During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. ...However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic-epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.
Age‐associated changes in the DNA methylation state can be used to assess the pace of aging. However, it is not understood what mechanisms drive these changes and whether these changes affect the ...development of aging phenotypes and the aging process in general. This study was aimed at gaining a more comprehensive understanding of aging‐related methylation changes across the whole genome, and relating these changes to biological functions. It has been shown that skeletal muscle and blood monocytes undergo typical changes with aging. Using whole‐genome bisulfite sequencing, we sought to characterize the genome‐wide changes in methylation of DNA derived from both skeletal muscle and blood monocytes, and link these changes to specific genes and pathways through enrichment analysis. We found that methylation changes occur with aging at the locations enriched for developmental and neuronal pathways regulated in these two peripheral tissues. These results contribute to our understanding of changes in epigenome in human aging.
Human aging is associated with changes in DNA methylation, but the extent of methylation changes across the genome during aging are not understood. Using whole‐genome bisulfite sequencing, we have studied DNA methylation changes in healthy aging in skeletal muscle and monocytes.
Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic ...integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat, high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genome-wide DNA methylation analyses for each strain and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects revealed a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by epigenetic and transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the farnesoid X receptor pathway, and found that GW4064 exerts strain-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis. Furthermore, GW4064 treatment induced inflammatory-related gene expression changes in NOD, indicating a strain-specific effect in its associated toxicities as well as its therapeutic efficacy. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention and a mouse platform for guiding this approach.
Translocations of the KMT2A (MLL) gene define a biologically distinct and clinically aggressive subtype of acute myeloid leukaemia (AML), marked by a characteristic gene expression profile and few ...cooperating mutations. Although dysregulation of the epigenetic landscape in this leukaemia is particularly interesting given the low mutation frequency, its comprehensive analysis using whole genome bisulphite sequencing (WGBS) has not been previously performed. Here we investigated epigenetic dysregulation in nine MLL-rearranged (MLL-r) AML samples by comparing them to six normal myeloid controls, using a computational method that encapsulates mean DNA methylation measurements along with analyses of methylation stochasticity. We discovered a dramatically altered epigenetic profile in MLL-r AML, associated with genome-wide hypomethylation and a markedly increased DNA methylation entropy reflecting an increasingly disordered epigenome. Methylation discordance mapped to key genes and regulatory elements that included bivalent promoters and active enhancers. Genes associated with significant changes in methylation stochasticity recapitulated known MLL-r AML expression signatures, suggesting a role for the altered epigenetic landscape in the transcriptional programme initiated by MLL translocations. Accordingly, we established statistically significant associations between discordances in methylation stochasticity and gene expression in MLL-r AML, thus providing a link between the altered epigenetic landscape and the phenotype.
Epigenetic modifications confer stable transcriptional patterns in the brain, and both normal and abnormal brain function involve specialized brain regions. We examined DNA methylation by ...whole-genome bisulfite sequencing in neuronal and non-neuronal populations from four brain regions (anterior cingulate gyrus, hippocampus, prefrontal cortex, and nucleus accumbens) as well as chromatin accessibility in the latter two. We find pronounced differences in both CpG and non-CpG methylation (CG-DMRs and CH-DMRs) only in neuronal cells across brain regions. Neuronal CH-DMRs were highly associated with differential gene expression, whereas CG-DMRs were consistent with chromatin accessibility and enriched for regulatory regions. These CG-DMRs comprise ~12 Mb of the genome that is highly enriched for genomic regions associated with heritability of neuropsychiatric traits including addictive behavior, schizophrenia, and neuroticism, thus suggesting a mechanistic link between pathology and differential neuron-specific epigenetic regulation in distinct brain regions.
Twin studies are powerful models to elucidate epigenetic modifications resulting from gene-environment interactions. Yet, commonly a limited number of clinical twin samples are available, leading to ...an underpowered situation afflicted with false positives and hampered by low sensitivity. We investigated genome-wide DNA methylation data from two small sets of monozygotic twins representing different phases during the progression of rheumatoid arthritis (RA) to find novel genes for further research.
We implemented a robust statistical methodology aimed at investigating a small number of samples to identify differential methylation utilizing the comprehensive CHARM platform with whole blood cell DNA from two sets of twin pairs discordant either for ACPA (antibodies to citrullinated protein antigens)-positive RA versus ACPA-negative healthy or for ACPA-positive healthy (a pre-RA stage) versus ACPA-negative healthy. To deconvolute cell type-dependent differential methylation, we assayed the methylation patterns of sorted cells and used computational algorithms to resolve the relative contributions of different cell types and used them as covariates.
To identify methylation biomarkers, five healthy twin pairs discordant for ACPAs were profiled, revealing a single differentially methylated region (DMR). Seven twin pairs discordant for ACPA-positive RA revealed six significant DMRs. After deconvolution of cell type proportions, profiling of the healthy ACPA discordant twin-set revealed 17 genome-wide significant DMRs. When methylation profiles of ACPA-positive RA twin pairs were adjusted for cell type, the analysis disclosed one significant DMR, associated with the EXOSC1 gene. Additionally, the results from our methodology suggest a temporal connection of the protocadherine beta-14 gene to ACPA-positivity with clinical RA.
Our biostatistical methodology, optimized for a low-sample twin design, revealed non-genetically linked genes associated with two distinct phases of RA. Functional evidence is still lacking but the results reinforce further study of epigenetic modifications influencing the progression of RA. Our study design and methodology may prove generally useful in twin studies.
