Reduced Representation Bisulfite Sequencing Research Articles

131 DNA methylation profile in bovine fetal liver is affected by maternal vitamin and mineral supplementation during early gestation

Abstract Early gestation is the critical period for successful pregnancy establishment. During this period, maternal nutrition affects fetal development with potentially long-lasting consequences on offspring performance. Despite the known effects of vitamins and minerals on embryonic development, supplementation is still not a widely adopted practice. Here we focused on the effects of maternal vitamin and mineral supplementation from pre-breeding to d 83 of gestation. We hypothesized that the DNA methylation pattern of genes involved with fetal hepatic metabolism and function would be altered in response to vitamin and mineral supplementation during early gestation. Sixteen Angus crossbred heifers (~16 mo of age) were randomly assigned to either a treatment (vitamin and mineral supplementation, VTM, n = 8) or control (corn-based carrier without supplementation, NoVTM, n = 8) group. Supplemented heifers received 0.45 kg •heifer-1•d-1 of to provide 113 g of vitamin-mineral premix (Purina Wind & Rain Storm All-Season 7.5 Complete, Land O’Lakes, Inc., Arden Hills, MN) from d 71 to 148 before breeding to d 83 of gestation. All heifers received 100% of NRC recommended nutritional requirements targeting 0.28 kg •heifer-1•d-1 of gain. The heifers were bred through artificial insemination following a 7-d Co-Synch + CIDR estrus synchronization protocol. All pregnant heifers were surgically ovariohysterectomized on d 83, and liver samples were collected from the fetuses. DNA methylation profiles from 16 fetuses (8 per treatment) were determined using Reduced Representation Bisulfite Sequencing (RRBS). The analysis was conducted using methylKit pipeline where data was normalized using principal components. We identified 794 differentially methylated cytosines (DMCs) for the control (NoVTM) vs VTM comparison. Most of the DMCs were found in introns followed by intergenic, promoter, and exonic regions. Among these DMCs, 60% (479) were hypermethylated and 40% (315) were hypomethylated, potentially regulating 739 nearby genes (FDR < 0.1). Biological processes over-represented by these genes included transport of ions through voltage gated channels (KCNK13, KCNT1, CACNA1B, KCNK9, LRRC55, KCNAB2, CACNA1S, KCNJ2, CACNG5, CLIC5, TPCN2, KCNQ1), which may be associated to pregnancy establishment by inducing pregnancy-associated relaxation and uterine artery dilatation. Some genes associated with structural development were also regulated by VTM supplementation (i.e., TIAM1, COL18A1, DYSF, BMP10, RXRA, EHMT1, COL4A2, PLEC, ZFAT, MTMR2, NAV1, ANHX, ACACB, and LIMK2). Collectively, these results suggest that periconceptual maternal vitamin and mineral supplementation affects the methylation pattern and potentially regulates the expression of genes involved in fetal liver development and ion transport.

Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid

BackgroundMechanistic understanding of transient exposures that lead to adverse health outcomes will enhance our ability to recognize biological signatures of disease. Here, we measured the transcriptomic and epigenomic alterations due to exposure to the metabolic reprogramming agent, dichloroacetic acid (DCA). Previously, we showed that exposure to DCA increased liver tumor incidence in B6C3F1 mice after continuous or early life exposures significantly over background level.MethodsUsing archived formalin-fixed liver samples, we utilized modern methodologies to measure gene expression and DNA methylation levels to link to previously generated phenotypic measures. Gene expression was measured by targeted RNA sequencing (TempO-seq 1500+ toxicity panel: 2754 total genes) in liver samples collected from 10-, 32-, 57-, and 78-week old mice exposed to deionized water (controls), 3.5 g/L DCA continuously in drinking water (“Direct” group), or DCA for 10-, 32-, or 57-weeks followed by deionized water until sample collection (“Stop” groups). Genome-scaled alterations in DNA methylation were measured by Reduced Representation Bisulfite Sequencing (RRBS) in 78-week liver samples for control, Direct, 10-week Stop DCA exposed mice.ResultsTranscriptomic changes were most robust with concurrent or adjacent timepoints after exposure was withdrawn. We observed a similar pattern with DNA methylation alterations where we noted attenuated differentially methylated regions (DMRs) in the 10-week Stop DCA exposure groups compared to the Direct group at 78-weeks. Gene pathway analysis indicated cellular effects linked to increased oxidative metabolism, a primary mechanism of action for DCA, closer to exposure windows especially early in life. Conversely, many gene signatures and pathways reversed patterns later in life and reflected more pro-tumorigenic patterns for both current and prior DCA exposures. DNA methylation patterns correlated to early gene pathway perturbations, such as cellular signaling, regulation and metabolism, suggesting persistence in the epigenome and possible regulatory effects.ConclusionLiver metabolic reprogramming effects of DCA interacted with normal age mechanisms, increasing tumor burden with both continuous and prior DCA exposure in the male B6C3F1 rodent model.

