Epigenetic Context Research Articles

Shifting epigenetic contexts influence regulatory variation and disease risk.

Epigenetic shifts are a hallmark of aging that impact transcriptional networks at regulatory level. These shifts may modify the effects of genetic regulatory variants during aging and contribute to disease pathomechanism. However, these shifts occur on the backdrop of epigenetic changes experienced throughout an individual’s development into adulthood; thus, the phenotypic, and ultimately fitness, effects of regulatory variants subject to developmental- versus aging-related epigenetic shifts may differ considerably. Natural selection therefore may act differently on variants depending on their changing epigenetic context, which we propose as a novel lens through which to consider regulatory sequence evolution and phenotypic effects. Here, we define genomic regions subjected to altered chromatin accessibility as tissues transition from their fetal to adult forms, and subsequently from early to late adulthood. Based on these epigenomic datasets, we examine patterns of evolutionary constraint and potential functional impacts of sequence variation (e.g., genetic disease risk associations). We find that while the signals observed with developmental epigenetic changes are consistent with stronger fitness consequences (i.e., negative selection pressures), they tend to have weaker effects on genetic risk associations for aging-related diseases. Conversely, we see stronger effects of variants with increased local accessibility in adult tissues, strongest in young adult when compared to old. We propose a model for how epigenetic status of a region may influence the effects of evolutionary relevant sequence variation, and suggest that such a perspective on gene regulatory networks may elucidate our understanding of aging biology.

Mining mutation contexts across the cancer genome to map tumor site of origin

The vast preponderance of somatic mutations in a typical cancer are either extremely rare or have never been previously recorded in available databases that track somatic mutations. These constitute a hidden genome that contrasts the relatively small number of mutations that occur frequently, the properties of which have been studied in depth. Here we demonstrate that this hidden genome contains much more accurate information than common mutations for the purpose of identifying the site of origin of primary cancers in settings where this is unknown. We accomplish this using a projection-based statistical method that achieves a highly effective signal condensation, by leveraging DNA sequence and epigenetic contexts using a set of meta-features that embody the mutation contexts of rare variants throughout the genome.

Tumors exploit FTO-mediated regulation of glycolytic metabolism to evade immune surveillance

The ever-increasing understanding of the complexity of factors and regulatory layers that contribute to immune evasion facilitates the development of immunotherapies. However, the diversity of malignant tumors limits many known mechanisms in specific genetic and epigenetic contexts, manifesting the need to discover general driver genes. Here, we have identified the m6A demethylase FTO as an essential epitranscriptomic regulator utilized by tumors to escape immune surveillance through regulation of glycolytic metabolism. We show that FTO-mediated m6A demethylation in tumor cells elevates the transcription factors c-Jun, JunB, and C/EBPβ, which allows the rewiring of glycolytic metabolism. Fto knockdown impairs the glycolytic activity of tumor cells, which restores the function of CD8+ Tcells, thereby inhibiting tumor growth. Furthermore, we developed a small-molecule compound, Dac51, that can inhibit the activity of FTO, block FTO-mediated immune evasion, and synergize with checkpoint blockade for better tumor control, suggesting reprogramming RNA epitranscriptome as a potential strategy for immunotherapy.

Forensics Meets Ecology – Environmental DNA Offers New Capabilities for Marine Ecosystem and Fisheries Research

Climatic changes and anthropogenic pressures affect biodiversity and community composition. These biodiversity shifts are recognized in marine ecosystems, but the underlying processes are barely understood so far. Importantly, human well-being highly relies on oceanic services, which are affected by anthropogenic pressures. Here, we review how interdisciplinary research approaches, with the incorporation of eDNA (environmental DNA) analyses, can help increase the understanding of complex ecosystem processes and dynamics, and how they affect ecosystem services. We discuss marine conservation issues in the light of life cycle aspects and conclude that eDNA can improve our ecological knowledge in some instances, for example, in tracking migration patterns. We also illustrate and discuss the application of eDNA analysis within the context of population genetics, epigenetics, geochemistry and oceanography. Embedded into an interdisciplinary context, eDNA can be exploited by a huge variety of methodological techniques, and can resolve spatio-temporal patterns of diversity, species, or even populations within ecological, evolutionary, and management frameworks.

