Contribution Of Epigenetic Modifications Research Articles

Abstract 5710: Identification of convergent gene repression mechanisms through integrative genomic and DNA methylation analysis in TRACERx

Abstract Introduction: Lung TRACERx is a prominent study employing multi-region and longitudinal multi-omics sequencing to unravel the evolutionary trajectories of lung cancer. Aberrant DNA methylation patterns have been widely described in nearly all human cancers, yet their interplay with DNA mutations in lung cancer is not well understood. Incorporating the contribution of epigenetic modifications to cancer evolution trajectories within TRACERx could improve our understanding of the intricate relationship between genetic and epigenetic changes in non-small cell lung cancer (NSCLC) evolution. Methods: Multi-region sampling from 38 TRACERx patients including 112 tumor regions and 37 matched normal adjacent tissue samples was performed. Reduced representation bisulfite sequencing (RRBS) was performed to assess DNA methylation and the CAMDAC (Larose-Cadieux et al, 2020) was applied to estimate purified tumor methylation rates and correct for copy number changes. Whole exome sequencing and somatic copy number alterations (SCNAs) were inferred using the ASCAT tool (Van Loo et al, 2010) and Methsig (Pan et al, 2021) was performed to discover new methylation driver genes. Results: Using multi-region sequencing, we identified ubiquitous hypermethylation of 29 known driver genes in both lung adenocarcinoma (LUAD) and squamous cell lung cancer (LUSC), together with an additional 9 and 27 genes exclusive to LUSC and LUAD, respectively. We also identified 13 and 7 driver genes non-ubiquitously hypermethylated exclusively in LUSC and LUAD, respectively. Using a differential methylation based approach, we describe a method to determine the extent of intra-tumor methylation heterogeneity akin to established ITH scores based on genomics data. In addition, we report the identification of novel subtype-specific methylation driver genes enriched in HOX family members which are related to cancer progression. Through integration of DNA methylation and genomic sequencing data, we identify parallel mechanisms contributing towards ubiquitous tumor suppressor gene alterations. At the patient level, multiple driver genes such as NSD1, GATA3 and MGA were subject to repression by both copy number loss and DNA hypermethylation. Finally, we describe dosage-compensation of genes such as the Notch ligands JAG2 and DLK1 that are proximal to amplified oncogenes and hypermethylated during tumor evolution. Conclusion: We describe the contribution of DNA methylation and genomic alterations to altering the landscape of NSCLC. Leveraging DNA methylation, we can determine the extent of convergent repression mechanisms in different regions of the same tumor, assess DNA methylation heterogeneity, and discover DNA methylation-based driver genes in NSCLC. Citation Format: Francisco Gimeno-Valiente, Carla Castignani, Elizabeth Larose-Cadieux, Kezhong Chen, Nana Mensah, Olga Chervova, Thomas Watkins, Pawan Dhami, Heli Vaikkinen, Andrew Feber, TRACERx Consortium, Jonas Demeulemeester, Miljana Tanic, Stephan Beck, Peter Van Loo, Charles Swanton, Nnennaya Kanu. Identification of convergent gene repression mechanisms through integrative genomic and DNA methylation analysis in TRACERx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5710.

Sex Steroid Regulation of Oxidative Stress in Bone Cells: An In Vitro Study

Environmental stimuli, including sex hormones and oxidative stress (OS), affect bone balance, modifying the epigenetic profiles of key osteogenic genes. Nonetheless, the interplay between sex steroids, epigenome and OS has yet be fully elucidated. This paper aims to study in vitro the role of sex steroids in OS-induced alteration in bone cells’ homeostasis, and to assess the possible contribution of epigenetic modifications. Toward this purpose, osteoblast (MC3T3-E1) and osteocyte (MLOY-4) cell lines were exposed to two different sources of free oxygen radicals, i.e., tert-butyl hydroperoxide and dexamethasone, and the protective effect of pre-treatment with androgens and estrogens was evaluated. In particular, we analyzed parameters that reflect bone cell homeostasis such as cell viability, cell migration, transcriptomic profile, transcriptional activity, and epigenetic signature. Our findings indicate that estrogens and androgens counteract OS effects. Using partially overlapping strategies, they reduce OS outcomes regarding cell viability, cell migration, the transcriptomic profile of gene families involved in bone remodeling, and epigenetic profile, i.e., H3K4me3 level. Additionally, we demonstrated that the protective effect of steroids against OS on bone homeostasis is partially mediated by the Akt pathway. Overall, these results suggest that the hormonal milieu may influence the mechanisms of age-related bone disease.