Age Prediction Using DNA Methylation Heterogeneity Metrics.

Dynamic changes in genomic DNA methylation patterns govern the epigenetic developmental programs and accompany the organism's aging. Epigenetic clock (eAge) algorithms utilize DNA methylation to estimate the age and risk factors for diseases as well as analyze the impact of various interventions. High-throughput bisulfite sequencing methods, such as reduced-representation bisulfite sequencing (RRBS) or whole genome bisulfite sequencing (WGBS), provide an opportunity to identify the genomic regions of disordered or heterogeneous DNA methylation, which might be associated with cell-type heterogeneity, DNA methylation erosion, and allele-specific methylation. We systematically evaluated the applicability of five scores assessing the variability of methylation patterns by evaluating within-sample heterogeneity (WSH) to construct human blood epigenetic clock models using RRBS data. The best performance was demonstrated by the model based on a metric designed to assess DNA methylation erosion with an MAE of 3.686 years. We also trained a prediction model that uses the average methylation level over genomic regions. Although this region-based model was relatively more efficient than the WSH-based model, the latter required the analysis of just a few short genomic regions and, therefore, could be a useful tool to design a reduced epigenetic clock that is analyzed by targeted next-generation sequencing.

Abstract LB103: Cell-free DNA methylation as a noninvasive prognostic biomarker in hepatocellular carcinoma

Abstract The current noninvasive prognostic evaluation of hepatocellular carcinoma (HCC) using markers such as imaging features and serum biomarkers like alpha-fetoprotein (AFP) is limited due to low sensitivity. Pathologic tumor features can enhance prognosis beyond imaging and AFP, yet pre-surgical percutaneous biopsies are invasive, not routinely pursued, and may not be representative of the entire tumor. There is a critical unmet need to develop noninvasive blood-based HCC biomarkers to guide therapeutic decisions. Epigenetic changes in circulating cell-free DNA (cfDNA) have shown promise in early cancer diagnosis and prognosis. Therefore, we aim to evaluate the potential of cfDNA methylation as a noninvasive predictor for pre-treatment prognostication in HCC patients. Using Illumina HumanMethylation450 array data of 377 HCC tumors and 50 adjacent normal tissues obtained from The Cancer Genome Atlas (TCGA), we systematically identified 158 HCC-related DNA methylation markers associated with overall survival (OS). Functional analyses of these markers indicated their roles in regulating gene expression and revealed biological pathways associated with HCC patient survival. We predicted an OS risk score utilizing the DNA methylation panel, referred to as “methRisk”, and validated its predictive power using the Reduced Representation Bisulfite Sequencing data of HCC tumor tissue samples from 29 surgically resected patients. To assess the noninvasive prognostic value of these markers, we collected plasma cfDNA samples from an independent validation cohort of 52 HCC patients, and profiled the methylation patterns of these samples using our newly developed cost-effective cfDNA methylome sequencing assay, cfMethyl-Seq. We utilized random survival forest models combined with 10-fold cross-validation to predict the methRisk using the circulating markers. The cfDNA-based methRisk effectively stratifies patients into clinically relevant risk groups and predicts patient survival with high accuracy (3-year AUC = 0.81, 95% CI: 0.68-0.94). Integrating the cfDNA-based methRisk with existing risk indices like the Barcelona clinic liver cancer (BCLC) staging greatly improves the noninvasive prognostic assessments (3-year AUC = 0.91, 95% CI: 0.80-0.99). In summary, these findings indicate that cfDNA methylation is a promising noninvasive prognostic biomarker for HCC, providing a proof-of-concept for its potential clinical utility. Incorporating cfDNA methylation analysis into the existing noninvasive prognostic framework for HCC patients holds great potential for enhancing the effectiveness of current staging systems and optimizing patient selection for curative therapies like liver transplantation. Citation Format: Ran Hu, Benjamin Tran, Shuo Li, Mary L. Stackpole, Weihua Zeng, Yonggang Zhou, Andrew Melehy, Xianghong Zhou, Wenyuan Li, Vatche Agopian. Cell-free DNA methylation as a noninvasive prognostic biomarker in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB103.