The Paradoxical Behavior of microRNA-211 in Melanomas and Other Human Cancers.

Cancer initiation, progression, and metastasis leverage many regulatory agents, such as signaling molecules, transcription factors, and regulatory RNA molecules. Among these, regulatory non-coding RNAs have emerged as molecules that control multiple cancer types and their pathologic properties. The human microRNA-211 (MIR211) is one such molecule, which affects several cancer types, including melanoma, glioblastoma, lung adenocarcinomas, breast, ovarian, prostate, and colorectal carcinoma. Previous studies suggested that in certain tumors MIR211 acts as a tumor suppressor while in others it behaves as an oncogenic regulator. Here we summarize the known molecular genetic mechanisms that regulate MIR211 gene expression and molecular pathways that are in turn controlled by MIR211 itself. We discuss how cellular and epigenetic contexts modulate the biological effects of MIR211, which exhibit pleiotropic effects. For example, up-regulation of MIR211 expression down-regulates Warburg effect in melanoma tumor cells associated with an inhibition of the growth of human melanoma cells in vitro, and yet these conditions robustly increase tumor growth in xenografted mice. Signaling through the DUSP6-ERK5 pathway is modulated by MIR211 in BRAFV600E driven melanoma tumors, and this function is involved in the resistance of tumor cells to the BRAF inhibitor, Vemurafenib. We discuss several alternate but testable models, involving stochastic cell-to-cell expression heterogeneity due to multiple equilibria involving feedback circuits, intracellular communication, and genetic variation at miRNA target sties, to reconcile the paradoxical effects of MIR211 on tumorigenesis. Understanding the precise role of this miRNA is crucial to understanding the genetic basis of melanoma as well as the other cancer types where this regulatory molecule has important influences. We hope this review will inspire novel directions in this field.

New Protocol for Long-Term Results With a Multi-Pulse Nd:YAG Laser for Melasma Treatment: A Descriptive Cohort Study.

Melasma is a photoaging condition with a typical clinical and histological pattern under a genetic and epigenetic context. Over the years, many treatments modalities have been proposed including topical agents, oral medication, procedures, and combination treatments aiming at different aspects of the pathogenesis including photodamage, inflammation, vascularity, and pigmentation. Recently, lasers have become an alternative for the treatment of melasma using thermal energy to target, in a selective way, the chromophore found in the skin such as melanin. This study aimed to show how we have implemented a multifactorial approach that not only treats the symptoms but addresses the primary underlying cause of melasma. Twenty female patients with dermal and mixed melasma were admitted to Medical Art between December 2017 and April 2018 and received a four-step Fotona laser protocol. Clinical evaluation of a clearance percentage of melasma was performed 1 month after the last session by a blinded dermatologist grading the images taken during the study. Follow-up evaluation was performed at 6 and 12 months following the completion of the laser treatment, which was 6 sessions. Patients were also asked following the end of the treatment about their satisfaction, which was evaluated with a 5-point scale. This descriptive cohort study included patients with melasma who underwent the four-step laser protocol treatment at 2-week intervals. The clearance percentage score showed 70% with excellent reduction, 25% with good reduction in appearance of melasma, none of the patients with a fair reduction, and 5% with a poor reduction. The mean score of patient satisfaction was 4,65 ± 0,93 (93%). Results of this study indicate that the four-step Fotona laser protocol is a safe and effective treatment option for melasma. J Drugs Dermatol. 2021;20(2):150-154. doi:10.36849/JDD.1221.

