Bisulfite Genomic Sequencing Research Articles

Elucidating the Role of Transcriptomic Networks and DNA Methylation in Collagen Deposition of Dezhou Donkey Skin.

DNA methylation represents a predominant epigenetic modification with broad implications in various biological functions. Its role is particularly significant in the process of collagen deposition, a fundamental aspect of dermal development in donkeys. Despite its critical involvement, the mechanistic insights into how DNA methylation influences collagen deposition in donkey skin remain limited. In this study, we employed whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) to investigate the epigenetic landscape and gene expression profiles in the dorsal skin tissues of Dezhou donkeys across three developmental stages: embryonic (YD), juvenile (2-year-old, MD), and mature (8-year-old, OD). Our analysis identified numerous differentially methylated genes that play pivotal roles in skin collagen deposition and overall skin maturation, including but not limited to COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, GLUL, SFRP2, FOSL1, SERPINE1, MMP1, MMP2, MMP9, and MMP13. Notably, we observed an inverse relationship between gene expression and DNA methylation proximal to transcription start sites (TSSs), whereas a direct correlation was detected in regions close to transcription termination sites (TTSs). Detailed bisulfite sequencing analyses of the COL1A1 promoter region revealed a low methylation status during the embryonic stage, correlating with elevated transcriptional activity and gene expression levels. Collectively, our findings elucidate key genetic markers associated with collagen deposition in the skin of Dezhou donkeys, underscoring the significant regulatory role of DNA methylation. This research work contributes to the foundational knowledge necessary for the genetic improvement and selective breeding of Dezhou donkeys, aiming to enhance skin quality attributes.

Hypomethylation-associated LINC00987 downregulation induced lung adenocarcinoma progression by inhibiting the phosphorylation-mediated degradation of SND1.

DNA methylation, an epigenetic regulatory mechanism dictating gene transcription, plays a critical role in the occurrence and development of cancer. However, the molecular underpinnings of LINC00987 methylation in the regulation of lung adenocarcinoma (LUAD) remain elusive. This study investigated LINC00987 expression in LUAD patients through analysis of The Cancer Genome Atlas data sets. Quantitative real-time polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization assays were used to assess LINC00987 expression in LUAD. The bisulfite genomic sequence PCR (BSP) assay was used to determine the methylation levels of the LINC00987 promoter. The interaction between LINC00987 and SND1 was elucidated via immunoprecipitation and RNA pull-down assays. The functional significance of LINC00987 and SND1 in Calu-3 and NCI-H1688 cells was evaluated in vitro through CCK-8, EdU, Transwell, flow cytometry, and vasculogenic mimicry (VM) tube formation assays. LINC00987 expression decreased in LUAD concomitant with hypermethylation of the promoter region, while hypomethylation of the LINC00987 promoter in LUAD tissues correlated with tumor progression. Treatment with 5-Aza-CdR augmented LINC00987 expression and inhibited tumor growth. Mechanistically, LINC00987 overexpression impeded LUAD progression and VM through direct binding with SND1, thereby facilitating its phosphorylation and subsequent degradation. Additionally, overexpression of SND1 counteracted the adverse effects of LINC00987 downregulation on cell proliferation, apoptosis, cell migration, invasion, and VM in LUAD in vitro. In conclusion, this pioneering study focuses on the expression and function of LINC00987 and reveals that hypermethylation of the LINC00987 gene may contribute to LUAD progression. LINC00987 has emerged as a potential tumor suppressor gene in tumorigenesis through its binding with SND1 to facilitate its phosphorylation and subsequent degradation.

Methylation entropy landscape of Chinese long-lived individuals reveals lower epigenetic noise related to human healthy aging.

