Sun Chong Wang

Epigenomic Profiling Reveals DNA-Methylation Changes Associated with Major Psychosis

Epigenetic misregulation is consistent with various non-Mendelian features of schizophrenia and bipolar disorder. To date, however, few studies have investigated the role of DNA methylation in major psychosis, and none have taken a genome-wide epigenomic approach. In this study we used CpG-island microarrays to identify DNA-methylation changes in the frontal cortex and germline associated with schizophrenia and bipolar disorder. In the frontal cortex we find evidence for psychosis-associated DNA-methylation differences in numerous loci, including several involved in glutamatergic and GABAergic neurotransmission, brain development, and other processes functionally linked to disease etiology. DNA-methylation changes in a significant proportion of these loci correspond to reported changes of steady-state mRNA level associated with psychosis. Gene-ontology analysis highlighted epigenetic disruption to loci involved in mitochondrial function, brain development, and stress response. Methylome network analysis uncovered decreased epigenetic modularity in both the brain and the germline of affected individuals, suggesting that systemic epigenetic dysfunction may be associated with major psychosis. We also report evidence for a strong correlation between DNA methylation in the MEK1 gene promoter region and lifetime antipsychotic use in schizophrenia patients. Finally, we observe that frontal-cortex DNA methylation in the BDNF gene is correlated with genotype at a nearby nonsynonymous SNP that has been previously associated with major psychosis. Our data are consistent with the epigenetic theory of major psychosis and suggest that DNA-methylation changes are important to the etiology of schizophrenia and bipolar disorder.

DNA methylation profiles in monozygotic and dizygotic twins

Twin studies have provided the basis for genetic and epidemiological studies in human complex traits. As epigenetic factors can contribute to phenotypic outcomes, we conducted a DNA methylation analysis in white blood cells (WBC), buccal epithelial cells and gut biopsies of 114 monozygotic (MZ) twins as well as WBC and buccal epithelial cells of 80 dizygotic (DZ) twins using 12K CpG island microarrays. Here we provide the first annotation of epigenetic metastability of ∼6,000 unique genomic regions in MZ twins. An intraclass correlation (ICC)-based comparison of matched MZ and DZ twins showed significantly higher epigenetic difference in buccal cells of DZ co-twins (P = 1.2 × 10−294). Although such higher epigenetic discordance in DZ twins can result from DNA sequence differences, our in silico SNP analyses and animal studies favor the hypothesis that it is due to epigenomic differences in the zygotes, suggesting that molecular mechanisms of heritability may not be limited to DNA sequence differences.

A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.

Complex disease, gender and epigenetics

Gender differences in susceptibility to complex disease such as asthma, diabetes, lupus, autism and major depression, among numerous other disorders, represent one of the hallmarks of non‐Mendelian biology. It has been generally accepted that endocrinological differences are involved in the sexual dimorphism of complex disease; however, specific molecular mechanisms of such hormonal effects have not been elucidated yet. This paper will review evidence that sex hormone action may be mediated via gene‐specific epigenetic modifications of DNA and histones. The epigenetic modifications can explain sex effects at DNA sequence polymorphisms and haplotypes identified in gender‐stratified genetic linkage and association studies. Hormone‐induced DNA methylation and histone modification changes at specific gene regulatory regions may increase or reduce the risk of a disease. The epigenetic interpretation of sexual dimorphism fits well into the epigenetic theory of complex disease, which argues for the primary pathogenic role of inherited and/or acquired epigenetic misregulation rather than DNA sequence variation. The new experimental strategies, especially the high throughput microarray‐based epigenetic profiling, can be used for testing the epigenetic hypothesis of gender effects in complex diseases.

5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary

The 5-methylcytosine (5-mC) derivative 5-hydroxymethylcytosine (5-hmC) is abundant in the brain for unknown reasons. Here we characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC by using glucosylation coupled with restriction-enzyme digestion and microarray analysis. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary in human and mouse. This boundary change was mainly due to 5-hmC in the brain but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent data sets and with single-molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC relative to alternatively spliced exons. Our study suggests a new role for 5-hmC in RNA splicing and synaptic function in the brain.