DOT1L-mediated murine neuronal differentiation associates with H3K79me2 accumulation and preserves SOX2-enhancer accessibility

During neuronal differentiation, the transcriptional profile and the epigenetic context of neural committed cells is subject to significant rearrangements, but a systematic quantification of global histone modification changes is still missing. Here, we show that H3K79me2 increases and H3K27ac decreases globally during in-vitro neuronal differentiation of murine embryonic stem cells. DOT1L mediates all three degrees of methylation of H3K79 and its enzymatic activity is critical to modulate cellular differentiation and reprogramming. In this context, we find that inhibition of DOT1L in neural progenitor cells biases the transcriptional state towards neuronal differentiation, resulting in transcriptional upregulation of genes marked with H3K27me3 on the promoter region. We further show that DOT1L inhibition affects accessibility of SOX2-bound enhancers and impairs SOX2 binding in neural progenitors. Our work provides evidence that DOT1L activity gates differentiation of progenitors by allowing SOX2-dependent transcription of stemness programs.

Epigenome-based splicing prediction using a recurrent neural network.

Alternative RNA splicing provides an important means to expand metazoan transcriptome diversity. Contrary to what was accepted previously, splicing is now thought to predominantly take place during transcription. Motivated by emerging data showing the physical proximity of the spliceosome to Pol II, we surveyed the effect of epigenetic context on co-transcriptional splicing. In particular, we observed that splicing factors were not necessarily enriched at exon junctions and that most epigenetic signatures had a distinctly asymmetric profile around known splice sites. Given this, we tried to build an interpretable model that mimics the physical layout of splicing regulation where the chromatin context progressively changes as the Pol II moves along the guide DNA. We used a recurrent-neural-network architecture to predict the inclusion of a spliced exon based on adjacent epigenetic signals, and we showed that distinct spatio-temporal features of these signals were key determinants of model outcome, in addition to the actual nucleotide sequence of the guide DNA strand. After the model had been trained and tested (with >80% precision-recall curve metric), we explored the derived weights of the latent factors, finding they highlight the importance of the asymmetric time-direction of chromatin context during transcription.

ICGEC: a comparative method for measuring epigenetic conservation of genes via the integrated signal from multiple histone modifications between cell types

BackgroundHistone post-translational modifications play crucial roles in epigenetic regulation of gene expression and are known to be associated with the phenotypic differences of different cell types. Therefore, it is of fundamental importance to dissect the genes and pathways involved in such a phenotypic variation at the level of epigenetics. However, the existing comparative approaches are largely based on the differences, especially the absolute difference in the levels of individual histone modifications of genes under contrasting conditions. Thus, a method for measuring the overall change in the epigenetic circumstance of each gene underpinned by multiple types of histone modifications between cell types is lacking.ResultsTo address this challenge, we developed ICGEC, a new method for estimating the degree of epigenetic conservation of genes between two cell lines. Different from existing comparative methods, ICGEC provides a reliable score for measuring the relative change in the epigenetic context of corresponding gene between two conditions and simultaneously produces a score for each histone mark. The application of ICGEC to the human embryonic stem cell line H1 and four H1-derived cell lines with available epigenomic data for the same 16 types of histone modifications indicated high robustness and reliability of ICGEC. Furthermore, the analysis of the epigenetically dynamic and conserved genes which were defined based on the ICGEC output results demonstrated that ICGEC can deepen our understanding of the biological processes of cell differentiation to overcome the limitations of traditional expression analysis. Specifically, the ICGEC-derived differentiation-direction-specific genes were shown to have putative functions that are well-matched with cell identity. Additionally, H3K79me1 and H3K27ac were found to be the main histone marks accounting for whether an epigenetically dynamic gene was differentially expressed between two cell lines.ConclusionsThe use of ICGEC creates a convenient and robust way to measure the overall epigenetic conservation of individual genes and marks between two conditions. Thus, it provides a basis for exploring the epigenotype-phenotype relationship. ICGEC can be deemed a state-of-the-art method tailored for comparative epigenomic analysis of changes in cell dynamics.