The transition from ordered to noisy is a significant epigenetic signature of aging and age-related disease. As a paradigm of healthy human aging and longevity, long-lived individuals (LLI, >90 years old) may possess characteristic strategies in coping with the disordered epigenetic regulation. In this study, we constructed high-resolution blood epigenetic noise landscapes for this cohort by a methylation entropy (ME) method using whole genome bisulfite sequencing (WGBS). Although a universal increase in global ME occurred with chronological age in general control samples, this trend was suppressed in LLIs. Importantly, we identified 38,923 genomic regions with LLI-specific lower ME (LLI-specific lower entropy regions, for short, LLI-specific LERs). These regions were overrepresented in promoters, which likely function in transcriptional noise suppression. Genes associated with LLI-specific LERs have a considerable impact on SNP-based heritability of some aging-related disorders (e.g., asthma and stroke). Furthermore, neutrophil was identified as the primary cell type sustaining LLI-specific LERs. Our results highlight the stability of epigenetic order in promoters of genes involved with aging and age-related disorders within LLI epigenomes. This unique epigenetic feature reveals a previously unknown role of epigenetic order maintenance in specific genomic regions of LLIs, which helps open a new avenue on the epigenetic regulation mechanism in human healthy aging and longevity.

567 Multigenerational impacts on DNA methylation signatures in autism spectrum disorder

OBJECTIVES/GOALS: to investigate the potential impact of grandparental factors and multigenerational epigenetic inheritance on the development of ASD METHODS/STUDY POPULATION: Our study recruited participants from the CHARGE (Child Autism Risks from Genetics and the Environment) study, including grandparents, parents, and children. A questionnaire was used to gather information about the participants’ exposure to environmental factors. Saliva samples werecollected from 349 participants. Newborn dried blood spotsfrom probands and parents are still being collected from the California New born Registry. DNA was extracted from 349 saliva samples from 85 families and subjected to whole genome bisulfite sequencing (WGBS) to analyze DNA methylation. Sequence alignments and bioinformatic analyses will be performed using R packages called DMRichR and Comethyl. RESULTS/ANTICIPATED RESULTS: Sequence alignments and bioinformatic analyses are ongoing, utilizing DMRichR to identify individual genomic loci associated with ASD in each of the three generations and Comethyl to compare correlation patterns between methylation marks and selected variables, including grand parental exposures. New born blood spot collections of parents and probands are ongoing and will be used to identify potential ASD epigenomic signatures that are tissue and life-stage independent. DISCUSSION/SIGNIFICANCE: This research will provide new insights into the increased prevalence and underlying etiology of ASD that should pave the way for future research in the field. DNA Methylation signatures can help create molecular biomarkers which can be used together with behavioral clinical tests for diagnosis of ASD.

Abstract 3: Multiomics analyses reveal functional DNA, methylation pattern changes in clinical transgenic T-cell receptor cell therapy products

Abstract Background: Transgenic T-cell receptor T-cells (TCR-T) directed against tumor-specific antigens are associated with robust initial clinical responses. However, these responses are often not durable, and patients relapse frequently. Therefore, there is a significant need to understand how these cells change at the epigenomic level over time following infusion into the patient. Methods: We analyzed MART-1 TCR-T products from 8 patients treated under a clinical trial at UCLA (NCT00910650). Samples from the baseline infusion product and samples from day +30 recovered from peripheral circulation were sorted for TCR+ cells via flow cytometry, and were then subjected to whole genome bisulfite sequencing (WGBS), ATACseq, and RNAseq in parallel. Differential chromatin accessibility and gene expression by the ATACseq and RNAseq datasets, respectively, were analyzed via DESeq2, with P adjusted less than 0.1. WGBS datasets were analyzed via Metilene, and differentially methylated regions (DMRs) were defined as those regions with CpG loci containing a methylation difference >= 10% and a P adjusted < 0.05. Functional DMRs were defined as those repressed over time (increased DNA methylation with decreased chromatin accessibility/gene expression relative to infusion product), or activated over time (decreased DNA methylation with increased chromatin accessibility/gene expression relative to infusion product). Functional DMRs were then analyzed via EnrichR to evaluate gene ontology and pathways significantly enriched in gene lists. Results: We collectively identified three genes with repressed DMR activity and 49 genes with activated DMR activity compared to baseline infusion products. Our findings revealed that genes associated with repressed DMRs included IL2RA, FBLN7, and ZNRF1. These genes were collectively associated with a decrease in T-cell activation, cytokine signaling, and migration activity, potentially impacting the functional capabilities of T-cells in the post-infusion environment. Conversely, genes associated with activated DMRs included PDCD1 (PD1), FGR, KIR3DL1, KIR2DL4, and KLF3, with pathway enrichment analysis indicating a significant shift in the T-cell landscape towards reduced anti-tumor activity, decreased T-cell proliferation, T-cell aging, and the promotion of immune escape tumor phenotypes. Conclusions: This comprehensive multi-omics approach showcases an atlas of genes in clinical TCR-T cell therapeutics which are subject to functional changes in DNA methylation over time in vivo. These findings shed light on the complex interplay between DNA methylation, chromatin accessibility, and gene expression in the context of TCR-T cell therapy, with implications for the development of more effective immunotherapeutic strategies. Citation Format: Giulia Protti, Cole Peters, Moe Kawakami, Conner Kidd, Varsha Subramanyam, Analynn Lechien, Andrew Dinh, Antoni Ribas, Matteo Pellegrini, Theodore S. Nowicki. Multiomics analyses reveal functional DNA, methylation pattern changes in clinical transgenic T-cell receptor cell therapy products [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 3.