Epigenome-wide association study identifies DNA methylation loci associated with handgrip strength in Chinese monozygotic twins.

Background: The decline in muscle strength and function with aging is well recognized, but remains poorly characterized at the molecular level. Here, we report the epigenetic relationship between genome-wide DNA methylation and handgrip strength (HGS) among Chinese monozygotic (MZ) twins. Methods: DNA methylation (DNAm) profiling was conducted in whole blood samples through Reduced Representation Bisulfite Sequencing method. Generalized estimating equation was applied to regress the DNAm of each CpG with HGS. The Genomic Regions Enrichment of Annotations Tool was used to perform enrichment analysis. Differentially methylated regions (DMRs) were detected using comb-p. Causal inference was performed using Inference about Causation through Examination of Familial Confounding method. Finally, we validated candidate CpGs in community residents. Results: We identified 25 CpGs reaching genome-wide significance level. These CpGs located in 9 genes, especially FBLN1, RXRA, and ABHD14B. Many enriched terms highlighted calcium channels, neuromuscular junctions, and skeletal muscle organ development. We identified 21 DMRs of HGS, with several DMRs within FBLN1, SLC30A8, CST3, and SOCS3. Causal inference indicated that the DNAm of 16 top CpGs within FBLN1, RXRA, ABHD14B, MFSD6, and TYW1B might influence HGS, while HGS influenced DNAm at two CpGs within FBLN1 and RXRA. In validation analysis, methylation levels of six CpGs mapped to FLBN1 and one CpG mapped to ABHD14B were negatively associated with HGS weakness in community population. Conclusion: Our study identified multiple DNAm variants potentially related to HGS, especially CpGs within FBLN1 and ABHD14B. These findings provide new clues to the epigenetic modification underlying muscle strength decline.

Blood leukocytes as a non-invasive diagnostic tool for thyroid nodules: a prospective cohort study

BackgroundThyroid nodule (TN) patients in China are subject to overdiagnosis and overtreatment. The implementation of existing technologies such as thyroid ultrasonography has indeed contributed to the improved diagnostic accuracy of TNs. However, a significant issue persists, where many patients undergo unnecessary biopsies, and patients with malignant thyroid nodules (MTNs) are advised to undergo surgery therapy.MethodsThis study included a total of 293 patients diagnosed with TNs. Differential methylation haplotype blocks (MHBs) in blood leukocytes between MTNs and benign thyroid nodules (BTNs) were detected using reduced representation bisulfite sequencing (RRBS). Subsequently, an artificial intelligence blood leukocyte DNA methylation (BLDM) model was designed to optimize the management and treatment of patients with TNs for more effective outcomes.ResultsThe DNA methylation profiles of peripheral blood leukocytes exhibited distinctions between MTNs and BTNs. The BLDM model we developed for diagnosing TNs achieved an area under the curve (AUC) of 0.858 in the validation cohort and 0.863 in the independent test cohort. Its specificity reached 90.91% and 88.68% in the validation and independent test cohorts, respectively, outperforming the specificity of ultrasonography (43.64% in the validation cohort and 47.17% in the independent test cohort), albeit with a slightly lower sensitivity (83.33% in the validation cohort and 82.86% in the independent test cohort) compared to ultrasonography (97.62% in the validation cohort and 100.00% in the independent test cohort). The BLDM model could correctly identify 89.83% patients whose nodules were suspected malignant by ultrasonography but finally histological benign. In micronodules, the model displayed higher specificity (93.33% in the validation cohort and 92.00% in the independent test cohort) and accuracy (88.24% in the validation cohort and 87.50% in the independent test cohort) for diagnosing TNs. This performance surpassed the specificity and accuracy observed with ultrasonography. A TN diagnostic and treatment framework that prioritizes patients is provided, with fine-needle aspiration (FNA) biopsy performed only on patients with indications of MTNs in both BLDM and ultrasonography results, thus avoiding unnecessary biopsies.ConclusionsThis is the first study to demonstrate the potential of non-invasive blood leukocytes in diagnosing TNs, thereby making TN diagnosis and treatment more efficient in China.