Abstract A22: Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1

Abstract The initial stages of pancreatic ductal adenocarcinoma (PDA) involve an oncogenic Kras-driven metaplastic transition of acinar cells to a more duct-like phenotype referred to as acinar to ductal metaplasia (ADM). The exact mechanism by which Kras reprograms acinar cells to a more duct-like cell at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells in other contexts and rely on appropriate chromatin context for their activity. Deletion of Bmi1, a component of the Polycomb Repressor Complex 1, completely represses Kras-driven ADM and neoplasia in genetically engineered mouse models of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used a genetically engineered mouse model of early pancreatic neoplasia bearing an acinar cell-specific tamoxifen inducible Cre-recombinase under the elastase promoter (Ela-CreER) combined with the Kras G12D oncogenic allele (Kras−LSL-G12D/+). We added the conditional Bmi1 knockout allele (Bmi1−fl) and the Rosa26 tdTomato reporter to generate Ela-CreER, Kras−LSL-G12D/+, Bmi1−fl/fl, R26 tdTomato mice and their litter mate controls with and without KrasG12D and Bmi1. We activated the Cre-recombinase in 6- to 8-week-old mice with 5 daily tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (eight doses per day for two days) one week after the first tamoxifen dose. We used FACS to sort tdTomato+ cells one week after pancreatitis induction and analyzed RNA expression levels of 26 TFs, previously reported to play a role in pancreatic development, homeostasis and neoplasia. Expression levels of three lineage markers (amylase, elastase and cytokeratin 19) were also measured by quantitative RT-PCR. Tamoxifen gavage induced high tdTomato expression in the acinar compartment of Ela-CreER+ mice. RT-qPCR of tdTomato+ cells indicated a loss of acinar specific TFs in mice expressing mutant Kras one week after induction of pancreatitis. This is consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase expression at the mRNA level. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in expression of three key acinar-specific fate TFs, Mist1, Hnf1a and Nr5a2. The mRNA expression levels of other acinar TFs like Pdx1 and Ptf1a/p48 did not recover with loss of Bmi1 and mutant Kras expression. Together, our data suggest that oncogenic Kras-driven ADM is controlled by changes in master TF gene regulatory networks. Bmi1 deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Jack Blumberg, Osama Alkahili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A22.

Abstract C56: Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia

Abstract Rare acinar cells expressing Bmi1, a component of the Polycomb Repressor Complex 1 (PRC1), represent a reservoir of cells contributing to pancreatic repair during injury and stress. This is marked by acinar-ductal metaplasia (ADM), a transition of acinar cells to a more duct-like phenotype, which is seen in pancreatitis and early stages of Kras-driven pancreatic neoplasia. The mechanism behind Kras-driven ADM at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells and rely on appropriate chromatin context for their activity. Deletion of Bmi1 represses Kras-driven ADM and neoplasia in genetically engineered mouse models (GEMMs) of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used GEMMs to study the expression of Bmi1 in the acinar compartment and its role in early pancreatic neoplasia. We combined an acinar cell-specific tamoxifen-inducible Cre-recombinase under the elastase promoter (Ela-CreER) with the Kras G12D oncogenic allele and the conditional Bmi1 knockout allele and the Rosa26 tdTomato reporter to generate Ela-CreER, KrasLSL-G12D/+, Bmi1, R26 tdTomato mice and their littermate controls. We also generated Ela-CreER, Bmi1GFP, R26tdTomato and Bmi1-CreER, R26 tdTomato mice to quantify the fraction of acinar cells expressing Bmi1. We activated the Cre-recombinase in 6- to 8-week-old Ela-CreER mice with 5 tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (8 hourly injections x 2 days) one week after the first tamoxifen dose. We FACS sorted tdTomato+ cells from untreated mice (0 hr) and one week (168 hr) after pancreatitis induction and analyzed RNA levels of 8 TFs, reported to play a role in pancreatic development, homeostasis, and neoplasia. Expression levels of two lineage markers (amylase and elastase) were also measured by RT-qPCR. Lineage tracing in Bmi1-CreER and Ela-CreER, Bmi1GFP mice revealed expression of Bmi1 in vast majority of acinar cells. RT-qPCR of tdTomato+ cells in the Ela-CreER models indicated a loss of acinar-specific TFs in mice expressing mutant Kras one week after induction of pancreatitis consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase mRNA levels. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in RNA expression of three key acinar-specific fate TFs—Mist1, Hnf1a, and Nr5a2. The mRNA levels of other key acinar TFs—Pdx1 and Ptf1a/p48—did not recover with the loss of Bmi1 in the presence of KrasG12D. Together, our data suggest that Bmi1 is expressed in the majority of acinar cells and regulates oncogenic Kras-driven ADM by altering master TF gene regulatory networks. Its deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Emily Wu, Osama Alkhalili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C56.