Abstract 7565: Tumor classification and deconvolution in liquid biopsy using enriched methylation sequencing

Abstract Background: Cell-free DNA (cfDNA) detected in proximal body fluids has demonstrated potential for cancer detection using minimally invasive methodology. Our past work showed that tumor cfDNA is present in the cerebrospinal fluid (CSF) and other body fluids of patients with inconclusive standard of care testing. However, past work measuring copy number aberrations or somatic mutations was limited in cancer classification. To facilitate reliable classification, even at low tumor fractions and with fragmented DNA, we developed XR-methylSeq, a methylation sequencing platform to enrich for cell type-specific markers. Methods: We benchmarked XR-methylSeq with the K562 cell line and correlated the methylation values with gold standard measurement − whole genome bisulfite sequencing (WGBS). Methylation classifiers were applied for at least 22 cytology-positive body fluids, incorporating methylation array data from public references. T-distributed stochastic neighbor embedding (t-SNE) analysis was used for visualization in R. Deconvolution of cell type fractions for at least 29 (seven cytology-negative) body fluids and plasma samples was conducted using wgbstools. Cell type-specific markers were identified from a human DNA methylation atlas. Results: Benchmarks: XR-methylSeq has a 5-fold enrichment of the cell type-specific markers compared with WGBS. XR-methylSeq at 20 ng input highly correlates with WGBS at 2 μg (Pearson’s r = 0.97). Body Fluids: Thirteen cytology-positive CSF samples had copy number aberrations, 77% of them had concordant tumor classification, while the remaining 23% clustered with low tumor fraction samples. All nine lung primaries, including a low tumor fraction case that did not originally classify, showed a consistent cell-of-origin through deconvolution, as indicated by increased contributions from lung alveolar epithelial cells. Among six other body fluids, three exhibited the highest fractions aligning with the clinically identified cancer cell-of-origin. Additionally, the plasma cfDNA of a patient with acute liver injury had a higher fraction of hepatocyte signatures (22%) than the healthy control (8%). Conclusions: This research highlights the potential of XR-methylSeq as an enriched methylation profiling method useful for liquid biopsy applications. Citation Format: Jingru Yu, Lauren S. Ahmann, Yvette Y. Yao, Angus Toland, Alicia Snowden, Chandler Ho, Benjamin Pinsky, Hannes Vogel, Ruben Y. Luo, Linlin Wang, Brooke Howitt, Brittany Holmes, Alarice C. Lowe, Wei Gu. Tumor classification and deconvolution in liquid biopsy using enriched methylation sequencing [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 7565.

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.

Cold exposure impacts DNA methylation patterns in cattle sperm.