Toxic and Teratogenic Effects of Prenatal Alcohol Exposure on Fetal Development, Adolescence, and Adulthood.

Prenatal alcohol exposure (PAE) can have immediate and long-lasting toxic and teratogenic effects on an individual’s development and health. As a toxicant, alcohol can lead to a variety of physical and neurological anomalies in the fetus that can lead to behavioral and other impairments which may last a lifetime. Recent studies have focused on identifying mechanisms that mediate the immediate teratogenic effects of alcohol on fetal development and mechanisms that facilitate the persistent toxic effects of alcohol on health and predisposition to disease later in life. This review focuses on the contribution of epigenetic modifications and intercellular transporters like extracellular vesicles to the toxicity of PAE and to immediate and long-term consequences on an individual’s health and risk of disease.

Contribution of Regulatory T Cell Methylation Modifications to the Pathogenesis of Allergic Airway Diseases.

Regulatory T (Treg) cells are a subtype of CD4+ T cells that play a significant role in the protection from autoimmunity and the maintenance of immune tolerance via immune regulation. Epigenetic modifications of Treg cells (i.e., cytosine methylation at the promoter region of the transcription factor, Forkhead Box P3) have been found to be closely associated with allergic diseases, including allergic rhinitis, asthma, and food allergies. In this study, we highlighted the recent evidence on the contribution of epigenetic modifications in Treg cells to the pathogenesis of allergic diseases. Moreover, we also discussed directions for future clinical treatment approaches, with a particular emphasis on Treg cell-targeted therapies for allergic disorders.

Abstract WP216: Epigenome-Wide Association Study of Cerebral White Matter Hyperintensities

Background: Cerebral white matter hyperintensities (WMH) on MRI are typical markers of small vessel disease and strong radiological correlates of stroke and age-related cognitive decline. Despite a recognized high heritability, the molecular basis of WMH has not been fully characterized. In particular, the contribution of epigenetic modifications, such as DNA methylation, has not been explored. Methods: We conducted a meta-analysis of epigenome-wide association studies of WMH burden in up to 5777 elderly participants of European and African ancestry from 9 population-based cohorts using blood-derived DNA methylation measured on the Infinium HumanMethylation450 BeadChip. Cohort-specific associations between burden of WMH and DNA methylation beta values were estimated using linear mixed-effect models and combined in a sample-size weighted fixed-effect meta-analysis. In addition, we used two different approaches to identify differentially methylated regions (DMRs), which may be more informative than individual loci. Bonferroni correction and False Discovery Rates were used to account for the multiple tests. Results: Single-site analyses identified a CpG site within the CLDN5 gene significantly associated with WMH (P= 2.3x10 -7 ). Claudin-5 is an endothelial-specific component in the brain vasculature and regulates blood brain barrier permeability. Region-based analyses, which leverage the correlations between nearby CpG sites identified several DMRs significantly associated with WMH burden including: PRMT1, BTBD17, and IFITM10 (P=1.4x10 -10 , 2.3x10 -8 , and 3.6x10 -7 , respectively). PRMT1 encodes the Protein Arginine N-methylase 1, which methylates histones in genes involved in glioblastomagenesis. Mice lacking this gene are characterized by severe defects in oligodendrocyte maturation processes. The function of BTBD17 and IFITM10 is not characterized but both are expressed in the brain and exhibit changes in expression in response to viral infection. Conclusions: Consistent with our previously reported genetic association studies, this genome-wide DNA methylation analysis supports a role of genes involved in glial cell function in WMH etiology. It also suggests a novel role of genes involved in viral response.

The functional ghost in the genome machine: Holistic mapping of environmentally induced changes in the epigenome of a soil sentinel

The realization that toxicants and other stressors can remodel or corrupt the epigenome opens an exciting new dimension for to our understanding of (eco)toxicology. Apart from elucidating mechanisms whereby populations evolve adaptations to local environmental challenges, the functional interplay between the epigenome and the genome underpins answers to important questions relating to ecotoxicology (mechanism and persistence of effects) and field-based risk assessment (potential for plasticity and adaptation). Earthworms possess an unusually high DNA methylation level (∼13%) suggesting an important regulatory role for the epigenome. Interestingly, some experts have questioned the validity of ‘traditional’ laboratory model invertebrates (e.g. nematodes and fruit flies) for epigenetic studies because these have no or very low levels of DNA methylation. Earthworms, as indicated by their high genome methylation status, however, offer to be ecologically relevant targets for epigenomic study. We have explored the epigenetic modifications focusing on alterations in miRNAs and DNA methylation induced in response to environmental cues, including chemical (heavy metals) and physical (temperature and hypoxia/hypercapnia) stress in a number of sentinel earthworm species. These observations have provided key insights into the contribution of epigenetic modification in long term (micro-evolution/adaptation) and short term (acclimatisation) response to chemical and physical challenges. Informatic linkage of contaminated modified epigenetic alterations to modulation in the global transcriptome provides insights into the mechanisms basis that underpins an organism's toxicological response.