MA17.01 Cell Lineage and Chromatin Landscape of Lung Cancer Are Controlled by GATA6

Thoracic malignancies are histologically and biologically heterogeneous. The underlying causes of this heterogeneity are believed to be linked to the complex cellular ontogenies of lung cancers and their relationship to pulmonary development. Lineage selective transcription factors (TFs) are critical determinants of airway cell differentiation and homeostasis, but their biological requirements are often conditional. Analogously, several developmental TFs can paradoxically enhance or inhibit lung cancer progression, depending on cellular and epigenetic contexts which remain largely undefined. In this study, we report a novel function for the endodermal and pulmonary specifying TF GATA6 in lung cancer. To understand the role of GATA6 in lung tumorigenesis we used a genetically engineer mouse model (GEMMs) harboring Kras p53 mutations using progenitor cell specific gene targeting. We combined GEMMs biology with integrated analysis of the transcriptome and the chromatin landscape of lung cells derived from GATA6 deficient tumors. In this study, we uncover a conditional role for the endodermal and pulmonary specifying TF GATA6 during the initiation and progression of Kras mutant lung adenocarcinoma (LUAD). Inhibition of Gata6 in genetically engineered mouse models dampens the proliferation and increases the differentiation of LUAD tumors. These effects are influenced by the epithelial cell type that is targeted for transformation, demonstrating that GATA6 expression is an important molecular determinant of the cell of origin in Kras mutant lung cancer. In LUAD cells derived from surfactant protein C expressing progenitors, we identify multiple genomic loci that are bound by GATA6. Moreover, suppression of Gata6 in these cells significantly alters chromatin accessibility, particularly at distal enhancer elements. Analogous to its paradoxical activity in the developing lungs, GATA6 expression fluctuates during different stages of LUAD progression and can epigenetically control diverse lineage programs associated with cell proliferation, alveolar specification, BMP signaling, and epithelial plasticity. In summary: 1) GATA6 expression varies during different stages of disease progression in the lung adenocarcinoma (LUAD) subtype 2) Suppression of Gata6 can diminish the proliferation and progression of LUAD in a manner that is influenced by the transforming progenitor of origin 3) GATA6 differentially modulates chromatin accessibility across the genome of LUAD cells 4) This epigenomic mechanism results in the activation of different lineage specific programs, including the BMP signaling pathway. These findings reveal how GATA6 can modulate the chromatin landscape of lung cancer cells to control their divergent lineage dependencies during tumor progression. D.X.N. has received research funding from AstraZeneca, Inc.

F8 Inversions at Xq28 Causing Hemophilia A Are Associated With Specific Methylation Changes: Implication for Molecular Epigenetic Diagnosis.

Diverse DNA structural variations (SVs) in human cancers and several other diseases are well documented. For genomic inversions in particular, the disease causing mechanism may not be clear, especially if the inversion border does not cross a coding sequence. Understanding about the molecular processes of these inverted genomic sequences, in a mainly epigenetic context, may provide additional information regarding sequence-specific regulation of gene expression in human diseases. Herein, we study one such inversion hotspot at Xq28, which leads to the disruption of F8 gene and results in hemophilia A phenotype. To determine the epigenetic consequence of this rearrangement, we evaluated DNA methylation levels of 12 CpG rich regions with the coverage of 550 kb by using bisulfite-pyrosequencing and next-generation sequencing (NGS)-based bisulfite re-sequencing enrichment assay. Our results show that this inversion prone area harbors widespread methylation changes at the studied regions. However, only 5/12 regions showed significant methylation changes, specifically in case of intron 1 inversion (two regions), intron 22 inversion (two regions) and one common region in both inversions. Interestingly, these aberrant methylated regions were found to be overlapping with the inversion proximities. In addition, two CpG sites reached 100% sensitivity and specificity to discriminate wild type from intron 22 and intron 1 inversion samples. While we found age to be an influencing factor on methylation levels at some regions, covariate analysis still confirms the differential methylation induced by inversion, regardless of age. The hemophilia A methylation inversion “HAMI” assay provides an advantage over conventional PCR-based methods, which may not detect novel rare genomic rearrangements. Taken together, we showed that genomic inversions in the F8 (Xq28) region are associated with detectable changes in methylation levels and can be used as an epigenetic diagnostic marker.

