Genome-wide Methylation Analysis Research Articles

Abstract B012: Genomic and epigenomic drivers of double-negative metastatic prostate cancer

Abstract Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen receptor (AR) signaling. Androgen deprivation therapy and second-generation AR-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by whether the tumor expresses either AR or neuroendocrine (NE) markers. Among the subtypes of mCRPC, the molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterize genomic and epigenomic features of treatment-emergent mCRPC subtypes in 210 tumors by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing. We show that AR-/NE- tumors exhibit a clinically and molecularly distinct phenotype. Patients with AR-/NE- mCRPC tumors have the shortest survival, and these tumors preferentially harbor amplification of the chromatin remodeler CHD7 and loss of PTEN. We demonstrate that methylation changes in CHD7 candidate enhancers are linked to elevated CHD7 expression in AR-/NE+ tumors. Moreover, we use genome-wide methylation analysis to nominate the Krüppel-like factor gene KLF5 as a driver of the AR-/NE- phenotype and link its activity to loss of the tumor suppressor RB1. These observations reveal the aggressiveness of the AR-/NE- tumors and elucidate genomic and epigenomic drivers of mCRPC subtypes, which may facilitate the identification of novel therapeutic targets in this highly aggressive disease. Citation Format: Arian Lundberg, Meng Zhang, Rahul R. Aggarwal, Haolong Li, Li Zhang, Adam Foye, Martin Sjöström, Jonathan Chou, Kevin Chang, Thaidy Moreno-Rodriguez, Raunak Shrestha, Avi Baskin, Xiaolin Zhu, Alana S. Weinstein, Noah Younger, Joshi J. Alumkal, Tomasz M. Beer, Kim N. Chi, Christopher P. Evans, Martin Gleave, Primo N. Lara, Robert E. Reiter, Matthew B. Rettig, Owen N. Witte, Alexander W. Wyatt, Felix Y. Feng, Eric J. Small, David A. Quigley. Genomic and epigenomic drivers of double-negative metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B012.

Folic Acid Supplementation Promotes Hypomethylation in Both the Inflamed Colonic Mucosa and Colitis-Associated Dysplasia.

The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

IL-23/IL-23R Promote Macrophage Pyroptosis and T Helper 1/T Helper 17 Cell Differentiation in Mycobacterial Infection

Pathogen-induced epigenetic modifications can reshape anti-infection immune processes and control the magnitude of host responses. DNA methylation profiling has identified crucial aberrant methylation changes associated with diseases, thus providing biological insights into the roles of epigenetic factors in mycobacterial infection. In this study, we performed a genome-wide methylation analysis of skin biopsies from patients with leprosy and healthy controls. T helper 17 differentiation pathway was found to be significantly associated with leprosy through functional enrichment analysis. As a key gene in this pathway, IL-23R was found to be critical to mycobacterial immunity in leprosy, according to integrated analysis with DNA methylation, RNA sequencing, and GWASs. Functional analysis revealed that IL-23/IL-23R-enhanced bacterial clearance by activating caspase-1/GSDMD-mediated pyroptosis in a manner dependent on NLRP3 through signal transducer and activator of transcription 3 signaling in macrophages. Moreover, IL23/IL-23R promoted T helper 1 and T helper 17 cell differentiation and proinflammatory cytokine secretion, thereby increasing host bactericidal activity. IL-23R knockout attenuated the effects and increased susceptibility to mycobacterial infection mentioned earlier. These findings illustrate the biological functions of IL-23/IL-23R in modulating intracellular bacterial clearance in macrophages and further support their regulatory effects in T helper cell differentiation. Our study highlights that IL-23/IL-23R might serve as potential targets for the prevention and treatment of leprosy and other mycobacterial infections.

Multimodal analysis of genome-wide methylation, copy number aberrations, and end motif signatures enhances detection of early-stage breast cancer.