DNA methylation is influenced by various exogenous factors such as nutrition, temperature, toxicants, and stress. Bulls from the Pacific Northwest region of the United States and other northern areas are exposed to extreme cold temperatures during winter. However, the effects of cold exposure on the methylation patterns of bovine sperm remain unclear. To address, DNA methylation profiles of sperm collected during late spring and winter from the same bulls were analyzed using whole genome bisulfite sequencing (WGBS). Bismark (0.22.3) were used for mapping the WGBS reads and R Bioconductor package DSS was used for differential methylation analysis. Cold exposure induced 3,163 differentially methylated cytosines (DMCs) with methylation difference ≥10% and a q-value < 0.05. We identified 438 differentially methylated regions (DMRs) with q-value < 0.05, which overlapped with 186 unique genes. We also identified eight unique differentially methylated genes (DMGs) (Pax6, Macf1, Mest, Ubqln1, Smg9, Ctnnb1, Lsm4, and Peg10) involved in embryonic development, and nine unique DMGs (Prmt6, Nipal1, C21h15orf40, Slc37a3, Fam210a, Raly, Rgs3, Lmbr1, and Gan) involved in osteogenesis. Peg10 and Mest, two paternally expressed imprinted genes, exhibited >50% higher methylation. The differential methylation patterns of six distinct DMRs: Peg10, Smg9 and Mest related to embryonic development and Lmbr1, C21h15orf40 and Prtm6 related to osteogenesis, were assessed by methylation-specific PCR (MS-PCR), which confirmed the existence of variable methylation patterns in those locations across the two seasons. In summary, cold exposure induces differential DNA methylation patterns in genes that appear to affect embryonic development and osteogenesis in the offspring. Our findings suggest the importance of replicating the results of the current study with a larger sample size and exploring the potential of these changes in affecting offspring development.

Epigenomic analyses identify FOXM1 as a key regulator of anti-tumor immune response in esophageal adenocarcinoma

Unlike most cancer types, the incidence of esophageal adenocarcinoma (EAC) has rapidly escalated in the western world over recent decades. Using whole genome bisulfite sequencing (WGBS), we identify the transcription factor (TF) FOXM1 as an important epigenetic regulator of EAC. FOXM1 plays a critical role in cellular proliferation and tumor growth in EAC patient-derived organoids and cell line models. We identify ERBB2 as an upstream regulator of the expression and transcriptional activity of FOXM1. Unexpectedly, gene set enrichment analysis (GSEA) unbiased screen reveals a prominent anti-correlation between FOXM1 and immune response pathways. Indeed, syngeneic mouse models show that FOXM1 inhibits the infiltration of CD8+ T cells into the tumor microenvironment. Consistently, FOXM1 suppresses CD8+ T cell chemotaxis in vitro and antigen-dependent CD8+ T cell killing. This study characterizes FOXM1 as a significant EAC-promoting TF and elucidates its novel function in regulating anti-tumor immune response.

Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T) results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2 mg folic acid/kg diet) and assess the effects of folic acid supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing and whole-genome bisulfite sequencing (WGBS). Reproductive parameters and locus-specific imprinted gene methylation were unaffected by genotype or diet. Using WGBS, sperm from 677TT mice had 360 differentially methylated tiles as compared to 677CC mice, predominantly hypomethylation (60% of tiles). Folic acid supplementation mostly caused hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic.

Comprehensive analysis of differentially methylated regions in colorectal cancer (CRC).

73 Background: Epigenetic alterations have been shown to govern many disease states, including cancer. A key epigenetic mark is the DNA methylation of CpGs. Large studies, like The Cancer Genome Atlas, have shown that DNA methylation can differentiate between cancer status and type. Here, differentially methylated genomic patterns were defined between patients with CRC and healthy individuals by utilizing patterns of co-methylation of adjacent CpG loci. Methods: Deep, whole genome bisulfite sequencing (WGBS) was conducted on DNA samples from 100 healthy individuals (median effective coverage of 352x±38x) and from 75 patients with CRC (median effective coverage of 48x±18x). The two cohorts were chosen to be balanced in terms of age (CRC: 58±11 years; healthy: 57±8 years) and to represent major ethnicities in the USA, and the CRC cohort was chosen to represent all stages, histologies and morphologies, and microsatellite instability (MSI, 23%). A novel machine learning model was developed to discover differentially methylated regions between the healthy and CRC cohorts as well as between subpopulations within the CRC cohort, including age, sex, stage, histology, and MSI vs microsatellite stability (MSS). Results: In total, we discovered 132,911 differentially methylated regions between healthy and CRC cohorts, spanning more than 1.14 million CpGs. 76% of all discovered regions were hypermethylated (70% CpG islands, 1.3% CpG shelves, 21% CpG shores) and the rest were hypomethylated (7.3% CpG islands, 8.6% CpG shelves, 25% CpG shores). We also discovered 220 hypermethylated regions that were differentially methylated between CRC MSI samples and CRC MSS samples and healthy cfDNA samples. Furthermore, we discovered subtype-specific differentially methylated regions, such as 62 hyper-methylated regions that were differentially methylated between mucinous adenocarcinoma samples and the rest of the cohort. Conclusions: Based on deep WGBS data from heterogeneous cohorts, representative of the US population eligible for CRC screening, we identified many differentially methylated regions as strong biomarker candidates for the early detection of cancer. Compared to recurrently mutated driver genes in CRC, we find that differential methylation profiles of CRC samples are generally much more homogenous and are therefore ideal biomarkers for targeted sequencing panels with the purpose of early cancer detection. Further, analysis of these patterns based on clinicopathologic factors enabled discovery of subtype specific markers, which can be incorporated to ensure equivalent performance for underrepresented subtypes or subpopulations.