Insights Into de novo Mutation Variation in Lithuanian Exome.

In the last decade, one of the biggest challenges in genomics research has been to distinguish definitive pathogenic variants from all likely pathogenic variants identified by next-generation sequencing. This task is particularly complex because of our lack of knowledge regarding overall genome variation and pathogenicity of the variants. Therefore, obtaining sufficient information about genome variants in the general population is necessary as such data could be used for the interpretation of de novo mutations (DNMs) in the context of patient’s phenotype in cases of sporadic genetic disease. In this study, data from whole-exome sequencing of the general population in Lithuania were directly examined. In total, 84 (VarScan) and 95 (VarSeqTM) DNMs were identified and validated using different algorithms. Thirty-nine of these mutations were considered likely to be pathogenic based on gene function, evolutionary conservation, and mutation impact. The mutation rate estimated per position pair per generation was 2.74 × 10-8 [95% CI: 2.24 × 10-8–3.35 × 10-8] (VarScan) and 2.4 × 10-8 [95% CI: 1.96 × 10-8–2.99 × 10-8] (VarSeqTM), with 1.77 × 10-8 [95% CI: 6.03 × 10-9–5.2 × 10-8] de novo indels per position per generation. The rate of germline DNMs in the Lithuanian population and the effects of the genomic and epigenetic context on DNM formation were calculated for the first time in this study, providing a basis for further analysis of DNMs in individuals with genetic diseases. Considering these findings, additional studies in patient groups with genetic diseases with unclear etiology may facilitate our ability to distinguish certain pathogenic or adaptive DNMs from tolerated background DNMs and to reliably identify disease-causing DNMs by their properties through direct observation.

PO-348 The other 98%: making sense of non-coding variation

IntroductionDespite the ever-increasing availability of whole genome sequencing datasets, our ability to understand functional impact of genetic variation remains largely restricted to the coding portion of the genome. Although a selection of methods exist to estimate variant damage potential, these methods are heavily biassed towards coding variation and have limited ability to detect potentially functional variants in non-coding regions. Furthermore, these methods either ignore tissue context of the variant or combine epigenetic annotations from multiple tissues, often dominated by blood cell lines, and are therefore poorly suited for predicting functional impact of variants in regulatory elements that act in a tissue-specific manner.Material and methodsIn order to address these limitations, we have developed a machine learning classifier aimed at prioritising non-coding variants while taking into account relevant tissue context. We defined ’functional impact’ as the propensity of a variant to cause allele-specific chromatin accessibility. DNase Hypersensitive Site (DHS) variants found to display preferential accessibility, as well as a matched set of negative variants, were annotated with a range of features including a set of core epigenetic marks from the ROADMAP Epigenomics project matched to the tissue of origin of the positive variant set. We then combined several machine learning algorithms to train an ensemble classifier. On a balanced test set (equal number of positive and negative variants) our model achieved area under curve (AUC) of ~75%. On a realistic dataset, where the negative variants outnumbered the positive ones 100:1, we achieved AUC of 90%.Results and discussionsTo demonstrate the importance of tissue context when estimating functional impact of non-coding variants, we compared the classifier’s performance on the same set of variants annotated with epigenetic information from either a closely matched or an unrelated tissue. We observed a notable drop in performance when using epigenetic context from a mismatched tissue. We show that our method drastically outperforms existing damage estimation tools in its ability to predict allele-specific chromatin accessibility. Finally, we demonstrate the utility of our method by successfully prioritising experimentally validated regulatory variants among the large number of variants within the same linkage disequilibrium block.ConclusionThe above method will aid in interpretation of whole genome sequencing datasets generated in cancer and rare disease studies.