Breast cancer causes the most cancer-related death in women and is the costliest cancer in the US regarding medical service and prescription drug expenses. Breast cancer screening is recommended by health authorities in the US, but current screening efforts are often compromised by high false positive rates. Liquid biopsy based on circulating tumor DNA (ctDNA) has emerged as a potential approach to screen for cancer. However, the detection of breast cancer, particularly in early stages, is challenging due to the low amount of ctDNA and heterogeneity of molecular subtypes. Here, we employed a multimodal approach, namely Screen for the Presence of Tumor by DNA Methylation and Size (SPOT-MAS), to simultaneously analyze multiple signatures of cell free DNA (cfDNA) in plasma samples of 239 nonmetastatic breast cancer patients and 278 healthy subjects. We identified distinct profiles of genome-wide methylation changes (GWM), copy number alterations (CNA), and 4-nucleotide oligomer (4-mer) end motifs (EM) in cfDNA of breast cancer patients. We further used all three signatures to construct a multi-featured machine learning model and showed that the combination model outperformed base models built from individual features, achieving an AUC of 0.91 (95% CI: 0.87-0.95), a sensitivity of 65% at 96% specificity. Our findings showed that a multimodal liquid biopsy assay based on analysis of cfDNA methylation, CNA and EM could enhance the accuracy for the detection of early- stage breast cancer.

Genome-wide DNA methylation analysis in families with multiple individuals diagnosed with schizophrenia and intellectual disability

Objectives Schizophrenia (SZ) and intellectual disability (ID) are both included in the continuum of neurodevelopmental disorders (NDDs). DNA methylation is known to be important in the occurrence of NDDs. The family study is conducive to eliminate the effects of relative epigenetic backgrounds, and to screen for differentially methylated positions (DMPs) and regions (DMRs) that are truly associated with NDDs. Methods Four monozygotic twin families were recruited, and both twin individuals suffered from NDDs (either SZ, ID, or SZ plus ID). Genome-wide methylation analysis was performed in all samples and each family. DMPs and DMRs between NDD patients and unaffected individuals were identified. Functional and pathway enrichment analyses were performed on the annotated genes. Results Two significant DMPs annotated to CYP2E1 were found in all samples. In Family One, 1476 DMPs mapped to 880 genes, and 162 DMRs overlapping with 153 unique genes were recognised. Our results suggested that the altered methylation levels of FYN, STAT3, RAC1, and NR4A2 were associated with the development of SZ and ID. Neurodevelopment and the immune system may participate in the occurrence of SZ and ID. Conclusions Our findings suggested that DNA methylation participated in the development of NDDs by affecting neurodevelopment and the immune system.

Abstract LB046: Characterizing the role of inflammation-induced epigenetic alterations in modulating the immune microenvironment during lung cancer initiation

Abstract One of the most important goals of developing timely detection and effective intervention strategies for lung cancer is to understand the biology and molecular mechanisms involved in early-stage evolution of this disease. Chronic inflammation is a major driver of lung cancer and contributes to its pathogenesis by inducing genetic and epigenetic abnormalities, as well as shifts in key immune cell populations. However, the evolution of these changes and the mechanisms underlying the early stages in development of pre-neoplasias and progression to invasive tumors have not been well delineated. Monolayer cell culture systems, while providing valuable insights into lung cancer development, lack the complexity of an intact organ system. This study aims to overcome these limitations by using a 3D lung organoid model to delineate the role of epigenetic alterations in driving initiation of non-small cell lung cancer. The stem cell enriched nature of the organoids makes it an ideal system to evaluate the implications of epigenetic alterations in these key cell types and their role in cancer initiation. To better identify the epigenetic alterations that play key roles in lung cancer initiation, cigarette smoke condensate (CSC) was applied to normal lung organoids to mimic chronic inflammatory exposure. Epigenomic and transcriptomic alterations were evaluated by genome-wide DNA methylation analysis, ATAC-seq, and RNA-seq. Flow cytometry and confocal microscopy were used to evaluate changes in composition of cell populations comprising the organoids. Long-term CSC exposure caused distinct morphological changes in organoid structure and cellular composition, accompanied by an increased proliferative potential. Analysis of changes in cell composition revealed increases in Krt14 expression in CSC treated organoids, leading to key shifts in basal stem cell populations from a TP63+KRT5+ to a TP63+KRT5+KRT14+ population. This was accompanied by reduction in differentiated cell types, as analyzed by qRT-PCR. Analysis of genome wide DNA methylation showed increases in promoter DNA methylation in key genes associated with lung tumorigenesis. Most importantly, co-culture studies involving culturing CSC treated and control organoids with key immune cells revealed that chronic CSC treatment caused modulation of the microenvironment from a pro- to an anti-inflammatory state. Our results suggest that chronic inflammation causes key shifts in populations of lung stem cells with an associated decrease in differentiation potential. These changes are accompanied by DNA methylation and gene expression changes suggestive of an increased tumorigenic potential. Finally, these changes in the CSC treated organoids are associated with the ability to modulate the function of key immune cells associated with lung tumorigenesis from a pro- to an anti-inflammatory phenotype. Results from our study, will aid in developing novel biomarker-based methodologies to distinguish and treat lung cancer in its early stages. Citation Format: Na Wang, Ray-Whay Chiu Yen, Hamza Khan, Malcolm V. Brock, Hariharan Easwaran, Stephen B. Baylin, Michelle Vaz. Characterizing the role of inflammation-induced epigenetic alterations in modulating the immune microenvironment during lung cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB046.