In cancer, linking epigenetic alterations to drivers of transformation has been difficult, in part because DNA methylation analyses must capture epigenetic variability, which is central to tumour ...heterogeneity and tumour plasticity. Here, by conducting a comprehensive analysis, based on information theory, of differences in methylation stochasticity in samples from patients with paediatric acute lymphoblastic leukaemia (ALL), we show that ALL epigenomes are stochastic and marked by increased methylation entropy at specific regulatory regions and genes. By integrating DNA methylation and single-cell gene-expression data, we arrived at a relationship between methylation entropy and gene-expression variability, and found that epigenetic changes in ALL converge on a shared set of genes that overlap with genetic drivers involved in chromosomal translocations across the disease spectrum. Our findings suggest that an epigenetically driven gene-regulation network, with UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a central node, links genetic drivers and epigenetic mediators in ALL.
DNA methylation dynamics in the brain are associated with normal development and neuropsychiatric disease and differ across functionally distinct brain regions. Previous studies of genome-wide ...methylation differences among human brain regions focus on limited numbers of individuals and one to two brain regions.
Using GTEx samples, we generate a resource of DNA methylation in purified neuronal nuclei from 8 brain regions as well as lung and thyroid tissues from 12 to 23 donors. We identify differentially methylated regions between brain regions among neuronal nuclei in both CpG (181,146) and non-CpG (264,868) contexts, few of which were unique to a single pairwise comparison. This significantly expands the knowledge of differential methylation across the brain by 10-fold. In addition, we present the first differential methylation analysis among neuronal nuclei from basal ganglia tissues and identify unique CpG differentially methylated regions, many associated with ion transport. We also identify 81,130 regions of variably CpG methylated regions, i.e., variable methylation among individuals in the same brain region, which are enriched in regulatory regions and in CpG differentially methylated regions. Many variably methylated regions are unique to a specific brain region, with only 202 common across all brain regions, as well as lung and thyroid. Variably methylated regions identified in the amygdala, anterior cingulate cortex, and hippocampus are enriched for heritability of schizophrenia.
These data suggest that epigenetic variation in these particular human brain regions could be associated with the risk for this neuropsychiatric disorder.
Epigenetic mechanisms such as altered DNA methylation have been suggested to play a role in autism, beginning with the classical association of Prader-Willi syndrome, an imprinting disorder, with ...autistic features.
Here we tested for the relationship of paternal sperm DNA methylation with autism risk in offspring, examining an enriched-risk cohort of fathers of autistic children.
We examined genome-wide DNA methylation (DNAm) in paternal semen biosamples obtained from an autism spectrum disorder (ASD) enriched-risk pregnancy cohort, the Early Autism Risk Longitudinal Investigation (EARLI) cohort, to estimate associations between sperm DNAm and prospective ASD development, using a 12-month ASD symptoms assessment, the Autism Observation Scale for Infants (AOSI). We analysed methylation data from 44 sperm samples run on the CHARM 3.0 array, which contains over 4 million probes (over 7 million CpG sites), including 30 samples also run on the Illumina Infinium HumanMethylation450 (450K) BeadChip platform (∼485 000 CpG sites). We also examined associated regions in an independent sample of post-mortem human brain ASD and control samples for which Illumina 450K DNA methylation data were available.
Using region-based statistical approaches, we identified 193 differentially methylated regions (DMRs) in paternal sperm with a family-wise empirical P-value family-wise error rate (FWER) <0.05 associated with performance on the Autism Observational Scale for Infants (AOSI) at 12 months of age in offspring. The DMRs clustered near genes involved in developmental processes, including many genes in the SNORD family, within the Prader-Willi syndrome gene cluster. These results were consistent among the 75 probes on the Illumina 450K array that cover AOSI-associated DMRs from CHARM. Further, 18 of 75 (24%) 450K array probes showed consistent differences in the cerebellums of autistic individuals compared with controls.
These data suggest that epigenetic differences in paternal sperm may contribute to autism risk in offspring, and provide evidence that directionally consistent, potentially related epigenetic mechanisms may be operating in the cerebellum of individuals with autism.
Abstract
Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brainstem tumor, driven by the H3K27M mutation leading to epigenetic dysregulation. Epigenomic analyses of DIPG have shown ...global loss of repressive chromatin marks accompanied by DNA hypomethylation and altered gene expression. However, most investigations of the epigenetic landscape of DIPG only provide a static view of the epigenome. Such studies are not capable of adequately capturing the regulatory underpinnings of DIPG intratumoral heterogeneity and phenotypic plasticity. To address this, we performed Whole-Genome Bisulfite Sequencing (WGBS) on 23 primary patient DIPG specimens, 4 patient-derived DIPG neurosphere cell lines, and 5 normal controls. To analyze this data set, we applied a novel framework for analysis of DNA methylation variability, called informME analysis, which permits the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG exhibits a markedly disordered epigenome, with increased stochasticity localizing to key regulatory elements and genes. We demonstrate that epigenetic instability maps onto key genes regulating pluripotency and developmental identity and hypothesize that these changes might enable tumor cells to sample a diverse range of transcriptional programs and phenotypes. Given this, we evaluated the responsiveness of the DIPG epigenetic landscape to pharmacologic modulation. We found that treatment with the DNA hypomethylating agent decitabine produced profound genome-wide demethylation and reduced DNA methylation stochasticity at active enhancers, bivalent promoters, and promoters of key developmental genes. Decitabine treatment also elicited changes in gene expression, including upregulation of genes related to immune signaling. Finally, treatment with DAC sensitized DIPG cells to the effects of histone deacetylase inhibition. This study suggests the application of epigenetic therapies to constrain cellular heterogeneity and plasticity in DIPG.
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