Towards annotating the plant epigenome: the Arabidopsis thaliana small RNA locus map

Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species.

A site specific model and analysis of the neutral somatic mutation rate in whole-genome cancer data

BackgroundDetailed modelling of the neutral mutational process in cancer cells is crucial for identifying driver mutations and understanding the mutational mechanisms that act during cancer development. The neutral mutational process is very complex: whole-genome analyses have revealed that the mutation rate differs between cancer types, between patients and along the genome depending on the genetic and epigenetic context. Therefore, methods that predict the number of different types of mutations in regions or specific genomic elements must consider local genomic explanatory variables. A major drawback of most methods is the need to average the explanatory variables across the entire region or genomic element. This procedure is particularly problematic if the explanatory variable varies dramatically in the element under consideration.ResultsTo take into account the fine scale of the explanatory variables, we model the probabilities of different types of mutations for each position in the genome by multinomial logistic regression. We analyse 505 cancer genomes from 14 different cancer types and compare the performance in predicting mutation rate for both regional based models and site-specific models. We show that for 1000 randomly selected genomic positions, the site-specific model predicts the mutation rate much better than regional based models.We use a forward selection procedure to identify the most important explanatory variables. The procedure identifies site-specific conservation (phyloP), replication timing, and expression level as the best predictors for the mutation rate. Finally, our model confirms and quantifies certain well-known mutational signatures.ConclusionWe find that our site-specific multinomial regression model outperforms the regional based models. The possibility of including genomic variables on different scales and patient specific variables makes it a versatile framework for studying different mutational mechanisms. Our model can serve as the neutral null model for the mutational process; regions that deviate from the null model are candidates for elements that drive cancer development.

Abstract IA21: Systematic, network-based elucidation of synthetic lethal proteins and synergistic compounds

Abstract Given the complex nature of cancer and the cell's ability to effectively compensate for individual perturbation through evolutionarily honed adaptive mechanisms, there is strong expectation that combination therapy approaches may be required to address the limitations that have emerged from single agent therapy. Synthetic lethality provides strong biological rationale supporting this observation, although mechanisms of compound synergy and sensitization are in general more complex than individual gene-pair interactions. Synthetic lethal genes have been elucidated mainly via large-scale screens in model organisms, or by pooled shRNA/CRISPR screens in cells harboring specific genetic events, or based on mechanistic understanding of specific pathways. These methodologies are laborious, expensive, and prone to significant false positive and negative rates due to the nature of pooled screens. More importantly, results are often idiosyncratic, depending on the specific genetic and epigenetic context of the cellular models used in the analysis. To address these challenges, we have developed network-based methods to identify master regulator proteins that are responsible for maintaining tumor state, despite being rarely mutated using only patient-derived data. Master regulators are highly enriched in synthetic lethal interactions that can be elucidated computationally, via protein-target analysis. Furthermore, the approach can be trivially extended to elucidate synergistic drug interaction, based on perturbational assays of individual compounds. In this presentation, we will address the discovery of synthetic lethal genes and synergistic compounds in several tumor contexts, including lymphoma, breast cancer, prostate cancer, and glioma, which has led to the design and implementation of novel clinical trials. We will also discuss integration of these methodologies into an N of 1 protocol, which is currently enrolling 260 patients across 14 distinct malignancies. Citation Format: Andrea Califano. Systematic, network-based elucidation of synthetic lethal proteins and synergistic compounds [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA21.