Brain region-specific genome-wide deoxyribonucleic acid methylation analysis in patients with Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuropathology and cognitive decline and associated with age. The comprehensive deoxyribonucleic acid methylation (DNAm)-transcriptome profile association analysis conducted in this study aimed to establish whole-genome DNAm profiles and explore DNAm-related genes and their potential functions. More appropriate biomarkers were expected to be identified in terms of AD. Illumina 450KGSE59685 dataset AD (n = 54) and HC (n = 21) and ribonucleic-acid-sequencing data GSE118553 dataset AD patients (n = 21) and HCs (n = 13) were obtained from the gene expression omnibus database before a comprehensive DNAm-transcriptome profile association analysis, and we performed functional enrichment analysis by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses (KEGG). Three transgenic mice and three wild-type mice were used to validate the hub genes. A total of 18,104 DNAm sites in healthy controls (n = 21) and AD patients (n = 54) were surveyed across three brain regions (superior temporal gyrus, entorhinal cortex, and dorsolateral prefrontal cortex). With the addition of the transcriptome analysis, eight hypomethylated-related highly expressed genes and 61 hypermethylated-related lowly expressed genes were identified. Based on 69 shared differentially methylated genes (DMGs), the function enrichment analysis indicated Guanosine triphosphate enzymes (GTPase) regulator activity, a synaptic vesicle cycle, and tight junction functioning. Following this, mice-based models of AD were constructed, and five hub DMGs were verified, which represented a powerful, disease-specific DNAm signature for AD. The results revealed that the cross-brain region DNAm was altered in those with AD. The alterations in DNAm affected the target gene expression and participated in the key biological processes of AD. The study provides a valuable epigenetic resource for identifying DNAm-based diagnostic biomarkers, developing effective drugs, and studying AD pathogenesis.

DNA METHYLATION AND ITS INFLUENCE ON THE PATHOGENESIS OF OSTEOARTHRITIS: A SYSTEMATIC LITERATURE REVIEW

There is an evolving body of evidence that demonstrates the role of epigenetic mechanisms, such as DNA-methylation in the pathogenesis of OA. This systematic review aims to summarize the current evidence of DNA methylation and its influence on the pathogenesis of OA.A pre-defined protocol in alignment with the PRISMA guidelines was employed to systematically review eight bibliographic databases, to identify associations between DNA-methylation of articular chondrocytes and osteoarthritis. A search of Medline (Ovid), Embase, Web-of-Science, Scopus, PubMed, Cinahl (EBSCOhost), Cochrane Central and Google Scholar was performed between 1st January 2015 to 31st January 2021. Data extraction was performed by two independent reviewers.During the observation period, we identified 15 gene specific studies and 24 genome wide methylation analyses. The gene specific studies mostly focused on the expression of pro-inflammatory markers, such as IL8 and MMP13 which are overexpressed in OA chondrocytes. DNA hypomethylation in the promoter region resulted in overexpression, whereas hypermethylation was seen in non-OA chondrocytes. Others reported on the association between OA risk genes and the DNA methylation pattern close to RUNX2, which is an important OA signal. The genome wide methylation studies reported mostly on differentially methylated regions comparing OA chondrocytes and non-OA chondrocytes. Clustering of the regions identified genes that are involved in skeletal morphogenesis and development. Differentially methylated regions were seen in hip OA and knee OA chondrocytes, and even within different regions of an OA affected knee joint, differentially methylated regions were identified depending on the disease stage.This systematic review demonstrates the growing evidence of epigenetic mechanisms, such as DNA methylation, in the pathogenesis of OA. In recent years, there has been a focus on the interplay between OA risk genes and DNA methylation changes which revealed a reactivation of genes responsible for endochondral ossification during development. These are important findings and may help to identify eventual future therapeutic targets. However, the current body of literature is mostly showing the differences in DNA methylation of OA chondrocytes and non-OA chondrocytes, but a true longitudinal analysis demonstrating the DNA methylation changes actually happening is still not available.

