Differential Methylation Patterns Research Articles

Leukocyte-specific DNA methylation biomarkers and their implication for pathological epigenetic analysis

BackgroundDistinct cell types can be identified by their DNA methylation patterns. Much research over the last decade has focused on DNA methylation changes in cancer or the use of cell-free circulating DNA in plasma to identify damaged tissue in cases of trauma or organ transplantation. However, there has been little research into the differential methylation patterns between leukocytes and other tissues and how they can be used as a detection tool for immune activity in a range of contexts.ResultsWe have identified several loci that are fully methylated in leukocytes but virtually devoid of methylation in a range of other mesoderm-, ectoderm-, and endoderm-derived tissues. We validated these biomarkers using amplicon-bisulphite-sequencing on saliva and in vitro mixing of peripheral blood mononuclear cells and intestinal organoid cells combined at a defined range of ratios. Interestingly, these methylation biomarkers have previously been identified as altered in various inflammatory diseases, including Alzheimer disease, inflammatory bowel disease, and psoriasis. We hypothesise this is due to leukocyte infiltration rather than being a feature of the diseased cells themselves. Moreover, we show a positive linear relationship between infiltrating leukocytes and DNA methylation levels at the HOXA3 locus in six cancer types, indicative of further immune cell infiltration.ConclusionsOur data emphasise the importance of considering cellular composition when undertaking DNA methylation analysis and demonstrate the feasibility of developing new diagnostic tests to detect inflammation and immune cell infiltration.

Embryonic heat conditioning in chicks induces transgenerational heat/immunological resilience via methylation on regulatory elements

The question of whether behavioral traits are heritable is under debate. An obstacle in demonstrating transgenerational inheritance in mammals originates from the maternal environment's effect on offspring phenotype. Here, we used in ovo embryonic heat conditioning (EHC) of first-generation chicks, demonstrating heredity of both heat and immunological resilience, confirmed by a reduced fibril response in their untreated offspring to either heat or LPS challenge. Concordantly, transcriptome analysis confirmed that EHC induces changes in gene expression in the anterior preoptic hypothalamus (APH) that contribute to these phenotypes in the offspring. To study the association between epigenetic mechanisms and trait heritability, DNA-methylation patterns in the APH of offspring of control versus EHC fathers were evaluated. Genome-wide analysis revealed thousands of differentially methylated sites (DMSs), which were highly enriched in enhancers and CCCTC-binding factor (CTCF) sites. Overlap analysis revealed 110 differentially expressed genes that were associated with altered methylation, predominantly on enhancers. Gene-ontology analysis shows pathways associated with immune response, chaperone-mediated protein folding, and stress response. For the proof of concept, we focused on HSP25 and SOCS3, modulators of heat and immune responses, respectively. Chromosome conformational capture (3C) assay identified interactions between their promoters and methylated enhancers, with the strongest frequency on CTCF binding sites. Furthermore, gene expression corresponded with the differential methylation patterns, and presented increased CTCF binding in both hyper- and hypomethylated DMSs. Collectively, we demonstrate that EHC induces transgenerational thermal and immunological resilience traits. We propose that one of the mechanisms underlying inheritance depends on three-dimensional (3D) chromatin reorganization.

Abstract 3632: Differential DNA methylation in the benign and cancerous prostate tissue of African American and European American men