Abstract LB-213: Thrombospondin-1 regulates intestinal microbiota and bile acid metabolism in a murine model of colorectal cancer

Abstract Colon cancer is major public health concern with over 50,000 deaths annually in the US. Environmental and genetic factors influence colon cancer progression, and expression of thrombospondin-1 (TSP1) inversely correlates with colon cancer aggressiveness. TSP1 is a matricellular protein that regulates vascular physiology and tissue responses to stress. We previously demonstrated that deleting the TSP1 (Thbs1) gene in ApcMin/+ mice results in systemic metabolic changes as assessed in liver tissue, increased tumorigenesis, and lower survival of mice fed a low fat diet. We now show that TSP1 induces changes in intestinal microbiota that may cause shifts in secondary bile acid metabolism that influence colon carcinogenesis. Mice were maintained and bred at least two generations on a low fat western diet to equalize dietary effects on their epigenetic context and microbiomes. Mice were pair-fed a low fat or a 21% high-fat western diet beginning at weaning. Mouse fecal matter was collected 8 weeks after dietary exposures and subjected to 16S sequencing for bacterial population identification. Lactobacillaceae, Lachnopiraceae, Ruminococcacea, Verrucomicrobiaceae families were present in all groups. The family Verrucomicrobiaceae and its genus Akkemensi represented a high proportion of the bacterial population. The relative levels of Akkemensia were elevated in low fat fed mice in mice ApcMin/+ and Thbs1−/−:ApcMin/+ when compared to WT counterparts. Interestingly Akkenmensia abundance was reduced in ApcMin/+ mice fed a high-fat diet when compared to other groups. The relative population of the genus Ruminococcus was reduced in Thbs1−/− mice as well as in Thbs1−/−:ApcMin/+ mice fed low and high fat diets, suggesting that these population shifts are driven by genotype. Because intestinal bacteria can alter metabolism, particularly of bile acids, we collected large intestine tissue from mice and subjected it to metabolomics analysis. Our data showed primary and secondary bile metabolism were regulated by the absence of TSP1 in mice fed a high fat diet when compared to WT. High 6-beta-hydroxylithocholate levels were associated with onset of carcinogenesis since it was elevated in ApcMin/+ and Thbs1−/−:ApcMin/+ mice but not in WT or Thbs1−/−. 6-oxolithocholate, hyocholate and hyodeoxycholate were elevated only in ApcMin/+ mice fed a high fat diet but not in Thbs1−/−:ApcMin/+, suggesting that these metabolites contribute to the shift in survival and carcinogenesis observed between low and high fat diets. On the other hand levels of taurohyodeoxycholic acid, 3-dehydrocholate, 7-ketodeoxycholate were elevated in Thbs1−/−:ApcMin/+ and Thbs1−/− mice but not in ApcMin/+, indicating that changes in these metabolites are driven by the loss of Thbs1 and may pre-dispose mice to increased carcinogenesis when fed a high fat diet. Taken together our data shows an unexpected role of TSP1 in regulation of microbiota and bile acid metabolism and reveals potential targets for prevention and treatment of colorectal cancer. Citation Format: David R. Soto-Pantoja, John M. Sipes, Brian Westwood, Nicole Morris, Nancy J. Emenaker, David D. Roberts. Thrombospondin-1 regulates intestinal microbiota and bile acid metabolism in a murine model of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-213. doi:10.1158/1538-7445.AM2017-LB-213

Understanding epigenetics of schizophrenia in the backdrop of its antipsychotic drug therapy

The diatheses of gene and environment interaction in schizophrenia (SCZ) are becoming increasingly evident. Genetic and epigenetic backgrounds are being considered in stratifying and addressing phenotypic variation and drug response in SCZ. But how much of these epigenetic alterations are the primary contributing factor, toward disease pathogenesis and drug response, needs further clarity. Evidence indicates that antipsychotic drugs can also alter the epigenetic homeostasis thereby inducing pharmacoepigenomic effects. We re-examine the context of epigenetics in disease pathogenesis and antipsychotic drug therapy in SCZ to understand how much of these observations act as real indicators of the disease or therapeutic response. We propose that epigenetic viewpoint in SCZ needs to be critically examined under the genetic, epigenetic and pharmacoepigenetic background.