Abstract 3561: Recurrent genetic alterations in epigenetic pineoblastoma subtypes

Abstract Introduction: Genome-wide methylation analyses recently revealed novel epigenetic pineoblastoma (PBL) subtypes, but so far few data are available on recurrent cytogenetic alterations due to the rarity of these neoplasms. Therefore, our aim was to shed further light onto the molecular and genetic characteristics underlying the pathogenesis of pineoblastoma subtypes. Experimental procedures: Cytogenetic alterations of tumor samples of 60 patients with a diagnosis of PBL confirmed by reference neuropathology and methylation array profiling (450K or EPIC BeadChips (Illumina)) were analyzed by high-resolution genome-wide molecular inversion probe analysis. 52 cases could be screened for mutations by next-generation DNA panel sequencing. Survival data of 41 patients were available. All patients had surgery and older patients (≥4y) received RT followed by maintenance chemotherapy (n=37); infants (&amp;lt;4y) were primarily given a RT-sparing regimen that involved intensified chemotherapy dosage. Results: Of the recently published epigenetic consensus PBL subtypes (Liu et al., 2021), our cohort consisted of 37 PBL-miRNA1 (1A, 30; 1B, 7), 17 PBL-miRNA2, 2 PBL-MYC/FOXR2, and 4 PBL-RB1 samples. PBL-miRNA subtype tumors had characteristic alterations in microRNA-processing genes; DICER1 mutations (n=14; 4 in Grp1A/1 in Grp1B/9 in Grp2) were most abundant, followed by homozygous deletions of the DROSHA locus (n=11; 8/1/2) and DROSHA mutations (n=9; 3/3/3). Most frequent cytogenetic aberrations in these subtypes were chromosome (chr.) 7 gains (n=25) and chr. 14 losses (n=20, with all but one of miRNA2 cases). DICER1 mutations were significantly associated with chr. 14 losses (p&amp;lt;0.001). 17 cases showed gains of the chromosomal arm 14q, in 6 additional cases, focal gains were found of the OTX2 oncogene located on chr. 14q. In the miRNA subtypes we identified cases with a tetraploid phenotype (n=11; 7/4/0). Interestingly, none of the 6 patients with tetraploid tumors and survival data had relapses, while the remaining patients of the miRNA subtypes had a 5-year PFS of 72% and 5-year OS of 68% (10 of 31 with relapse/death) after a median follow-up time of 3.9 years. However, this did not reach statistical significance. A significant impact on survival could not be demonstrated for chr. 14q gains or focal OTX2 gains. The PBL-RB1 subtype cases had homozygous RB1 deletions (n=3) or biallelic RB1 mutations (n=1). Focal chromosomal aberrations were frequently found in this subtype, but rarely numerical alterations. Conclusion: The epigenetically defined PBL subtypes were characterized by distinct cytogenetic and mutational events. Although we could not demonstrate a prognostic impact of these events, this might be due to the small sample size. A possible prognostic role for a superior (e.g. tetraploidy) or inferior outcome (e.g. OTX2 gains) need studies in larger cohorts and ideally in prospective clinical trials. Citation Format: Tobias Goschzik, Mathias Yuan, Martin Mynarek, Elke Pfaff, Evelyn Dörner, David T. Jones, Stefan M. Pfister, Stefan Rutkowski, Torsten Pietsch. Recurrent genetic alterations in epigenetic pineoblastoma subtypes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3561.