Differential DNA methylation in iPSC-derived dopaminergic neurons: a step forward on the role of SNORD116 microdeletion in the pathophysiology of addictive behavior in Prader-Willi syndrome.

A microdeletion including the SNORD116 gene (SNORD116 MD) has been shown to drive the Prader-Willi syndrome (PWS) features. PWS is a neurodevelopmental disorder clinically characterized by endocrine impairment, intellectual disability and psychiatric symptoms such as a lack of emotional regulation, impulsivity, and intense temper tantrums with outbursts. In addition, this syndrome is associated with a nutritional trajectory characterized by addiction-like behavior around food in adulthood. PWS is related to the genetic loss of expression of a minimal region that plays a potential role in epigenetic regulation. Nevertheless, the role of the SNORD116 MD in DNA methylation, as well as the impact of the oxytocin (OXT) on it, have never been investigated in human neurons. We studied the methylation marks in induced pluripotent stem-derived dopaminergic neurons carrying a SNORD116 MD in comparison with those from an age-matched adult healthy control. We also performed identical neuron differentiation in the presence of OXT. We performed a genome-wide DNA methylation analysis from the iPSC-derived dopaminergic neurons by reduced-representation bisulfite sequencing. In addition, we performed RNA sequencing analysis in these iPSC-derived dopaminergic neurons differentiated with or without OXT. The analysis revealed that 153,826 cytosines were differentially methylated between SNORD116 MD neurons and control neurons. Among the differentially methylated genes, we determined a list of genes also differentially expressed. Enrichment analysis of this list encompassed the dopaminergic system with COMT and SLC6A3. COMT displayed hypermethylation and under-expression in SNORD116 MD, and SLC6A3 displayed hypomethylation and over-expression in SNORD116 MD. RT-qPCR confirmed significant over-expression of SLC6A3 in SNORD116 MD neurons. Moreover, the expression of this gene was significantly decreased in the case of OXT adjunction during the differentiation. SNORD116 MD dopaminergic neurons displayed differential methylation and expression in the COMT and SLC6A3 genes, which are related to dopaminergic clearance.

Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

LXRα Promotes Abdominal Aortic Aneurysm Formation Through UHRF1 Epigenetic Modification of miR-26b-3p.

Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

Abstract 2333: Methylation based phylogeny and timing in cancer

Abstract Knowing the chronological sequence of genetic alterations during cancer development and progression is crucial for understanding the process of transformation of normal tissue into malignant tumors. This can offer insights into the etiology and intrinsic cellular mechanisms of the process, presenting opportunities for early detection and treatment of initial disease stages, as well as highlight key events as potential therapeutic. Current methods of establishing progression histories of cancers rely either on direct sequencing of premalignant lesions, which are seldom available for tumors or on computational inference timing methods capable of reconstructing the history from primary tumor data. These methods require genome or exome sequencing data from a relatively large cohort of tumors and provide limited resolution due to overall scarcity of somatic mutations. We utilize a different source of genetic information, genome methylation, that also “records” historic information about acquisition and development of mutations. Over 28 million methylated CpG sites exist in the genome with several percent of them differentially methylated (DMS) between tissue types and tumor vs adjacent normal tissue. The several orders of magnitude higher density of distinctly methylated sites provides unprecedented resolution, needs fewer tumors, can be merged with driver mutation data and co-analyzed with “methylation clock” information that allows to establish early development, phylogeny and progression at unprecedented resolution. We developed methods for whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS) and Oxford Nanopore Technologies native methylation sequencing data. Somatic DMS sites have distinct properties from somatic point mutations, can appear and disappear in the process of tumor evolution and development allowing for unique configuration of methylated islands. Our tools perform single sample and cohort level “timing”, multi-sample phylogeny reconstruction and cancer population reconstruction by integrating methylation and mutation information from the same sample. These new methods will significantly contribute to our understanding of cancer initiation and progression at a resolution not available previously. Citation Format: Nataliya Guteneva, Eliezer Lichter, Ignaty Leshchiner. Methylation based phylogeny and timing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2333.