Pancreatic cancer acquires resistance to MAPK pathway inhibition by clonal expansion and adaptive DNA hypermethylation

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. It is marked by extraordinary resistance to conventional therapies including chemotherapy and radiation, as well as to essentially all targeted therapies evaluated so far. More than 90% of PDAC cases harbor an activating KRAS mutation. As the most common KRAS variants in PDAC remain undruggable so far, it seemed promising to inhibit a downstream target in the MAPK pathway such as MEK1/2, but up to now preclinical and clinical evaluation of MEK inhibitors (MEKi) failed due to inherent and acquired resistance mechanisms. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition, we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data.ResultsWe found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. Furthermore, MEKi resistance could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells.ConclusionOverall, the concept of acquired therapy resistance as a result of the expansion of a single cell clone with epigenetic plasticity sheds light on genetic, epigenetic and phenotypic patterns during evolvement of treatment resistance in a tumor with high adaptive capabilities and provides potential for reversion through epigenetic targeting.

Abstract C111: Whole genome bisulfite sequencing of human and mouse organoid models of PDAC progression identifies stage- and subtype-specific DNA methylation signatures

Abstract The genetic underpinnings of PDAC progression from normal ductal cells to pancreatic intraepithelial neoplasms (PanINs) and primary tumors have been uncovered through the identification of common somatic point mutations and copy number variants in exome sequencing studies. However, attempts to identify recurrent metastasis-specific genetic mutations have been unsuccessful, suggesting that late-stage disease progression is predominantly driven by epigenetic alterations. Previous genome-wide DNA methylation studies have made it increasingly clear that DNA methylation is an important regulator of PDAC molecular pathobiology with impacts on clinical phenotypes and patient survival. While providing invaluable insights into functional roles of aberrant DNA methylation in PDAC, these studies were limited due to use of microarrays which assay less than 5% of CpG sites in the human genome, use of primary tumor samples with a high stromal content that can interfere with detection of tumor cell specific DNA methylation changes, and/or a limited sample size. To assay a larger portion of the PDAC methylome without stromal interference, we performed whole genome bisulfite sequencing (WGBS) analysis on a panel of 35 patient-derived organoid (PDO) models and 12 mouse organoid models of PDAC. Thus, this study is the first comprehensive analysis of genome-wide DNA methylation landscape in PDAC progression. With this approach, we assayed over 70% of the CpGs in the human and mouse genome and provide novel insight into the role of DNA methylation in PDAC progression and subtype differentiation. First, we uncovered thousands of DMRs that distinguish metastases from primary tumors and preneoplastic tissue in mouse organoids, early-stage primary tumors from late-stage primary tumors in PDOs, and primary tumors from normal pancreata in PDOs. Second, we showed that these stage-specific DMRs are enriched in promoters and bivalent enhancers and are associated with genes related to neurodevelopment and Ras signaling. Third, we identified DMRs whose DNA methylation correlate with expression of genes that have prognostic value. Lastly, we demonstrated that squamous and progenitor core gene programs are differentially methylated and separate the two subtypes based on their DNA methylation profiles. Together, these results show that DNA methylation changes underlie the disease stages and molecular subtypes of PDAC, resulting in significant and reoccurring DNA methylation patterns with diagnostic and prognostic potential. Citation Format: Sarah S. Wang, Madison Hall, Eun Jung Lee, Neha Ramesh, Sang Hyub Lee, Jin-Young Jang, Ja-Lok Ku, Chang-Il Hwang. Whole genome bisulfite sequencing of human and mouse organoid models of PDAC progression identifies stage- and subtype-specific DNA methylation signatures [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C111.