Abstract 5352: Targeting DNA methylation in T cells to improve the efficacy of immunotherapy

Abstract T-cells are critical mediators of immunity and immunologic memory. Their cell fates are regulated in part through epigenetic mechanisms, including DNA methylation. Recent genome-wide methylation analyses have revealed dynamic alterations in the methylome at various stages of development and differentiation of T cells. At single cell level, it is not easy to simultaneously collect RNA-seq and RBBS methylation profiling. An important task is to understand the expression change of which genes and pathways are regulated by DNA methylations, especially for the ones that are associated with functional variations in the T cells from tumor microenvironment.In this study, we developed a computational approach based on our recently developed metabolic flux estimation to estimate cell-wise global DNA methylation activity level by using scRNA-seq data. We also hypothesize that the global DNA methylation activity level in one cell determines most of the DNA methylation level in gene-specific DNA methylations. Hence, the dependency between gene-specific DNA methylation and expression could be imputed by the dependency between predicted global DNA methylation input level and the gene expression. We validated our method to impute cell-wise global DNA methylation level by using four independent sets of paired gene expression and DNA methylation data. Noted, our prediction of global DNA methylation activity is from a pure metabolic perspective. We found that two metabolic reaction rates, named metabolic flux from methionine to SAM and SAM to SAH, purely predicted by using gene expression data can accurately impute DNA methylation activity in all validating data sets. Our method enables further identification of the disease/cell context specific contributor of DNA methylation, i.e., the genes high contribution to DNA methylations in each individual cell or cell groups. We applied our method on scRNA-seq data of different T cell types extracted from TME of lung, liver, and colon cancer. We have seen that exhausted T cells, especially the ones with decreased Granzymes and PRF1 are associated with increased global DNA methylation level and related genes, suggesting the potential clinical implications in targeting DNA methylation to improve the efficacy of immunotherapies. Citation Format: Pengtao Dang, Xiao Wang, Haiqi Zhu, Jia Wang, Tingbo Guo, Xinyu Zhou, Paveethran Swaminathan, Chi Zhang, Sha Cao. Targeting DNA methylation in T cells to improve the efficacy of immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5352.

Genome-wide methylation analysis of circulating tumor DNA: A new biomarker for recurrent glioblastom

BackgroundGlioblastoma (GBM) is a malignant tumor with a short survival and poor prognosis and a lack of clinically validated biomarkers for diagnosis and prognosis. MethodsWe collected cerebrospinal fluid (CSF) samples and normal CSF sample from recurrent GBM patients and paired tissue samples. Methylation profiles of CSF circulating tumor DNA (ctDNA) and transcriptional profiles of tumor tissues were analyzed. The China Glioma Genome Atlas (CGGA) database and Gene Expression Omnibus (GEO) was used for data analysis. ResultsLasso analysis and multiplex Cox analysis were performed using intersecting genes of differentially methylated regions and differentially expressed genes. 8 hub genes were screened to construct diagnostic and prognostic models. Based on these 8 hub genes, the diagnostic (AUC = 0.944) and prognostic (3-years, AUC = 0.876) models were accurate. ConclusionsIn this study, 8 hub genes were identified for the diagnosis and prognosis of recurrent GBM, providing new biomarkers for the clinical study of recurrent GBM.

Comparative and integrative analysis of transcriptomic and epigenomic-wide DNA methylation changes in African American prostate cancer

ABSTRACT African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To date, PCa genomic studies have largely under-represented tumour samples from AA men. We measured genome-wide DNA methylation in benign and tumor prostate tissues from AA men using the Illumina Infunium 850 K EPIC array. mRNA expression database from a subset of the AA biospecimen were used to assess correlation of transcriptome and methylation datasets. Genome-wide methylation analysis identified 11,460 probes that were significant (p < 0.01) and differentially methylated in AA PCa compared to normal prostate tissues and showed significant (p < 0.01) inverse-correlation with mRNA expression. Ingenuity pathway analysis and Gene Ontology analysis in our AA dataset compared with TCGA dataset showed similarities in methylation patterns: top candidate genes with significant hypermethylation and corresponding down-regulated gene expression were associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthesis, and cell communication. In addition, top candidate genes with significant hypomethylation and corresponding up-regulated gene expression were associated with biological pathways in macrophage differentiation, cAMP-dependent protein kinase activity, protein destabilization, transcription co-repression, and fatty acid biosynthesis. In contrast, differences in genome-wide methylation in our AA dataset compared with TCGA dataset were enriched for genes in steroid signalling, immune signalling, chromatin structure remodelling and RNA processing. Overall, differential methylation of AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL, and FGF6 were significant and uniquely associated with PCa progression in our AA cohort.