Abstract In this study, we compare patterns of differential DNA methylation (DNAm) distinguishing tumor and benign prostate tissue in European American (EA) and African American (AA) men to improve our understanding of biological mechanisms that may underlie prostate cancer (PCa) disparities. In the United States, AA men are more likely to be diagnosed with and to die of PCa compared with EA men. Changes in DNAm accompany tumor development and progression, and differences in DNAm across ancestry groups may be an important lens through which to study PCa disparities. We collected paired tumor and histologically benign formalin-fixed paraffin-embedded tissue blocks from 151 (76 AA, 75 EA) PCa patients undergoing prostatectomy at the University of Chicago. DNA was extracted and genome-wide DNAm was measured at ~850,000 CpG sites using the Illumina MethylationEpic Array. After quality control, 682,694 CpGs remained for analysis. We conducted differential methylation analyses, adjusting for batch, age, and individual and identified 29,236 and 38,035 tumor-associated CpGs in AA and EA respectively (p<5x10-8 and Δβ>0.3) with 25,263 CpGs common to both sets. There was a depletion of differentially methylated CpGs in CpG islands and promoters in both groups, but depletion was stronger in AA. Tumor-associated CpGs within islands and in promoters were more likely to be hypermethylated for both groups, though this was stronger in EA. We identified 2,392 genes with differentially methylated promoters common to both ancestry groups; 223 and 1,045 were unique to AA and EA respectively. Shared gene regions included GSTP1, APC, RARB, and GRASP, which have been previously reported. We assigned CpG sites to genes and used Gene Set Enrichment Analysis to identify differentially methylated pathways. The identified pathways include several previously associated with cancer development including the epithelial mesenchymal transition. We also identified ancestry-specific pathways including the early and late estrogen response pathways in AA. Examining the predictive ability of our differentially methylated CpGs, we found that relatively few CpGs (~10) were sufficient to distinguish tumor and benign tissue in AA and EA men (AUC>0.9). Scores constructed from differentially methylated CpGs in EA were able to predict tumor vs benign in AA (and vice versa) with high accuracy. Finally, we examined the association between ancestry and DNAm in both tumor and benign tissue. Here, 89 and 423 ancestry-associated CpGs were identified in tumor and benign respectively. Overall, we find that differential methylation patterns distinguishing tumor and benign tissue are generally similar for EA and AA men. However, differenced in tumor-associated DNAm as well as the presence of ancestry-associated CpGs provide a rich area for future studies exploring the impact of these differences on cancer development in AA and EA men. Citation Format: Meytal B. Chernoff, Marc Gillard, Kathryn Demanelis, Dayana Delgado, Anthony Williams, Donald Vander Griend, Brandon L. Pierce. Differential DNA methylation in the benign and cancerous prostate tissue of African American and European American men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3632.

Abstract 3675: Epigenetic variations associated with therapy resistance among prostate tumors from Puerto Rican men

Abstract Prostate cancer (PCa) is the leading cause of cancer-related death in Puerto Rican (PR) men, a population at higher risk of developing the disease than other Hispanic/Latino groups. But the relationship between this phenomenon and epigenetics, and how this relationship explains PCa racial and ethnic health disparities are controversial. The purpose of this study was to expand on the knowledge regarding epigenetic differences in terms of DNA methylation to enable risk stratification and treatment selection in this genetically admixed population. In addition, we aim to assess differential DNA methylation patterns associated with drug resistance in PCa in PR Hispanic/Latino men. Since epigenetic changes and modifications represent critical components of carcinogenesis and progression, we are expecting differential methylation levels between aggressive vs. indolent, and resistant vs. non-resistant cases. Prostate tumors from PR men were stratified based on Gleason score. Methylation levels were measured by Illumina Infinium Methylation EPIC BeadChip DNA methylation platform, and bioinformatic analyses. In parallel, DNA methylation profiles were also analyzed using genomic DNA from PCa cells selected for resistance to androgen depletion, enzalutamide resistance, or docetaxel resistance. We identified 1,225 differentially methylated genes in prostate tumors compared with normal adjacent tissues, and 141 differentially methylated genes between aggressive vs. indolent tumors. Additionally, 723 differentially methylated genes were found in therapy resistant cell lines compared to parental controls. Analysis of the methylated genes obtained from aggressive prostate tumors from Puerto Rican patients and therapy-resistant PCa cell lines revealed four overlapping methylated genes: RNF220, FAM65B, PRDM16, and DGKH. Further analysis of PCa cells resistant to androgen depletion, enzalutamide, or docetaxel demonstrated impaired cellular migration of resistant phenotypes upon DNA methyltransferase (DNMT) inhibition using 5-azacytidine or zebularine compared to parental cell lines. DNMT1 was associated with resistance in PCa cell lines, and future studies will include silencing of methyltransferases will evaluation of drug re-sensitization. These findings provide critical insight into the epigenetic landscape of aggressive prostate tumors among PR men and highlight several candidate methylation biomarkers for assessing risk in this population. Citation Format: Carlos J. Diaz Osterman, Anders Berglund, Shannalee Martinez, Camila Luis, Julie Dutil, Jaime Matta, Jarline Encarnacion-Medina, Carmen Ortiz-Sanchéz, Liang Wang, Jasreman Dhillon, Youngchul Kim, Hyun Y. Park, Gilbert Ruiz-Deya, Kosj Yamoah, Jong Y. Park. Epigenetic variations associated with therapy resistance among prostate tumors from Puerto Rican men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3675.