Abstract 6225: Tumor evolution through selection by ECM stiffness

Abstract In this study, we used experimental evolution to investigate the extent to which the mechanical stiffness of the tumor extracellular matrix (ECM) exerts selection pressure on a genetically diverse population of breast cancer cells. Polyacrylamide hydrogels were fabricated and conjugated with collagen type I to create the ECM with different stiffness. MDA-MB-231 cells were continuously cultured on the hydrogels of the same corresponding stiffness. Growth rate was determined at certain time points by harvesting and counting the cells. Abundance of clonal MDA-MB-231 cells labeled with heritable DNA barcodes underwent the same selection was identified with sequencing of DNA barcode tags. Cellular phenotypes were characterized through immunofluorescence staining and confocal microscopy. 3D migration of the cancer cells was examined using Boyden chamber assay. Cellular contractility was measured through traction force microscopy. Whole genome transcriptome sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing, and reduced representation bisulfite sequencing were performed for transcriptomic and epigenetic analysis. Sustained culture of MDA-MB-231 breast cancer cells overall three months on soft ECM resulted in an increased proliferation rate and beta1 integrin-dependent spreading area of the cells. Such changes did not occur in clonal cells with low genetic variation and did not occur in ancestral cells on stiff substrates. Barcode analysis revealed two distinct surviving populations post-genetic selection, with one demonstrating robust proliferation on both soft and stiff ECM, and the other exhibiting preferential adaptation to the soft matrix. The soft-selected populations exhibited nuclear localization of the transcriptional regulator yes-associated protein and less wrinkled nuclei compared with ancestral populations when both populations were grown on soft ECM. Aggressive migration was observed in the soft-selected populations, indicating a more malignant selected phenotype. The soft-selected cells showed increased RhoA activity and contractile tension on a soft substrate compared to the ancestral cells. RNA-seq analysis revealed systematic differences between ancestral, stiff-selected, and soft-selected populations corresponding to enriched biological processes related to cell adhesion and ECM organization. Significant differences in DNA methylation and chromatin accessibility were observed in soft selected populations which were correlated with changes in RNA expression. ECM stiffness exerts selection pressure on genetically variable cancer cell populations, selecting specific genetic variants in a cell population optimally adapted for that stiffness. Selected variants on soft ECM tend to be highly migratory, proliferative and generate high traction even on soft ECM. Our results highlight the importance of ECM stiffness-driven evolution in cancer development. Citation Format: Ting-Ching Wang, Suchitaa Sawhney, Daylin Morgan, Richa Rashmi, Marcos R. Estecio, Amy Brock, Irtisha Singh, Richard L. Bennett, Charles F. Baer, Jonathan D. Licht, Tanmay P. Lele. Tumor evolution through selection by ECM stiffness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6225.

Abstract 1722: Subtype classification of small cell lung cancer (SCLC) tissues using the EpiCheck methylation sensitive restriction-based PCR platform