The role of DNA methylation reprogramming during sex determination and sex reversal in the Pacific oyster Crassostrea gigas

DNA methylation is instrumental in vertebrate sex reversal. However, the mechanism of DNA methylation regulation regarding sex reversal in invertebrates is unclear. In this study, we used whole genome bisulfite sequencing (WGBS) to map single-base resolution methylation profiles of the Pacific oyster, including female-to-male (FMa-to-FMb) and male-to-female (MFa-to-MFb) sex reversal, as well as sex non-reversed males and females (MMa-to-MMb and FFa-to-FFb). The results showed that global DNA methylation levels increase during female-to-male sex reversals, with a particular increase in the proportion of high methylation levels (mCGs >0.75) and a decrease in the proportion of intermediate methylation levels (0.25 < mCGs <0.75). This increase in DNA methylation was mainly associated with the elevated expression of DNA methylase genes. Genome-wide methylation patterns of females were accurately remodeled to those of males after sex reversal, while the opposite was true for the male-to-female reversal. Those findings directly indicate that alterations in DNA methylation play a significant role in sex reversal in Pacific oysters. Comparative analysis of the DNA methylomes of pre- and post- sex reversal gonadal tissues (FMb-vs-FMa or MFb-vs-MFa) revealed that differentially methylated genes were mainly involved in the biological processes of sex determination or gonadal development. However critical genes such as Dmrt1, Foxl2 and Sox-like, which are involved in the putative sex determination pathway in Pacific oysters, showed almost an absence of methylation modifications, varying greatly from vertebrates. Additionally, comparative analysis of the DNA methylomes of sexual reversal and sex non-reversal (FMa-vs-FFa or MFa-vs-MMa) revealed that heat shock protein genes, such as Hsp68-like and Hsp70B, were important for the occurrence of sex reversal. These findings shed light on the epigenetic mechanisms underlying the maintenance of gonadal plasticity and the reversal of organ architecture in oysters.

SarCTAB: an efficient and cost-effective DNA isolation protocol from geophytes.

Geophytes are herbaceous plants that grow anew from underground buds and are excellent models to study storage organ formation. However, molecular studies involving geophytes are constrained due to the presence of a wide spectrum of polysaccharides and polyphenols that contaminate the genomic DNA. At present, several protocols exist for the extraction of genomic DNA from different plant species; however, isolating high-quality DNA from geophytes is challenging. Such challenges are further complexed by longer incubation time and multiple precipitation steps involved in existing DNA isolation methods. To overcome such problems, we aimed to establish a DNA extraction method (SarCTAB) which is an economical, quick, and sustainable way of DNA isolation from geophytes. We improved the traditional CTAB method by optimizing key ingredients such as sarcosine, β-mercaptoethanol, and high molar concentration of sodium chloride (NaCl), which resulted in high concentration and good-quality DNA with lesser polysaccharides, proteins, and polyphenols. This method was evaluated to extract DNA from storage organs of six different geophytes. The SarCTAB method provides an average yield of 1755ng/µl of high-quality DNA from 100mg of underground storage tissues with an average standard purity of 1.86 (260/280) and 1.42 (260/230). The isolated genomic DNA performed well with Inter-simple sequence repeat (ISSR) amplification, restriction digestion with EcoRI, and PCR amplification of plant barcode genes viz. matK and rbcL. Also, the cost involved in DNA isolation was low when compared to that with commercially available kits. Overall, SarCTAB method works effectively to isolate high-quality genomic DNA in a cost-effective manner from the underground storage tissues of geophytes, and can be applied for next-generation sequencing, DNA barcoding, and whole genome bisulfite sequencing.

DNA methylation remodeled amino acids biosynthesis regulates flower senescence in carnation (Dianthus caryophyllus).