Abstract 2499: Identifying DNA methylation signatures of retinoblastoma via aqueous humor, a novel ocular liquid biopsy

Abstract Introduction: Retinoblastoma (Rb) is the most common childhood intraocular cancer. Tissue biopsy of Rb can cause tumor spread, so it is contraindicated. We demonstrated that aqueous humor (AH), an ocular fluid, is a high yield liquid biopsy enabling in vivo detection of tumor-derived cell free DNA (cfDNA) thus overcoming the contraindication to biopsy. Prognostic genomic cfDNA biomarkers in Rb AH enables objective real time disease monitoring. In ~13% of Rb, tumor progression is driven by epigenetic deregulation of tumor-promoting pathways without detectable genomic alterations. However, epigenetic studies have been done only on tumor from surgically removed eyes. The frequency and effect of epigenomic regulation in eyes that have been saved with therapy is not clear due to no access to in vivo tumor. Therefore, epigenetic analysis of AH cfDNA is highly desired to understand the broader spectrum of Rb tumorigenesis and prognosis. Materials and Methods: 16 AH samples and 4 Rb tumors from 12 patients with 14 Rb eyes were included in the study. We conducted global DNA methylation profiling of tumor tissues from surgically removed Rb eyes, AH samples collected from different clinical stages with different treatment outcomes using the Illumina Infinium EPIC DNA methylation BeadArray platform. Publicly available DNA methylation data of normal retina, Rb eyes, and Rb patients were obtained from Gene Expression Omnibus (GEO, GSE57362) for cell type DNA methylation comparisons. Results: Our preliminary studies revealed a high degree of concordance in genome-wide differential DNA methylation patterns between paired AH and tumor samples. Integrating our data with large public datasets, we identified reliable RB DNA methylation signatures in cfDNA that have potential diagnostic and prognostic values. We also identified hypermethylation at RB1 promoter, which suggested RB1 is druggable to DNA methylation inhibitors. By integrating DNA methylation data with gene expression data, we identified over 300 differentially expressed genes potentially regulated by DNA methylation change. Pathway analysis indicated several tumor suppressor pathways (e.g., RB1 and P53 pathways) were suppressed and some oncogenic pathways were activated (e.g., E2F pathways). Conclusions: Our study sets the stage for exploiting epigenetic markers in AH and identifying potential therapeutic targets to improve the clinical management of patients with RB. Citation Format: Liya Xu, Hongtao Li, Dan Weisenberger, Gangning Liang, Jesse Berry. Identifying DNA methylation signatures of retinoblastoma via aqueous humor, a novel ocular liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2499.

Cell-Free DNA in Dermatology Research

In various diseases, particularly cancer, cell-free DNA (cfDNA) has been widely studied as a marker of disease prognosis or to facilitate the detection of therapeutic targets. In dermatology, most studies have focused on melanoma; other skin diseases such as vascular malformations and psoriasis have also been examined. Genetic alterations unique to the tissue of origin such as sequence variations, copy number alterations, chromosomal rearrangements, differential DNA methylation patterns, and fragmentation patterns can be identified in circulation providing information on patient disease status. These alterations can be detected either by PCR-based methods or next-generation sequencing depending on the target of interest. In this article, we discuss the origins of cfDNA, the most common methods of detection, current studies assessing cfDNA as a biomarker, and cfDNA's potential clinical applications in melanoma and other skin diseases. In addition, we provide important factors to consider during blood processing and DNA extraction as well as limitations for each assay.

Moderate DNA methylation changes associated with nitrogen remobilization and leaf senescence in Arabidopsis.

The lifespan of plants is restricted by environmental and genetic components. Following the transition to reproductive growth, leaf senescence ends cellular life in monocarpic plants to remobilize nutrients to storage organs. In Arabidopsis, we initially observed altered leaf to seed ratios, faster senescence progression, altered leaf nitrogen recovery after transient nitrogen removal, and ultimately enhanced nitrogen remobilization from the leaves in two methylation mutants (ros1 and the triple dmr1/2 cmt3 knockout). Analysis of the DNA methylome in wild type Col-0 leaves identified an initial moderate decline of cytosine methylation with progressing leaf senescence, predominantly in the CG context. Late senescence was associated with moderate de novo methylation of cytosines, primarily in the CHH context. Relatively few differentially methylated regions, including one in the ROS1 promoter linked to down-regulation of ROS1, were present, but these were unrelated to known senescence-associated genes. Differential methylation patterns were identified in transcription factor binding sites, such as the W-boxes that are targeted by WRKYs. Methylation in artificial binding sites impaired transcription factor binding in vitro. However, it remains unclear how moderate methylome changes during leaf senescence are linked with up-regulated genes during senescence.