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy with poor survival rates. Despite molecular and clinical heterogeneity, SCLC is currently treated as a single entity without guidance of predictive biomarkers leading to expectedly poor outcomes. A recent study suggested classifying SCLC into four subtypes (“A”, “N”, “P” and “I”), each with unique molecular features and therapeutic vulnerabilities. While initially this classification was based on gene expression (RNA-seq) data, subsequent data suggest it can be recapitulated using reduced-representation bisulfite sequencing (RRBS) methylation profile. While the classification system accurately predicts responses to therapies, including immunotherapies, in retrospective analysis, both tissue-based RNAseq and RRBS are cumbersome, time-consuming and impractical for prospective treatment assignment in an aggressive malignancy. In this pilot study, we developed a methylation-based PCR assay for classification of SCLC subtypes based on the EpiCheck platform, which combines methylation-sensitive restriction endonuclease (MSRE) digestion with qPCR amplification to detect differential methylation at the DNA level. We developed a 13-marker assay and successfully classified, in a blinded study, 96.55% of FFPE tissue samples originating from SCLC patient of A, N and P subtypes. First, a bioinformatic analysis identified 41 candidate biomarkers that are differentially methylated between pairs of SCLC subtypes (A-N, A-P, and N-P) using RRBS data from 56 RNA-seq classified SCLC tumor samples (34 “A”, 19 “N”, 3 “P”). Development of primers and probes for these markers was followed by a biochemical selection process, identifying 13 markers that displayed optimal performance. A PCR EpiCheck assay, employing these 13 markers, was applied to a blinded set of 29 DNA samples from SCLC FFPE tumor tissues (21 “A”, 6 “N”, 2 “P”). Each sample received two ranks based on methylation levels of 5 A-N and 9 P-AN markers and was classified using determined thresholds for each rank. Finally, when comparing PCR-based and RNA-seq-based classifications, the 13-marker PCR assay classified 28/29 (96.55%) of the samples concordantly, misclassifying one "P" sample as "A". This pilot demonstrates the potential of a simple PCR EpiCheck-based assay to accurately differentiate between SCLC subtypes. For complete subtype classification, “I” markers need to be added to the panel, and the assay should be further validated using a larger cohort, particularly for the “P” subtype. Early detection and classification are critical for timely personalized therapy, and such an assay, performed on cfDNA samples, could potentially be used to shorten the time between a patient diagnosis and tailored treatment initiation or clinical study inclusion from a month in best case scenarios to a few days. Citation Format: Orna Savin, Dvir Netanely, Simon Heeke, Carl Michael Gay, Marcos Roberto Estecio, Shacade Danan, Sarah Zaouch, Aharona Shuali, Mathias Ehrich, Lauren Averett Byers, John Victor Heymach, Adam Wasserstrom, Danny Frumkin. Subtype classification of small cell lung cancer (SCLC) tissues using the EpiCheck methylation sensitive restriction-based PCR platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1722.

Trichloroethylene metabolite modulates DNA methylation-dependent gene expression in Th1 polarized CD4+ T cells from autoimmune-prone mice.

Trichloroethylene (TCE) is an industrial solvent and widespread environmental contaminant associated with CD4+ T cell activation and autoimmune disease. Prior studies showed that exposure to TCE in the drinking water of autoimmune-prone mice expanded effector/memory CD4+ T cells with an interferon-γ (IFN-γ)-secreting Th1-like phenotype. However, very little is known how TCE exposure skews CD4+ T cells towards this pro-inflammatory Th1 subset. As observed previously, TCE exposure was associated with hypermethylation of regions of the genome related to transcriptional repression in purified effector/memory CD4 T cells. We hypothesized that TCE modulates transcriptional and/or epigenetic programming of CD4+ T cells as they differentiate from a naïve to effector phenotype. In the current study, purified naïve CD4 T cells from both male and female autoimmune-prone MRL/MpJ mice were activated ex vivo and polarized towards a Th1 subset for 4 days in the presence or absence of the oxidative metabolite of TCE, trichloroacetaldehyde hydrate (TCAH) in vitro. An RNA-seq assessment and Reduced Representation Bisulfite sequencing for DNA methylation were conducted on Th1 cells or activated, non- polarized cells. The results demonstrated TCAH's ability to regulate key genes involved in the immune response and autoimmunity, including Ifng, by altering the level of DNA methylation at the gene promoter. Intriguing sex differences were observed and for the most part the effects were more robust in females compared to males. In conclusion, TCE via TCAH epigenetically regulates gene expression in CD4+ T cells. These results may have implications for mechanistic understanding or future therapeutics for autoimmunity.