N6-methyladenosine RNA is modified in the rat hippocampus following traumatic brain injury with hypothermia treatment.

Recent studies have suggested a role for N6-methyladenosine (m6A) modification in neurological diseases. Hypothermia, a commonly used treatment for traumatic brain injury, plays a neuroprotective role by altering m6A modifications. In this study, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was applied to conduct a genome-wide analysis of RNA m6A methylation in the rat hippocampus of Sham and traumatic brain injury (TBI) groups. In addition, we identified the expression of mRNA in the rat hippocampus after TBI with hypothermia treatment. Compared with the Sham group, the sequencing results of the TBI group showed that 951 different m6A peaks and 1226 differentially expressed mRNAs were found. We performed cross-linking analysis of the data of the two groups. The result showed that 92 hyper-methylated genes were upregulated, 13 hyper-methylated genes were downregulated, 25 hypo-methylated genes were upregulated, and 10 hypo-methylated genes were downregulated. Moreover, a total of 758 differential peaks were identified between TBI and hypothermia treatment groups. Among these differential peaks, 173 peaks were altered by TBI and reversed by hypothermia treatment, including Plat, Pdcd5, Rnd3, Sirt1, Plaur, Runx1, Ccr1, Marveld1, Lmnb2, and Chd7. We found that hypothermia treatment transformed some aspects of the TBI-induced m6A methylation landscape of the rat hippocampus.

The role of N6-methyladenosine modification in benzene-induced testicular damage and the protective effect of melatonin

Benzene is a universal ambient pollutant. Population-based studies have shown that benzene exposure affects male fertility. However, the mechanism of benzene-induced reproductive toxicity is unknown. Here, we established a dynamic inhalation model and exposed C57BL/6J mice to 0, 10, and 50 ppm benzene (6 h/day, 6 days/week, 7 weeks). Our study revealed that benzene exposure caused testicular injury, including structural damage to spermatogenic tubules, reduced semen quality, and decreased testosterone levels. In addition, the decrease in the global level of N6-Methyladenosine (m6A) and the change of m6A important regulatory enzymes in mice testes suggested that m6A was involved in the benzene-induced testicular injury. Further genome-wide m6A methylation analysis showed that 1469 differential m6A peaks were present in the testes of control and benzene groups, indicating that benzene exposure modulated m6A methylation in testes. Furthermore, the comprehensive analysis of m6A-sequencing and transcriptome revealed that hypermethylated Rara and its consequent reduced expression impaired the sperm production process. In particular, melatonin alleviated benzene-induced testicular injury by modulating m6A-related genes. Overall, our research provides a new idea and fundamental knowledge into the possible mechanisms of m6A modifications in benzene-induced testicular impairment, as well as a new experimental basis for benzene-induced male fertility therapy.

Genome-wide methylation analysis of treatment resistant schizophrenia

Various studies have investigated the relationship between genetic polymorphisms of antipsychotic drug-metabolizing agents and drug response. DNA methylation is a form of epigenetic modification that regulates gene expression. Few studies have analyzed the relationship between genome-wide methylation patterns and treatment resistance schizophrenia. The primary aim of this pilot study is to investigate the association between treatment resistance status and genome-wide DNA methylation in schizophrenia patients. Treatment resistance status was determined for 109 patients with schizophrenia. Treatment resistance was the primary outcome variable in a model, including methylation status of white blood cells using the Illumina 450 array. The genome-wide DNA methylation levels in 109 Schizophrenia subjects did not show that DNA methylation sties were associated with resistance status. From our study, it is evident the importance of continuing to investigate the relationship between DNA methylation and antipsychotic response to personalize treatment in schizophrenia. Future studies require larger prescription databases to build on the results presented in this pilot study.