Integrated single cell RNA‐seq and ATAC‐seq of iPSC‐derived kidney organoids reveals the complexity of TGFβ signalling to chromatin during the origin of fibroblasts

Differential methylation patterns and changes in post‐translational histone modifications are observed in patients with or without progressive diabetic kidney disease (DKD). TGFβ1 plays a central regulatory role in the determination of renal cell fate and the progression of DKD. Using ChIP‐seq, we identified an association between SMAD3 and the histone methyltransferase, EZH2, in iPSCs and in iPSC derived nephron progenitors. Using the 10X Genomics platform, we performed single cell RNA‐seq and ‐ATAC‐seq on human iPSC‐derived kidney organoids treated with the EZH2 specific inhibitor, GSK343, prior to treatment with TGFβ1. Integration of single cell RNAseq and ‐ATACseq identified de novo ACTA2+ve/POSTN+ve myofibroblasts in TGFβ1‐treated kidney organoids, characterised by increased SMAD3‐dependent cis chromatin accessibility and the expression of genes associated with fibroblast activation, which are similarly overexpressed in DKD patients. This fibrosis‐associated regulon is characterised by enrichment of SMAD3 and AP‐1, corresponding to FOS/JUN motifs at accessible loci identified by scATACseq. Treatment with GSK343 blocked SMAD3‐dependent cis co‐accessibility and inhibited myofibroblast activation. We propose that that the enzymatic function of the polycomb repressive complex is necessary for TGFβ1 induced increase in chromatin accessibility and its subsequent gene regulatory functions. Manipulation of the association between SMAD3 and EZH2 may be a useful therapeutic strategy for the resolution of renal fibrosis.

Methylomic and transcriptomic characterization of postoperative systemic inflammatory dysregulation

In this study, we define and validate a state of postoperative systemic inflammatory dysregulation (PSID) based on postoperative phenotypic extremes of plasma C-reactive protein concentration following major abdominal surgery. PSID manifested clinically with significantly higher rates of sepsis, complications, longer hospital stays and poorer short, and long-term outcomes. We hypothesized that PSID will be associated with, and potentially predicted by, altered patterns of genome-wide peripheral blood mononuclear cell differential DNA methylation and gene expression. We identified altered DNA methylation and differential gene expression in specific immune and metabolic pathways during PSID. Our findings suggest that dysregulation results in, or from, dramatic changes in differential DNA methylation and highlights potential targets for early detection and treatment. The combination of altered DNA methylation and gene expression suggests that dysregulation is mediated at multiple levels within specific gene sets and hence, nonspecific anti-inflammatory treatments such as corticosteroids alone are unlikely to represent an effective therapeutic strategy.

An integrated analysis of the effects of maternal broccoli sprouts exposure on transcriptome and methylome in prevention of offspring mammary cancer.

Broccoli sprouts (BSp), a cruciferous vegetable, has shown promising effects on prevention of many types of cancer including breast cancer (BC). BC has a developmental foundation, and maternal nutrition status may influence an offspring’s risk to BC later in life. What is less understood, however, is the influence of maternal nutrition intervention on reversing epigenomic abnormalities that are essential in BC programming during early development. Our research focused on how maternal exposure to BSp diet prevents offspring BC and investigation of possible epigenetic mechanisms during these processes. Our results showed that maternal feeding of BSp can prevent mammary tumor development in the offspring of a transgenic mouse model. Through comprehensive integrated multi-omics studies on transcriptomic and methylomic analysis, we identified numerous target genes exhibiting significantly differential gene expression and DNA methylation patterns in the offspring mammary tumor. We discovered that maternal exposure to BSp diet can induce both gene and methylation changes in several key genes such as Avpr2, Cyp4a12b, Dpp6, Gria2, Pcdh9 and Tspan11 that are correlated with pivotal biological functions during carcinogenesis. In addition, we found an impact of maternal BSp treatment on DNA methyltransferase and histone deacetylases activity. Our study provides knowledgeable information regarding how maternal BSp diet influences key tumor-related gene expression and the epigenetic changes using a genome-wide perspective. Additionally, these findings provide mechanistic insights into the effectiveness of maternal BSp administration on the prevention of BC in the offspring later in life, which may lead to an early-life BC chemopreventive strategy that benefits the progenies’ long-term health.

DNA Methylation Associates With Clinical Courses of Atypical Meningiomas: A Matched Case-Control Study.