Dynamic DNA methylation regulatory networks are involved in many biological processes. However, how DNA methylation patterns change during flower senescence and their relevance with gene expression and related molecular mechanism remain largely unknown. Here, we used whole genome bisulfite sequencing to reveal a significant increase of DNA methylation in the promoter region of genes during natural and ethylene-induced flower senescence in carnation (Dianthus caryophyllus L.), which was correlated with decreased expression of DNA demethylase gene DcROS1. Silencing of DcROS1 accelerated while overexpression of DcROS1 delayed carnation flower senescence. Moreover, among the hypermethylated differentially expressed genes during flower senescence, we identified two amino acid biosynthesis genes, DcCARA and DcDHAD, with increased DNA methylation and reduced expression in DcROS1 silenced petals, and decreased DNA methylation and increased expression in DcROS1 overexpression petals, accompanied by decreased or increased amino acids content. Silencing of DcCARA and DcDHAD accelerates carnation flower senescence. We further showed that adding corresponding amino acids could largely rescue the senescence phenotype of DcROS1, DcCARA and DcDHAD silenced plants. Our study not only demonstrates an essential role of DcROS1-mediated remodeling of DNA methylation in flower senescence but also unravels a novel epigenetic regulatory mechanism underlying DNA methylation and amino acid biosynthesis during flower senescence.

DNA methylation and gene expression profiling reveal potential association of retinol metabolism related genes with hepatocellular carcinoma development.

Aberrant DNA methylation patterns play a critical role in the development of hepatocellular carcinoma (HCC). However, the molecular mechanisms associated with these aberrantly methylated genes remain unclear. This study aimed to comprehensively investigate the methylation-driven gene expression alterations in HCC using a multi-omics dataset. Whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) techniques were used to assess the methylation and gene expression profiles of HCC tissues (HCCs) and normal adjacent tissues (NATs). The candidate genes' potential function was further investigated using single-cell RNA sequencing (scRNA seq) data. We observed widespread hypomethylation in HCCs compared to NATs. Methylation levels in distinct genomic regions exhibited significant differences between HCCs and NATs. We identified 247,632 differentially methylated regions (DMRs) and 4,926 differentially expressed genes (DEGs) between HCCs and NATs. Integrated analysis of DNA methylation and RNA-seq data identified 987 methylation-driven candidate genes, with 970 showing upregulation and 17 showing downregulation. Four genes involved in the retinol metabolic pathway, namely ADH1A, CYP2A6, CYP2C8, and CYP2C19, were identified as hyper-downregulated genes. Their expression levels could stratify HCCs into three subgroups with distinct survival outcomes, immune cell infiltration, and tumor microenvironments. Validation of these findings in an independent dataset yielded similar outcomes, confirming the high concordance and potential prognostic value of these genes. ScRNA seq data revealed the low expression of these genes in immune cells, emphasizing their role in promoting malignant cell proliferation and migration. In conclusion, this study provides insights into the molecular characteristics of HCC, revealing the involvement of retinol metabolism-related genes in the development and progression of HCC. These findings have implications for HCC diagnosis, prognosis prediction, and the development of therapeutic targets.

Multi ‐ omics studies of eye and lens development

Early eye development involves formation of a symmetric pair of optic vesicles from the anterior neuroectoderm and parallel formation of closely apposed lens placodes. Their reciprocal invaginations generate the 3D‐foundation of the optic cups. Embryonic lens development is initiated within the border between the anterior neuroectoderm and naïve ectoderm, called the anterior pre‐placodal region. The earliest stages of lens development require coordinated actions of DNA‐binding transcription factor Pax6, signalling growth factor BMP4 and a pair of chromatin remodelling enzymes p300 and CBP. Studies in model vertebrates postulated a common Pax6+ progenitor cell population to generate both the lens and olfactory placodes. Recent progress in technologies to probe chromatin structure, landscape of transcription factor binding, DNA methylation, coding (mRNA) and noncoding (e.g. eRNAs and ncRNAs) transcriptomes and chromatin conformation have been shown to generate unprecedented mechanistic insights into the earliest cell fate decisions and tissue organization. To examine lens development at E8.5, E9.5 and E10.5 mouse embryos, single cell RNA‐seq (scRNA‐seq) experiments using the 10XGenomics Chromium platform were conducted. E9.5 Pax6 null embryos and controls were also included. Whole genome bisulfite sequencing (WGBS) was performed in E14.5 and P0.5 microdissected mouse lenses to map DNA methylation and integrate the data with corresponding ATAC‐ and RNA‐seq data sets. Open chromatin and DNA methylation changes were defined through identification of differentially accessible regions (DARs), unmethylated/low methylated regions (UMRs/LMRs) coupled with differential gene expression during lens differentiation. To probe 3D‐organization of lens chromatin and to identify temporally and spatially regulated lens‐specific distal enhancers, Hi‐C was used in conjunction with ChIP‐seq analysis of transcription factor CTCF in microdissected newborn lenses. Previous ChIP‐seq data using whole newborn lenses include H3K27ac peaks to identify super‐enhancers. Pax6 proteins were found both in open and closed chromatin domains, including both closed and methylated regions. In vitro binding of Pax6 proteins was not inhibited by 5‐methylcytosines within one or two CpG dinucleotides present in the Pax6 binding sites. In summary, cell fate decisions are governed by complex changes in chromatin landscape controlled by sequence‐specific DNA‐binding transcription factors and their recruitment of chromatin remodelling complexes. A combination of different –omics is a powerful approach to examine molecular mechanisms governing eye development. These studies also reveal additional levels of genetic code complexity and stimulate follow up mechanistic studies of the individual cellular and molecular processes of lens morphogenesis and their underlying GRNs and identification of cataract‐causing mutation in non‐coding regulatory sequences.