Integrating genome-wide methylation and transcriptome-wide analyses to reveal the genetic mechanism of milk traits in Kazakh horses

Horse Milk has important quantitative characteristics and high economic value. However, the DNA methylation regulators involved in horse milk traits have not been clarified. To explore the important role of genome-wide DNA methylation in regulating equine milk yield, this study systematically investigated the genome-wide DNA methylation profiles of Kazakh horse blood by comparing a high-production group (HP, average daily milk yield of 7.5 kg) and low-production group (LP, average daily milk yield of 3.2 kg) using deep whole-genome bisulfite sequencing. First, both groups showed similar proportions of methylation at CpG sites. Subsequently, we identified 26,677 differential methylated regions (DMRs) of CG, 15 DMRs of CHG, 480 DMRs of CHH and 8268 DMR-related genes (DMGs). GO and KEGG analyses revealed that some DMGs were involved in regulating milk and milk component formation. By combining the WGBS-seq and the previous RNA-seq data, a total of 94 overlapping genes were obtained. Finally, we found that 9 DMGs are likely involved in milk production by Kazakh horses.

Canalization of genome-wide transcriptional activity in Arabidopsis thaliana accessions by MET1-dependent CG methylation

BackgroundDespite its conserved role on gene expression and transposable element (TE) silencing, genome-wide CG methylation differs substantially between wild Arabidopsis thaliana accessions.ResultsTo test our hypothesis that global reduction of CG methylation would reduce epigenomic, transcriptomic, and phenotypic diversity in A. thaliana accessions, we knock out MET1, which is required for CG methylation, in 18 early-flowering accessions. Homozygous met1 mutants in all accessions suffer from common developmental defects such as dwarfism and delayed flowering, in addition to accession-specific abnormalities in rosette leaf architecture, silique morphology, and fertility. Integrated analysis of genome-wide methylation, chromatin accessibility, and transcriptomes confirms that MET1 inactivation greatly reduces CG methylation and alters chromatin accessibility at thousands of loci. While the effects on TE activation are similarly drastic in all accessions, the quantitative effects on non-TE genes vary greatly. The global expression profiles of accessions become considerably more divergent from each other after genome-wide removal of CG methylation, although a few genes with diverse expression profiles across wild-type accessions tend to become more similar in mutants. Most differentially expressed genes do not exhibit altered chromatin accessibility or CG methylation in cis, suggesting that absence of MET1 can have profound indirect effects on gene expression and that these effects vary substantially between accessions.ConclusionsSystematic analysis of MET1 requirement in different A. thaliana accessions reveals a dual role for CG methylation: for many genes, CG methylation appears to canalize expression levels, with methylation masking regulatory divergence. However, for a smaller subset of genes, CG methylation increases expression diversity beyond genetically encoded differences.

Exploration of genome-wide DNA methylation profiles in night shift workers

ABSTRACT The past decades, studies indicated that night shift work is associated with adverse health effects, however, molecular mechanisms underlying these effects are poorly understood. A few previous studies have hypothesized a role for DNA-methylation (DNAm) in this relationship. We performed a cross-sectional epigenome-wide association study, to investigate if night shift work is associated with genome-wide DNAm changes and DNAm-based biological age acceleration, based on previously developed so-called ‘epigenetic clocks.’ Short term (2–6 years) and intermediate term (10–16 years) night shift workers, along with age and sex matched dayworkers (non-shift workers) were selected from the Lifelines Cohort Study. For genome-wide methylation analysis the Infinium Methylation EPIC array (Ilumina) was used. Linear regression analyses were used to detect differences in methylation at individual CpG-sites associated with night shift work. Pathway analysis was performed based on KEGG pathways and predictions of age acceleration in night shift workers were performed based on four previously developed epigenetic age calculators. Only in women, differences in methylation at individual CpG-sites were observed between night shift workers and non-shift workers. Most of these differentially methylated positions (DMPs) were observed in intermediate term night shift workers. Pathway analysis shows involvement of pathways related to circadian rhythm and cellular senescence. Increased age acceleration was observed only in short-term night shift workers (men and women). This might be indicative of adaptation to night shift work or a so-called healthy worker effect. In conclusion, these results show that DNA methylation changes are associated with night shift work, specifically in women.