BackgroundAccounting for 15–20% of all meningiomas, WHO grade II meningiomas represent an intermediate group regarding risk of tumor recurrence. However, even within this subgroup varying clinical courses are observed with potential occurrence of multiple recurrences. Recently, DNA methylation profiles showed their value for distinguishing biological behaviors in meningiomas. Therefore, aim of this study was to investigate DNA methylation profiles in WHO grade II meningiomas.MethodsAll patients that underwent resection of WHO grade II meningiomas between 1993 and 2015 were screened for a dismal course clinical course with ≥2 recurrences. These were matched to control cases with benign clinical courses without tumor recurrence. DNA methylation was assessed using the Infinium Methylation EPIC BeadChip microarray. Unsupervised hierarchical clustering was performed for identification of DNA methylation profiles associated with such a dismal clinical course.ResultsOverall, 11 patients with WHO grade II meningiomas with ≥2 recurrences (Group dismal) and matched 11 patients without tumor recurrence (Group benign) were identified. DNA methylation profiles revealed 3 clusters—one comprising only patients of group dismal, a second cluster comprising mainly patients from group benign and a third cluster comprising one group dismal and one group benign patient. Based on differential methylation pattern associations with the Wnt and the related cadherin signaling pathway was observed.ConclusionDNA methylation clustering showed remarkable differences between two matched subgroups of WHO grade II meningiomas. Thus, DNA methylation profiles may have the potential to support prognostic considerations regarding meningioma recurrence and radiotherapeutic treatment allocation after surgical resection.

The progress on the estimation of DNA methylation level and the detection of abnormal methylation

BackgroundDNA methylation is a key heritable epigenetic modification that plays a crucial role in transcriptional regulation and therefore a broad range of biological processes. The complex patterns of DNA methylation highlight the significance of the profiling the DNA methylation landscape.ResultsIn this review, the main high‐throughput detection technologies are summarized, and then the three trends of computational estimation of DNA methylation levels were analyzed, especially the expanding of the methylation data with lower coverage. Furthermore, the detection methods of differential methylation patterns for sequencing and array data were presented.ConclusionsMore and more research indicated the great importance of DNA methylation changes across different diseases, such as cancers. Although a lot of enormous progress has been made in understanding the role of DNA methylation, only few methylated genes or functional elements serve as clinically relevant cancer biomarkers. The bottleneck in DNA methylation advances has shifted from data generation to data analysis. Therefore, it is meaningful to develop machine learning models for computational estimation of methylation profiling and identify the potential biomarkers.

Abstract P3-05-06: Genome-wide DNA methylation analysis identifies novel biomarkers associated with risk of relapse beyond oncotype DX recurrence-score risk assessment within HR+/HER2- early-stage breast cancer patients