Functional Validation and Promoter DNA Methylation Analysis of the OfPAO Gene of Osmanthus fragrans ‘Yinbi Shuanghui’

Osmanthus fragrans ‘Yinbi Shuanghui’ is a colored leaf variety of O. fragrans. To study the mechanism of leaf color formation in O. fragrans ‘Yinbi Shuanghui’, we selected green and colored leaves with two different parts, namely yellow and green, as our research materials. We analyzed the expression changes related to leaf color in genes by performing qRT-PCR in the different leaf parts, finding that OfPAO was significantly up-regulated in the yellow part of colored leaves, and we initially determined that OfPAO was the key gene involved in the formation of colored leaves. Then, we constructed an OfPAO overexpression vector, before transforming it into tobacco through an Agrobacterium-mediated transformation to obtain transgenic plants. We found that the transgenic tobacco leaf color of OfPAO was lighter than that of the null carrier, the chlorophyll content in leaves decreased, and the expression of genes involved in the chlorophyll degradation pathway in OfPAO transgenic tobacco was up-regulated, suggesting that OfPAO regulates chlorophyll degradation, leading to changes in leaf color. According to the results of transcriptome sequencing and the genome data of O. fragrans ‘Rixianggui’, we cloned CDS and the promoter sequence of OfPAO, and the promoter regions 901-1307 of the OfPAO were sequenced through bisulfite genomic sequencing PCR (BSP), finding that the methylation level of CHH in the yellow part of colored leaves was lowest in colored and green leaves at 145 bp. The methylation of CHH in the promoter of OfPAO in O. fragrans ‘Yinbi Shuanghui’ was negatively correlated with the gene expression level, suggesting that the methylation of the promoter of OfPAO may regulate the expression of OfPAO, affecting chlorophyll degradation in the leaves.

High-resolution DNA methylation screening of the major histocompatibility complex in multiple sclerosis.

The HLA-DRB1 gene in the major histocompatibility complex (MHC) region in chromosome 6p21 is the strongest genetic factor identified as influencing multiple sclerosis (MS) susceptibility. DNA methylation changes associated with MS have been consistently detected at the MHC region. However, understanding the full scope of epigenetic regulations of the MHC remains incomplete, due in part to the limited coverage of this region by standard whole genome bisulfite sequencing or array-based methods. We developed and validated an MHC capture protocol coupled with bisulfite sequencing and conducted a comprehensive analysis of the MHC methylation landscape in blood samples from 147 treatment naïve MS study participants and 129 healthy controls. We identified 132 differentially methylated region (DMRs) within MHC region associated with disease status. The DMRs overlapped with established MS risk loci. Integration of the MHC methylome with human leukocyte antigen (HLA) genetic data indicate that the methylation changes are significantly associated with HLA genotypes. Using DNA methylation quantitative trait loci (mQTL) mapping and the causal inference test (CIT), we identified 643 cis-mQTL-DMRs paired associations, including 71 DMRs possibly mediating causal relationships between 55 single nucleotide polymorphisms (SNPs) and MS risk. The results describe MS-associated methylation changes in MHC region and highlight the association between HLA genotypes and methylation changes. Results from the mQTL and CIT analyses provide evidence linking MHC region variations, methylation changes, and disease risk for MS.