Publications

Epigenetic association studies have been carried out to test the hypothesis that environmental perturbations trigger cellular reprogramming, with downstream effects on cellular function and phenotypes. There have now been numerous studies of the potential molecular mediators of epigenetic changes by epigenome-wide association studies (EWAS). However, a challenge for the field is the interpretation of the results obtained. We describe a second-generation EWAS approach, which focuses on the possible cellular models of epigenetic perturbations, studied by rigorous analysis and interpretation of genomic data. Thus refocused, epigenetics research aligns with the field of functional genomics to provide insights into environmental and genetic influences on phenotypic variation in humans.

The field described as 'epigenetics' has captured the imagination of scientists and the lay public. Advances in our understanding of chromatin and gene regulatory mechanisms have had impact on drug development, fueling excitement in the lay public about the prospects of applying this knowledge to address health issues. However, when describing these scientific advances as 'epigenetic', we encounter the problem that this term means different things to different people, starting within the scientific community and amplified in the popular press. To help researchers understand some of the misconceptions in the field and to communicate the science accurately to each other and the lay audience, here we review the basis for many of the assumptions made about what are currently referred to as epigenetic processes.

Methyl-CpG binding protein 2 (MeCP2) is a multifunctional protein that guides neuronal development through its binding to DNA, recognition of sites of methyl-CpG (mCpG) DNA modification, and interaction with other regulatory proteins. Our study explores the relationship between mCpG and hydroxymethyl-CpG (hmCpG) recognition mediated by its mCpG binding domain (MBD) and binding cooperativity mediated by its C-terminal polypeptide. Previous study of the isolated MBD of MeCP2 documented an unusual mechanism by which ion uptake is required for discrimination of mCpG and hmCpG from CpG. MeCP2 binding cooperativity suppresses discrimination of modified DNA and is highly sensitive to both the total ion concentration and the type of counterions. Higher than physiological total ion concentrations completely suppress MeCP2 binding cooperativity, indicating a dominant electrostatic component to the interaction. Substitution of SO4(2-) for Cl(-) at physiological total ion concentrations also suppresses MeCP2 binding cooperativity, This effect is of particular note as the intracellular Cl(-) concentration changes during neuronal development. A related effect is that the protein-stabilizing solutes, TMAO and glutamate, reduce MeCP2 (but not isolated MBD) binding affinity by 2 orders of magnitude without affecting the apparent binding cooperativity. These observations suggest that polypeptide flexibility facilitates DNA binding by MeCP2. Consistent with this view, nuclear magnetic resonance (NMR) analyses show that ions have discrete effects on the structure of MeCP2, both MBD and the C-terminal domains. Notably, anion substitution results in changes in the NMR chemical shifts of residues, including some whose mutation causes the autism spectrum disorder Rett syndrome. Binding cooperativity makes MeCP2 an effective competitor with histone H1 for accessible DNA sites. The relationship between MeCP2 binding specificity and cooperativity is discussed in the context of chromatin binding, neuronal function, and neuronal development.

Epigenome-wide association studies represent one means of applying genome-wide assays to identify molecular events that could be associated with human phenotypes. The epigenome is especially intriguing as a target for study, as epigenetic regulatory processes are, by definition, heritable from parent to daughter cells and are found to have transcriptional regulatory properties. As such, the epigenome is an attractive candidate for mediating long-term responses to cellular stimuli, such as environmental effects modifying disease risk. Such epigenomic studies represent a broader category of disease -omics, which suffer from multiple problems in design and execution that severely limit their interpretability. Here we define many of the problems with current epigenomic studies and propose solutions that can be applied to allow this and other disease -omics studies to achieve their potential for generating valuable insights.