Novel compound heterozygous mutations in UHRF1 are associated with atypical immunodeficiency, centromeric instability and facial anomalies syndrome with distinctive genome-wide DNA hypomethylation.

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is in most cases caused by mutations in either DNA methyltransferase (DNMT)3B, zinc finger and BTB domain containing 24, cell division cycle associated 7 or helicase lymphoid-specific. However, the causative genes of a few ICF patients remain unknown. We, herein, identified ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) as a novel causative gene of one such patient with atypical symptoms. This patient is a compound heterozygote for two previously unreported mutations in UHRF1: c.886C > T (p.R296W) and c.1852C > T (p.R618X). The R618X mutation plausibly caused nonsense-mediated decay, while the R296W mutation changed the higher order structure of UHRF1, which is indispensable for the maintenance of CG methylation along with DNMT1. Genome-wide methylation analysis revealed that the patient had a centromeric/pericentromeric hypomethylation, which is the main ICF signature, but also had a distinctive hypomethylation pattern compared to patients with the other ICF syndrome subtypes. Structural and biochemical analyses revealed that the R296W mutation disrupted the protein conformation and strengthened the binding affinity of UHRF1 with its partner LIG1 and reduced ubiquitylation activity of UHRF1 towards its ubiquitylation substrates, histone H3 and proliferating cell nuclear antigen -associated factor 15 (PAF15). We confirmed that the R296W mutation causes hypomethylation at pericentromeric repeats by generating the HEK293 cell lines that mimic the patient's UHRF1 molecular context. Since proper interactions of the UHRF1 with LIG1, PAF15 and histone H3 are essential for the maintenance of CG methylation, the mutation could disturb the maintenance process. Evidence for the importance of the UHRF1 conformation for CG methylation in humans is, herein, provided for the first time and deepens our understanding of its role in regulation of CG methylation.

Abstract A011: Epigenetic vulnerabilities in patient-derived colorectal cancer organoids

Abstract Colorectal cancer (CRC) is a major cause of cancer-related deaths worldwide. Here, we established human patient-derived colorectal cancer organoid cultures (PDOs) to decipher the patient-specific DNA methylation profile and drug sensitivity towards 5-aza-2’-deoxycytidine, a DNA methyltransferase inhibitor. Organoid lines (n=15) were generated from adjacent normal mucosa and tumor tissue located in different anatomical sites of the colon and rectum. The derived PDOs were characterized on a histopathological level and reproduced the grade and differentiation capacity of their parental tumors. Additional genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. However, the stability of the DNA methylation landscape of human cancer organoids remains largely unknown. In genome-wide methylation analysis of long-term organoid cultures (up to 6 months in culture), we observed surprisingly stable methylation signatures that recapitulates the patient’s profile whilst demonstrating the vast intertumoral heterogeneity among patients. Strikingly, we identified a tumor-specific methylation signature that consisted of 39 CpG sites, which were unmethylated in normal epithelial cells but highly (91-96%) methylated in all tumor cells analyzed. This signature allows for the estimation of the percentage of tumor content in resected tissues and might represent potent biomarkers for early CRC diagnostics. Notably, it has been demonstrated that PDOs have the potential to predict clinical outcome and response to chemo- and radiation therapy in patients. Herein, drug screening with 5-aza-2’-deoxycytidine revealed heterogeneous responses and a clustering into drug sensitive, median and resistant organoid lines. ATAC- and RNA-seq analyses identified pathways rendering drug sensitivity and resistance. Our findings highlight the utility of PDOs as an advanced model system to study the role of the epigenome, especially DNA methylation, and its impact on tumor burden and vulnerability towards epigenetic modifiers. Citation Format: Loan Tran, Raheleh Sheibani Tezerji, Carlos Uziel Perez Malla, Anna Malzer, Ajna Logo, Katarina Misura, Theresia Mair, Thomas Dillinger, Madeleine Kuroll, Velina Atanasova, Julijan Kabiljo, Helga Schachner, Katharina Wöran, Judith Stift, Helmut Dolznig, Walter Pulverer, Michael Bergmann, Gerda Egger. Epigenetic vulnerabilities in patient-derived colorectal cancer organoids. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A011.