Abstract 1611: DNA methylation co-operates with genomic alterations during non-small cell lung cancer evolution

Abstract Aberrant DNA methylation patterns have been described in nearly all human cancers, yet their interplay with genomic alterations during tumor evolution is poorly understood. To explore this crosstalk, we performed reduced representation bisulfite sequencing on 217 tumor regions from 59 patients from the TRACERx study, all with matched adjacent normal tissue, to deconvolve tumor methylation rates. We combined two new metrics for integrative evolutionary analysis with DNA and RNA sequencing data. I-TMD (intra-tumoral methylation distance) quantifies intra-tumor DNA methylation heterogeneity. MR/MN classifies genes based on the rate of hypermethylation at regulatory (MR) versus non-regulatory (MN) CpGs, to identify driver genes exhibiting recurrent functional hypermethylation. We observed early DNA methylation of VIPR2 and ZNF714 co-occurring with driver mutations in CDKN2A and STK11, respectively. Finally, we found DNA methylation-linked dosage compensation of essential genes co-amplified with neighboring oncogenes. Our identification of hypermethylated driver genes under positive selection may open new avenues for therapeutic stratification of patients with non-small cell lung cancer. Citation Format: Francisco Gimeno-Valiente, Carla Castignani, Elizabeth Cadieux L. Cadieux, Nana E. Mensah, Olga Chervova, Mariam Jamal-Hanjani, Nicholas McGranahan, Stephan Beck, Jonas Demeulemeester, Miljana Tanic, Charles Swanton, Peter Van Loo, Nnennya Kanu. DNA methylation co-operates with genomic alterations during non-small cell lung cancer evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1611.

Investigating the potential of single-cell DNA methylation data to detect allele-specific methylation and imprinting

Allele-specific methylation (ASM) is an epigenetic modification whereby one parental allele becomes methylated and the other unmethylated at a specific locus. ASM is most often driven by the presence of nearby heterozygous variants that influence methylation, but also occurs somatically in the context of genomic imprinting. In this study, we investigate ASM using publicly available single-cell reduced representation bisulfite sequencing (scRRBS) data on 608 B cells sampled from six healthy B cell samples and 1,230 cells from 11 chronic lymphocytic leukemia (CLL) samples. We developed a likelihood-based criterion to test whether a CpG exhibited ASM, based on the distributions of methylated and unmethylated reads both within and across cells. Applying our likelihood ratio test, 65,998 CpG sites exhibited ASM in healthy B cell samples according to a Bonferroni criterion (p<8.4×10-9), and 32,862 CpG sites exhibited ASM in CLL samples (p<8.5×10-9). We also called ASM at the sample level. To evaluate the accuracy of our method, we called heterozygous variants from the scRRBS data, which enabled variant-based calls of ASM within each cell. Comparing sample-level ASM calls to the variant-based measures of ASM, we observed a positive predictive value of 76%-100% across samples. We observed high concordance of ASM across samples and an overrepresentation of ASM in previously reported imprinted genes and genes with imprinting binding motifs. Our study demonstrates that single-cell bisulfite sequencing is a potentially powerful tool to investigate ASM, especially as studies expand to increase the number of samples and cells sequenced.

Methylation changes of liver DNA during the formation of gallstones.

Aim: To explore the overall methylation changes in liver tissues during the formation of gallstones, as well as the key pathways and genes involved in the process. Methods: Reduced-representation bisulfite sequencing and RNA sequencing were conducted on the liver tissues of mice with gallstones and control normal mice. Results: A total of 8705 differentially methylated regions in CpG and 1410 differentially expressed genes were identified. The joint analysis indicated that aberrant DNA methylation may be associated with dysregulated gene expression in key pathways such as cholesterol metabolism and bile secretion. Conclusion: We propose for the first time that methylation changes in some key pathway genes in liver tissue may be involved in the formation of gallstones.

Strengths and limitations of reduced representation bisulfite sequencing (RRBS) in the perspective of DNA methylation analysis in fish: a case-study on rainbow trout spermatozoa.