Human papillomavirus (HPV)-associated cervical carcinoma is preceded by stages of cervical intra-epithelial neoplasia (CIN) that can variably progress to malignancy. Understanding the different molecular processes involved in the progression of pre-malignant CIN is critical to the development of improved predictive and interventional capabilities. We tested the role of regulators of transcription in both the development and the progression of HPV-associated CIN, performing the most comprehensive genomic survey to date of DNA methylation in HPV-associated cervical neoplasia, testing  2 million loci throughout the human genome in biopsies from 78 HPV+ women, identifying changes starting in early CIN and maintained through carcinogenesis. We identified loci at which DNA methylation is consistently altered, beginning early in the course of neoplastic disease and progressing with disease advancement. While the loss of DNA methylation occurs mostly at intergenic regions, acquisition of DNA methylation is at sites involved in transcriptional regulation, with strong enrichment for targets of polycomb repression. Using an independent cohort from The Cancer Genome Atlas, we validated the loci with increased DNA methylation and found that these regulatory changes were associated with locally decreased gene expression. Secondary validation using immunohistochemistry showed that the progression of neoplasia was associated with increasing polycomb protein expression specifically in the cervical epithelium. We find that perturbations of genomic regulatory processes occur early and persist in cervical carcinoma. The results indicate a polycomb-mediated epigenetic field defect in cervical neoplasia that may represent a target for early, topical interventions using polycomb inhibitors.

We present a capture-based approach for bisulfite-converted DNA that allows interrogation of pre-defined genomic locations, allowing quantitative and qualitative assessments of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CG dinucleotides and in non-CG contexts (CHG, CHH) in mammalian and plant genomes. We show the technique works robustly and reproducibly using as little as 500 ng of starting DNA, with results correlating well with whole genome bisulfite sequencing data, and demonstrate that human DNA can be tested in samples contaminated with microbial DNA. This targeting approach will allow cell type-specific designs to maximize the value of 5mC and 5hmC sequencing.

Osteosarcoma is the most common primary malignant bone tumor in children. Validated biological markers for disease prognosis available at diagnosis are lacking. No genome-wide DNA methylation studies linked to clinical outcomes have been reported in osteosarcoma to the best of our knowledge. To address this, we tested the methylome at over 1.1 million loci in 15 osteosarcoma biopsy samples obtained prior to the initiation of therapy and correlated these molecular data with disease outcomes. At more than 17% of the tested loci, samples obtained from patients who experienced disease relapse were more methylated than those from patients who did not have recurrence while patients who did not experience disease relapse had more DNA methylation at fewer than 1%. In samples from patients who went on to have recurrent disease, increased DNA methylation was found at gene bodies, intergenic regions and empirically-annotated candidate enhancers, whereas candidate gene promoters were unusual for a more balanced distribution of increased and decreased DNA methylation with 6.6% of gene promoter loci being more methylated and 2% of promoter loci being less methylated in patients with disease relapse. A locus at the TLR4 gene demonstrates one of strongest associations between DNA methylation and 5 y event-free survival (P-value = 1.7 × 10(-6)), with empirical annotation of this locus showing promoter characteristics. Our data indicate that DNA methylation information has the potential to be predictive of outcome in pediatric osteosarcoma, and that both promoters and non-promoter loci are potentially informative in DNA methylation studies.

BACKGROUND: RNA:DNA hybrids represent a non-canonical nucleic acid structure that has been associated with a range of human diseases and potential transcriptional regulatory functions. Mapping of RNA:DNA hybrids in human cells reveals them to have a number of characteristics that give insights into their functions.

RESULTS: We find RNA:DNA hybrids to occupy millions of base pairs in the human genome. A directional sequencing approach shows the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to their in vivo stability. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation, indicating potential transcriptional regulatory properties. Mass spectrometry studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation.

CONCLUSIONS: Overall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting the intriguing model of RNA generating these structures in trans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo.

The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA. We show that solution conditions with physiological or greater salt concentrations or the presence of nonspecific competitor DNA is necessary for the MBD to discriminate mCpG from CpG with high specificity. The specificity for mCpG over CpG is >100-fold under these solution conditions. In contrast, the MBD does not discriminate hydroxymethyl-CpG from CpG. The MBD is unusual among site-specific DNA binding proteins in that (i) specificity is not conferred by the enhanced affinity for the specific site but rather by suppression of its affinity for generic DNA, (ii) its specific binding to mCpG is highly electrostatic, and (iii) it takes up as well as displaces monovalent cations upon DNA binding. The MBD displays an unusually high affinity for single-stranded DNA independent of modification or sequence. In addition, the MBD forms a discrete dimer on DNA via a noncooperative binding pathway. Because the affinity of the second monomer is 1 order of magnitude greater than that of nonspecific binding, the MBD dimer is a unique molecular complex. The significance of these results in the context of neuronal function and development and MeCP2-related developmental disorders such as Rett syndrome is discussed.