Myocardin-Related Transcription Factor Mediates Epithelial Fibrogenesis in Polycystic Kidney Disease.

Polycystic kidney disease (PKD) is characterized by extensive cyst formation and progressive fibrosis. However, the molecular mechanisms whereby the loss/loss-of-function of Polycystin 1 or 2 (PC1/2) provokes fibrosis are largely unknown. The small GTPase RhoA has been recently implicated in cystogenesis, and we identified the RhoA/cytoskeleton/myocardin-related transcription factor (MRTF) pathway as an emerging mediator of epithelium-induced fibrogenesis. Therefore, we hypothesized that MRTF is activated by PC1/2 loss and plays a critical role in the fibrogenic reprogramming of the epithelium. The loss of PC1 or PC2, induced by siRNA in vitro, activated RhoA and caused cytoskeletal remodeling and robust nuclear MRTF translocation and overexpression. These phenomena were also manifested in PKD1 (RC/RC) and PKD2 (WS25/-) mice, with MRTF translocation and overexpression occurring predominantly in dilated tubules and the cyst-lining epithelium, respectively. In epithelial cells, a large cohort of PC1/PC2 downregulation-induced genes was MRTF-dependent, including cytoskeletal, integrin-related, and matricellular/fibrogenic proteins. Epithelial MRTF was necessary for the paracrine priming of the fibroblast-myofibroblast transition. Thus, MRTF acts as a prime inducer of epithelial fibrogenesis in PKD. We propose that RhoA is a common upstream inducer of both histological hallmarks of PKD: cystogenesis and fibrosis.

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.

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.

DNA unmethylome profiling by covalent capture of CpG sites.

Dynamic patterns of cytosine-5 methylation and successive hydroxylation are part of epigenetic regulation in eukaryotes, including humans, which contributes to normal phenotypic variation and disease risk. Here we present an approach for the mapping of unmodified regions of the genome, which we call the unmethylome. Our technique is based on DNA methyltransferase-directed transfer of activated groups and covalent biotin tagging of unmodified CpG sites followed by affinity enrichment and interrogation on tiling microarrays or next generation sequencing. Control experiments and pilot studies of human genomic DNA from cultured cells and tissues demonstrate that, along with providing a unique cross-section through the chemical landscape of the epigenome, the methyltransferase-directed transfer of activated groups-based approach offers high precision and robustness as compared with existing affinity-based techniques.

Quantitative leukocyte BDNF promoter methylation analysis in bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is a complex psychiatric phenotype with a high heritability and a multifactorial etiology. Multisite collaborative efforts using genome-wide association studies (GWAS) have identified only a portion of DNA sequence-based risk factors in BD. In addition to predisposing DNA sequence variants, epigenetic misregulation may play an etiological role in BD and account for monozygotic twin discordance, parental origin effects, and fluctuating course of BD. In this study, we investigated DNA methylation of the brain-derived neurotrophic factor (BDNF) gene in BD. METHODS: Fifty participants with BD were compared to the same number of age- and sex-matched controls for DNA methylation differences at BDNF promoters 3 and 5. DNA methylation reads were obtained using a mass spectrophotometer for 64 cytosine-guanine (CpG) sites in 36 CpG 'units' across three amplicons of BDNF promoters 3 and 5. RESULTS AND DISCUSSION: Methylation fractions differed between BD participants and controls for 11 of 36 CpG units. Five CpG units, mostly in promoter 5, remained significant after false discovery rate correction (FDR) (p values ≤ 0.004) with medium to large effect sizes (Cohen's d ≥ 0.61). Several of the significant CpGs overlapped with or were immediately adjacent to transcription factor binding sites (TFBSs) - including two of the FDR-significant CpG units in promoter 5. For the CpGs in promoter 3, there was a positive and significant correlation between age at sample collection and DNA methylation fraction (rho = 0.56, p = 2.8 ×10(-5)) in BD cases, but not in controls. Statistically significant differences in mean methylation fraction at 5/36 CpG units (after FDR), some at or immediately adjacent to TFBSs, suggest possible relevance for the current findings to BD etiopathogenesis. The positive correlation between age and methylation seen in promoter 3 is consistent with age-related decline in BDNF expression previously reported. Future studies should provide more exhaustive epigenetic study of the BDNF locus to better characterize the relationship between BDNF methylation differences and BD.

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.

Understanding bipolar disorder: the epigenetic perspective.

Bipolar disease (BPD) is a complex major psychiatric disorder that affects between 1% and 2% of the population and exhibits ?85% heritability. This has made BPD an appealing target for genetic studies yet, despite numerous attempts, the genetic basis of this disease remains elusive. Recently, it has come to light that epigenetic factors may also influence the development of BPD. These factors act via stable but reversible modifications of DNA and chromatin structure. In this chapter, we revisit the epidemiological, clinical, and molecular findings in BPD and reanalyze them from the perspective of inherited and acquired epigenetic misregulation. Epigenetic research has great potential to enhance our understanding of the molecular basis of BPD.

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.

Evolution of the Beckwith-Wiedemann syndrome region in vertebrates.

In the animal kingdom, genomic imprinting appears to be restricted to mammals. It remains an open question how structural features for imprinting evolved in mammalian genomes. The clustering of genes around imprinting control centers (ICs) is regarded as a hallmark for the coordinated imprinted regulation. Hence imprinted clusters might be structurally distinct between mammals and nonimprinted vertebrates. To address this question we compared the organization of the Beckwith Wiedemann syndrome (BWS) gene cluster in mammals, chicken, Fugu (pufferfish), and zebrafish. Our analysis shows that gene synteny is apparently well conserved between mammals and birds, and is detectable but less pronounced in fish. Hence, clustering apparently evolved during vertebrate radiation and involved two major duplication events that took place before the separation of the fish and mammalian lineages. A cross-species analysis of imprinting center regions showed that some structural features can already be recognized in nonimprinted amniotes in one of the imprinting centers (IC2). In contrast, the imprinting center IC1 is absent in chicken. This suggests a progressive and stepwise evolution of imprinting control elements. In line with that, imprinting centers in mammals apparently exhibit a high degree of structural and sequence variation despite conserved epigenetic marking.