DNA methylation in CpG dinucleotides is an important epigenetic mark in fish spermatozoa since it has been shown that some sperm methylome features are transmitted to the offspring. Reduced representation bisulfite sequencing (RRBS) is one genome-scale methods developed to assess DNA methylation at CpG sites. It allows the sequencing of a reduced fraction of the genome expected to be enriched in CpGs. The aim of this study is to characterize the extent of the CpG sites that can be identified in the RRBS-reduced sequenced fraction of rainbow trout spermatozoa, in order to evaluate the potential of RRBS for sperm DNA methylation studies. We observed that RRBS did provide a reduced amount of genomic data, the sum of the CpGs analyzed on 12 males spanning 9% of the total genomic CpGs. CpGs were only slightly enriched in the RRBS data (×1.7 times the sequenced nucleotides), the possible causes being linked to trout genome structure and sequenced fragments size. All genomic functional features were represented in our CpG dataset, with a noticeable enrichment in exons but, strikingly, not in promoters. The number of CpGs shared between biological replicates was low, but this proportion reached workable values from six biological replicates (46% of the analyzed cytosines) on. The choices that are to be made regarding fragment size selection and the options during bioinformatic data processing are discussed. In all, RRBS is a relevant first-approach method to scan the CpG DNA methylation status of spermatozoa along rainbow trout genome, although in a very reduced pattern among biological replicates.

DNA methylation-environment interactions in the human genome

Previously, we showed that a massively parallel reporter assay, mSTARR-seq, could be used to simultaneously test for both enhancer-like activity and DNA methylation-dependent enhancer activity for millions of loci in a single experiment (Lea et al., 2018). Here, we apply mSTARR-seq to query nearly the entire human genome, including almost all CpG sites profiled either on the commonly used Illumina Infinium MethylationEPIC array or via reduced representation bisulfite sequencing. We show that fragments containing these sites are enriched for regulatory capacity, and that methylation-dependent regulatory activity is in turn sensitive to the cellular environment. In particular, regulatory responses to interferon alpha (IFNA) stimulation are strongly attenuated by methyl marks, indicating widespread DNA methylation-environment interactions. In agreement, methylation-dependent responses to IFNA identified via mSTARR-seq predict methylation-dependent transcriptional responses to challenge with influenza virus in human macrophages. Our observations support the idea that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures—one of the tenets of biological embedding. However, we also find that, on average, sites previously associated with early life adversity are not more likely to functionally influence gene regulation than expected by chance.

DNA methylation-environment interactions in the human genome.

Previously, we showed that a massively parallel reporter assay, mSTARR-seq, could be used to simultaneously test for both enhancer-like activity and DNA methylation-dependent enhancer activity for millions of loci in a single experiment (Lea et al., 2018). Here, we apply mSTARR-seq to query nearly the entire human genome, including almost all CpG sites profiled either on the commonly used Illumina Infinium MethylationEPIC array or via reduced representation bisulfite sequencing. We show that fragments containing these sites are enriched for regulatory capacity, and that methylation-dependent regulatory activity is in turn sensitive to the cellular environment. In particular, regulatory responses to interferon alpha (IFNA) stimulation are strongly attenuated by methyl marks, indicating widespread DNA methylation-environment interactions. In agreement, methylation-dependent responses to IFNA identified via mSTARR-seq predict methylation-dependent transcriptional responses to challenge with influenza virus in human macrophages. Our observations support the idea that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures-one of the tenets of biological embedding. However, we also find that, on average, sites previously associated with early life adversity are not more likely to functionally influence gene regulation than expected by chance.

DNA hypomethylation promotes the expression of CASPASE-4 which exacerbates inflammation and amyloid-β deposition in Alzheimer’s disease

Alzheimer’s disease (AD) is the sixth leading cause of death in the USA. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release pro-inflammatory products such as IL-1β which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed reduced representation bisulfite sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed promotes the generation of IL-1β and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4 and CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4−/−). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-β (Aβ) and increased microglial production of IL-1β in 5xFAD mice. Utilizing RNA-sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1β from macrophages in response to cytosolic Aβ through cleavage of downstream effector Gasdermin D (GSDMD). Therefore, here we unravel the role for CASP11 and GSDMD in the generation of IL-1β in response to Aβ and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential DNA methylation in AD microglia contributes to the progression of AD pathobiology. Thus, we identify CASP4 as a potential target for immunotherapies for the treatment and prevention of AD.