The Contribution of Next Generation Sequencing Technologies to Epigenome Research of Stem Cell and Tumorigenesis

Epigenome contains another layer of genetic information, not as stable as genome. Dynamic epigenome can serve as an interface to explain the role of environmental factors. Stem cell and tumorigenesis are reported to be closely associated with epigenome modifications. Next generation sequencing (NGS) technologies have directly leaded to the recent advances in epigenome research of stem cell and cancer. DNA methylation and histone modification are two major epigenetic modifications. Four NGS-based approaches have been developed to identify these two epigenetic modifications, including whole genome bisulfite sequencing (WGBS), methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq), reduced representation bisulfite sequencing (RRBS) and chromatin immunoprecipitation sequencing (ChIP-Seq). This paper reviews the recent advances of WGBS, MeDIP-Seq and RRBS for DNA methylation and ChIP-Seq for histone modification in the field of stem cell. The potential contribution of epigenetic modifications to tumorigenesis is also described. At present, the epigenome research still faces the defects of current sampling strategy and unknown network regulation pattern. In future, worldwide collaboration and latest sequencing technologies application are expected to solve these problem and offer new insight into epigenome research.

MHC Class-II Expression Reveals Differential Regulation of BLIMP-1 Target Genes.

BLIMP-1 initiates plasma cell differentiation by inhibiting the expression of a limited set of transcription factor genes. BLIMP-1 mediates repression of its targets by promoter occupancy and recruitment of the Groucho corepressor, histone deacetylase and the G9a lysine methyltransferase. The relative contribution of these mechanisms to the stable silencing of BLIMP-1 target genes in plasma cells is not known. Epigenetic changes initiated by BLIMP-1 could be maintained by independent factors or alternatively may be dependent on continuous occupancy of the promoter by BLIMP-1. The aim of this study was to investigate the mechanisms operating at two well-defined BLIMP-1 target genes, MHC2TA and PAX-5. Repression of PAX-5 is essential for extinction of the B-cell phenotype, while repression of MHC2TA mediates the loss of MHC class-II expression characteristic of plasma cell differentiation. PAX-5 is silenced in virtually all cases of myeloma, whereas MHC class-II continues to be expressed in a subset of primary myeloma cells and many myeloma cell lines. Using chromatin immunoprecipitation we demonstrate that BLIMP-1 constitutively binds MHC2TA promoter-3 and is present equally in MHC2TA expressing and non-expressing myeloma cell lines. In expressing cells MHC2TA promoter-3 is associated with acetylation of histone H3 lysine-9, a marker of open chromatin, whereas histone H3 lysine-9 is neither acetylated nor methylated in non-expressing cells. In contrast the PAX-5 promoter is associated with trimethylation of histone H3 lysine-9, a marker of repressed heterochromatin, in all the cell lines examined, but constitutive BLIMP-1 occupancy is not detectable. Although BLIMP-1 does not silence MHC2TA expression in the MHC class-II positive U266 cell line, it is mediating transcriptional repression, since siRNA knockdown of BLIMP-1 is associated with elevated MHC2TA mRNA and MHC class-II surface expression. In contrast PAX-5 mRNA and its target CD19 continue to be repressed. MHC class-II re-expression was associated with loss of CD138 and the entry of the majority of cells into apoptosis. Our data demonstrate that MHC class-II expression in myeloma cell lines is associated with defective silencing of MHC2TA despite BLIMP-1 occupancy, and reveal differences in the contribution of epigenetic modifications and promoter occupancy in the maintenance of target gene silencing by BLIMP-1. Loss of BLIMP-1 results in a partial reversion of plasma cell phenotype that is associated with induction of apoptosis. As continual high levels of BLIMP-1 expression appear to be critical for plasma cell survival, suppression of BLIMP-1 represents a potential therapeutic pathway.