Abstract Background: Early-stage, node-negative, hormone receptor positive (HR+) HER2 negative (HER2-) breast cancer (BC) patients comprise a subset with good prognosis. Based on gene expression panels, these patients may skip chemotherapy and complete adjuvant endocrine therapy as an individual genomic basis assessment. However, up to 15% of patients categorized as non-high risk by Oncotype DX (Recurrence Score, RS≤25) may experience disease relapse. The field of cancer epigenomics is emerging as a dynamic tool to complement prognostic expression analyses and to provide further insight into the biological underpinnings of early HR+/HER2- BC. Methods: A genome-wide DNA methylation profiling in tumor tissue samples, from node-negative HR+/HER2- BC patients with available Oncotype DX RS data, was analyzed using the Illumina MethylationEPIC 850K BeadChip. We evaluated all differential methylation patterns independently of their position to establish a bioinformatic analytical pipeline. The online tools KM Plotter and ROC Plotter were used to assess the impact of survival outcomes (5-year relapse-free survival, RFS) and response to therapy of the candidate genes. Individual patient data from TCGA was used to analyze the correlation between methylation and mRNA levels in HR+/HER2- BC patients. Statistical significance was defined by p<0.05; no multiple testing correction was applied. Results: 31 patients were evaluated (mean age 51.4, SD 13.3). Four samples were discarded after quality control analysis. Sixteen tumor patients presented a RS ≤25, of which 6 patients had experienced relapse with a median follow-up of 7.1 years. Of the 773,193 loci studied, significant differential methylation was observed between the relapse and non-relapse groups at 19665 cgs (2,5%). Genes were ranked according to differential methylation patterns, and those with Delta ≥|0.25|were selected. Ninety-nine genes met these criteria. Of them, 85 (86%) were hypomethylated and 14 (14%) were hypermethylated in the relapse group. We studied methylation-expression correlations and the impact on RFS in independent datasets. Among the hypomethylated genes, VAC14 (HR 0.71, CI 0.63-0.82, p 9.7e-7), NDUFS6 (HR 0.68, CI 0.59-0.76, p 1.5e-8), and C7orf50 (HR 0.74, 0.6-0.91, p 0.0059) were found significantly associated with a worse RFS and exhibited a negative correlation between methylation levels and gene expression. Among the hypermethylated genes, MFSD7 (HR 1.31, CI 1.15-1.49, p 6.1e-5), PLA2G3 (HR 1.15, CI 1.09-1.40, p 0.0016), and FGFR2 (HR 1.27, CI 1.11-1.45, p 3.6e-4) were significantly associated to a better RFS and showed a strong negative correlation. Additionally, NDUFS6 mRNA levels were found to be increased in patients with worse response to adjuvant chemotherapy (p 0.012, 1.5x higher; ROC 0.733), while C7orf50 expression levels was associated with a better outcome following adjuvant ET (p 0.012, 1.3x higher, ROC 0.795). Conclusion: In this exploratory study, 6 differentially methylated genes were identified to discriminate patients with non-high RS who may be at a higher risk of relapse. An effort is ongoing towards the development of an analytical pipeline including a more extensive annotation of both experimental and validation cohorts. Citation Format: Jesus Fuentes-Antrás, Vanesa García-Barberán, Nicolas Costa-Fraga, Fernando Moreno, Aida Bao-Caamano, Aitor Rodríguez-Casanova, Alfonso López de Sá, Alicia De Luna, Igor Lopez-Cade, Carmen Ramirez-Ruda, Alejando Pascual, Pedro Perez-Segura, Balázs Győrffy, Alberto Ocaña, Angel Diaz-Lagares, Jose Angel Garcia-Saenz. Genome-wide DNA methylation analysis identifies novel biomarkers associated with risk of relapse beyond oncotype DX recurrence-score risk assessment within HR+/HER2- early-stage breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-05-06.

Early fish domestication affects methylation of key genes involved in the rapid onset of the farmed phenotype

ABSTRACT Animal domestication is a process of environmental modulation and artificial selection leading to permanent phenotypic modifications. Recent studies showed that phenotypic changes occur very early in domestication, i.e., within the first generation in captivity, which raises the hypothesis that epigenetic mechanisms may play a critical role on the early onset of the domestic phenotype. In this context, we applied reduced representation bisulphite sequencing to compare methylation profiles between wild Nile tilapia females and their offspring reared under farmed conditions. Approximately 700 differentially methylated CpG sites were found, many of them associated not only with genes involved in muscle growth, immunity, autophagy and diet response but also related to epigenetic mechanisms, such as RNA methylation and histone modifications. This bottom-up approach showed that the phenotypic traits often related to domestic animals (e.g., higher growth rate and different immune status) may be regulated epigenetically and prior to artificial selection on gene sequences. Moreover, it revealed the importance of diet in this process, as reflected by differential methylation patterns in genes critical to fat metabolism. Finally, our study highlighted that the TGF-β1 signalling pathway may regulate and be regulated by several differentially methylated CpG-associated genes. This could be an important and multifunctional component in promoting adaptation of fish to a domestic environment while modulating growth and immunity-related traits.

Identification of Putative Markers of Non-infectious Bud Failure in Almond [Prunus dulcis (Mill.) D.A. Webb] Through Genome Wide DNA Methylation Profiling and Gene Expression Analysis in an Almond × Peach Hybrid Population.