Epigenetics of personality traits: An illustrative study of identical twins discordant for risk-taking behavior

DNA methylation differences between identical twins could account for phenotypic twin discordance of behavioral traits and diseases. High throughput epigenomic microarray profiling can be a strategy of choice for identification of epigenetic differences in phenotypically different monozygotic (MZ) twins. Epigenomic profiling of a pair of MZ twins with quantified measures of psychometric discordance identified several DNA methylation differences, some of which may have developmental and behavioral implications and are consistent with the contrasting psychometric profiles of the twins. In particular, differential methylation of CpG islands proximal to the homeobox DLX1 gene could modulate stress responses and risk taking behavior, and deserve further attention as a potential marker of aversion to danger. The epigenetic difference detected at DLX1 of approximately 1.2 fold change was used to evaluate experimental design issues such as the required numbers of technical replicates. It also enabled us to estimate the power this technique would have to detect a functionally relevant epigenetic difference given a range of 1 to 50 twin pairs. We found that use of epigenomic microarray profiling in a relatively small number (15-25) of phenotypically discordant twin pairs has sufficient power to detect 1.2 fold epigenetic changes.

Epigenetic markers of prostate cancer in plasma circulating DNA

Epigenetic differences are a common feature of many diseases, including cancer, and disease-associated changes have even been detected in bodily fluids. DNA modification studies in circulating DNA (cirDNA) may lead to the development of specific non-invasive biomarkers. To test this hypothesis, we investigated cirDNA modifications in prostate cancer patients with locally confined disease (n = 19), in patients with benign prostate hyperplasias (n = 20) and in men without any known prostate disease (n = 20). This initial discovery screen identified 39 disease-associated changes in cirDNA modification, and seven of these were validated using the sodium bisulfite-based mapping of modified cytosines in both the discovery cohort and an independent 38-patient validation cohort. In particular, we showed that the DNA modification of regions adjacent to the gene encoding ring finger protein 219 distinguished prostate cancer from benign hyperplasias with good sensitivity (61%) and specificity (71%). We also showed that repetitive sequences detected in this study were meaningful, as they indicated a highly statistically significant loss of DNA at the pericentromeric region of chromosome 10 in prostate cancer patients (p = 1.8 × 10(-6)). Based on these strong univariate results, we applied machine-learning techniques to develop a multi-locus biomarker that correctly distinguished prostate cancer samples from unaffected controls with 72% accuracy. Lastly, we used systems biology techniques to integrate our data with publicly available DNA modification and transcriptomic data from primary prostate tumors, thereby prioritizing genes for further studies. These data suggest that cirDNA epigenomics are promising source for non-invasive biomarkers.

DNA Modification Study of Major Depressive Disorder: Beyond Locus-by-Locus Comparisons

BACKGROUND Major depressive disorder (MDD) exhibits numerous clinical and molecular features that are consistent with putative epigenetic misregulation. Despite growing interest in epigenetic studies of psychiatric diseases, the methodologies guiding such studies have not been well defined. METHODS We performed DNA modification analysis in white blood cells from monozygotic twins discordant for MDD, in brain prefrontal cortex, and germline (sperm) samples from affected individuals and control subjects (total N = 304) using 8.1K CpG island microarrays and fine mapping. In addition to the traditional locus-by-locus comparisons, we explored the potential of new analytical approaches in epigenomic studies. RESULTS In the microarray experiment, we detected a number of nominally significant DNA modification differences in MDD and validated selected targets using bisulfite pyrosequencing. Some MDD epigenetic changes, however, overlapped across brain, blood, and sperm more often than expected by chance. We also demonstrated that stratification for disease severity and age may increase the statistical power of epimutation detection. Finally, a series of new analytical approaches, such as DNA modification networks and machine-learning algorithms using binary and quantitative depression phenotypes, provided additional insights on the epigenetic contributions to MDD. CONCLUSIONS Mapping epigenetic differences in MDD (and other psychiatric diseases) is a complex task. However, combining traditional and innovative analytical strategies may lead to identification of disease-specific etiopathogenic epimutations.

High Precision DNA Modification Analysis of HCG9 in Major Psychosis

New epigenetic technologies may uncover etiopathogenic mechanisms of major psychosis. In this study, we applied padlock probe-based ultra-deep bisulfite sequencing for fine mapping of modified cytosines of the HLA complex group 9 (nonprotein coding) gene in the postmortem brains of individuals affected with schizophrenia or bipolar disorder and unaffected controls. Significant differences between patients and controls were detected in both CpG and CpH modifications. In addition, we identified epigenetic age effects, DNA modification differences between sense and anti-sense strands, and demonstrated how DNA modification data can be used in clustering of patient populations. Our findings revealed new epigenetic complexities but also highlighted the potential of DNA modification approaches in the search of heterogeneous causes of major psychiatric disease.