation distinguished these leukemias of distinct cell lineage, as expected, but that an integrative analysis combining the information from each platform revealed hundreds of additional differentially expressed genes that were missed by gene expression arrays alone. This integrated analysis also enhanced the detection and statistical significance of biological pathways dysregulated in AML and ALL. Integrative epigenomic studies are thus feasible using clinical samples and provide superior detection of aberrant transcriptional programming than single-platform microarray studies