Almond [Prunus dulcis (Mill.) D.A. Webb] is an economically important nut crop susceptible to the genetic disorder, Non-infectious Bud Failure (NBF). Despite the severity of exhibition in several prominent almond cultivars, no causal mechanism has been identified underlying NBF development. The disorder is hypothesized to be associated with differential DNA methylation patterns based on patterns of inheritance (i.e., via sexual reproduction and clonal propagation) and previous work profiling methylation in affected trees. Peach (Prunus persica L. Batsch) is a closely related species that readily hybridizes with almond; however, peach is not known to exhibit NBF. A cross between an NBF-exhibiting ‘Carmel’ cultivar and early flowering peach (‘40A17’) produced an F1 where ∼50% of progeny showed signs of NBF, including canopy die-back, erratic branching patterns (known as “crazy-top”), and rough bark. In this study, whole-genome DNA methylation profiles were generated for three F1 progenies exhibiting NBF and three progenies considered NBF-free. Subsequent alignment to both the almond and peach reference genomes showed an increase in genome-wide methylation levels in NBF hybrids in CG and CHG contexts compared to no-NBF hybrids when aligned to the almond genome but no difference in methylation levels when aligned to the peach genome. Significantly differentially methylated regions (DMRs) were identified by comparing methylation levels across the genome between NBF- and no-NBF hybrids in each methylation context. In total, 115,635 DMRs were identified based on alignment to the almond reference genome, and 126,800 DMRs were identified based on alignment to the peach reference genome. Nearby genes were identified as associated with the 39 most significant DMRs occurring either in the almond or peach alignments alone or occurring in both the almond and peach alignments. These DMR-associated genes include several uncharacterized proteins and transposable elements. Quantitative PCR was also performed to analyze the gene expression patterns of these identified gene targets to determine patterns of differential expression associated with differential DNA methylation. These DMR-associated genes, particularly those showing corresponding patterns of differential gene expression, represent key targets for almond breeding for future cultivars and mitigating the effects of NBF-exhibition in currently affected cultivars.

Regulation of Opsin Gene Expression by DNA Methylation and Histone Acetylation.

One important role of epigenetic regulation is controlling gene expression in development and homeostasis. However, little is known about epigenetics’ role in regulating opsin expression. Cell cultures (HEK 293, Y79, and WERI) producing different levels of opsins were treated with 5-aza-2’-deoxycytidine (5-Aza-dc) and/or sodium butyrate (SB) or suberoylanilide hydroxamic acid (SAHA) for 72 h. Global DNA methylation, site-specific methylation, and expressions of opsins were measured by LUMA assay, bisulfite pyrosequencing, and qPCR, respectively. Mouse retinal explants from wild-type P0/P1 pups were ex vivo cultured with/without 5-Aza-dc or SAHA for 6 days. The morphology of explants, DNA methylation, and expressions of opsins was examined. The drugs induced global DNA hypomethylation or increased histone acetylation in cells, including DNA hypomethylation of rhodopsin (RHO) and L-opsin (OPN1LW) and a concomitant increase in their expression. Further upregulation of RHO and/or OPN1LW in HEK 293 or WERI cells was observed with 5-Aza-dc and either SB or SAHA combination treatment. Mouse retinal explants developed normally but had drug-dependent differential DNA methylation and expression patterns of opsins. DNA methylation and histone acetylation directly regulate opsin expression both in vitro and ex vivo. The ability to manipulate opsin expression using epigenetic modifiers enables further study into the role of epigenetics in eye development and disease.

Epigenomics in trees: decoding the plastic lives of mangroves.

This article is a Commentary on Miryeganeh et al. (2022), 233: 2094–2110.

Tobacco carcinogen 4-[methyl(nitroso)amino]-1-(3-pyridinyl)-1-butanone (NNK) drives metabolic rewiring and epigenetic reprograming in A/J mice lung cancer model and prevention with diallyl sulphide (DAS).

Early detection of biomarkers in lung cancer is one of the best preventive strategies. Although many attempts have been made to understand the early events of lung carcinogenesis including cigarette smoking (CS) induced lung carcinogenesis, the integrative metabolomics and next-generation sequencing approaches are lacking. In this study, we treated the female A/J mice with CS carcinogen 4-[methyl(nitroso)amino]-1-(3-pyridinyl)-1-butanone (NNK) and naturally occurring organosulphur compound, diallyl sulphide (DAS) for 2 and 4 weeks after NNK injection and examined the metabolomic and DNA CpG methylomic and RNA transcriptomic profiles in the lung tissues. NNK drives metabolic changes including mitochondrial tricarboxylic acid (TCA) metabolites and pathways including Nicotine and its derivatives like nicotinamide and nicotinic acid. RNA-seq analysis and Reactome pathway analysis demonstrated metabolism pathways including Phase I and II drug metabolizing enzymes, mitochondrial oxidation and signaling kinase activation pathways modulated in a sequential manner. DNA CpG methyl-seq analyses showed differential global methylation patterns of lung tissues from week 2 versus week 4 in A/J mice including Adenylate Cyclase 6 (ADCY6), Ras-related C3 botulinum toxin substrate 3 (Rac3). Oral DAS treatment partially reversed some of the mitochondrial metabolic pathways, global methylation and transcriptomic changes during this early lung carcinogenesis stage. In summary, our result provides insights into CS carcinogen NNK's effects on driving alterations of metabolomics, epigenomics and transcriptomics and the chemopreventive effect of DAS in early stages of sequential lung carcinogenesis in A/J mouse model.

Risk-focused differences in molecular processes implicated in SARS-CoV-2 infection: corollaries in DNA methylation and gene expression

BackgroundUnderstanding the molecular basis of susceptibility factors to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global health imperative. It is well-established that males are more likely to acquire SARS-CoV-2 infection and exhibit more severe outcomes. Similarly, exposure to air pollutants and pre-existing respiratory chronic conditions, such as asthma and chronic obstructive respiratory disease (COPD) confer an increased risk to coronavirus disease 2019 (COVID-19).MethodsWe investigated molecular patterns associated with risk factors in 398 candidate genes relevant to COVID-19 biology. To accomplish this, we downloaded DNA methylation and gene expression data sets from publicly available repositories (GEO and GTEx Portal) and utilized data from an empirical controlled human exposure study conducted by our team.ResultsFirst, we observed sex-biased DNA methylation patterns in autosomal immune genes, such as NLRP2, TLE1, GPX1, and ARRB2 (FDR < 0.05, magnitude of DNA methylation difference Δβ > 0.05). Second, our analysis on the X-linked genes identified sex associated DNA methylation profiles in genes, such as ACE2, CA5B, and HS6ST2 (FDR < 0.05, Δβ > 0.05). These associations were observed across multiple respiratory tissues (lung, nasal epithelia, airway epithelia, and bronchoalveolar lavage) and in whole blood. Some of these genes, such as NLRP2 and CA5B, also exhibited sex-biased gene expression patterns. In addition, we found differential DNA methylation patterns by COVID-19 status for genes, such as NLRP2 and ACE2 in an exploratory analysis of an empirical data set reporting on human COVID-9 infections. Third, we identified modest DNA methylation changes in CpGs associated with PRIM2 and TATDN1 (FDR < 0.1, Δβ > 0.05) in response to particle-depleted diesel exhaust in bronchoalveolar lavage. Finally, we captured a DNA methylation signature associated with COPD diagnosis in a gene involved in nicotine dependence (COMT) (FDR < 0.1, Δβ > 0.05).ConclusionOur findings on sex differences might be of clinical relevance given that they revealed molecular associations of sex-biased differences in COVID-19. Specifically, our results hinted at a potentially exaggerated immune response in males linked to autosomal genes, such as NLRP2. In contrast, our findings at X-linked loci such as ACE2 suggested a potentially distinct DNA methylation pattern in females that may interact with its mRNA expression and inactivation status. We also found tissue-specific DNA methylation differences in response to particulate exposure potentially capturing a nitrogen dioxide (NO2) effect—a contributor to COVID-19 susceptibility. While we identified a molecular signature associated with COPD, all COPD-affected individuals were smokers, which may either reflect an association with the disease, smoking, or may highlight a compounded effect of these two risk factors in COVID-19. Overall, our findings point towards a molecular basis of variation in susceptibility factors that may partly explain disparities in the risk for SARS-CoV-2 infection.

Understanding the role of telomere attrition and epigenetic signatures in COVID-19 severity.

Within the past several decades, the emergence and spread of infectious diseases with pandemic potential have endangered human lives. Coronavirus disease 2019 (COVID-19) outbreak represents an unprecedented threat for all health systems worldwide. The clinical spectrum of COVID-19 is highly heterogeneous, ranging from asymptomatic and mild upper respiratory tract illness to severe interstitial pneumonia with respiratory failure and even death. Highly age-dependent patterns of immune response potentially explain the higher rates of the severe forms of COVID-19 in elderly patients. However, genetic and epigenetic architecture can influence multiple biological processes during the lifespan, therefore as far as our knowledge shows, vulnerability to viral infection concerning telomere length and epigenetic signature is not a new idea. This review aims is to summarize the current understanding of the role of telomere length and epigenetic mechanisms on the severity of COVID-19. The current knowledge highlights the significant association between the shorter telomere length and the higher risk of developing severe COVID-19. Differential DNA methylation patterns and miRNA expression profiles imply that these hallmarks can play a pivotal role in COVID- 19 pathogenesis. Understanding the causes of inter-individual variations in COVID-19 outcomes could provide clues to the development of the personalized therapeutic intervention.