Changes In Methylome Research Articles

Sodium arsenite-induced DNA methylation alterations exacerbated by p53 knockout in MCF7 cells

Epigenetic alterations are ubiquitous across human malignancies. Thus, functional characterization of epigenetic events deregulated by environmental pollutants should enhance our understanding of the mechanisms of carcinogenesis and inform preventive strategies. Recent reports showing the presence of known cancer-driving mutations in normal tissues have sparked debate on the importance of non-mutational stressors potentially acting as cancer promoters. Here, we aimed to test the hypothesis that the presence of mutations in p53, a commonly mutated gene in human malignancies, may influence cellular response to an environmental non-mutagenic agent, potentially involving epigenetic mechanism. We used the CRISPR-Cas9 system to generate knockouts of p53 in MCF7 and T47D breast cancer cell lines and characterized DNA methylome changes by targeted pyrosequencing and methylome-wide Infinium MethylationEPIC BeadChip arrays after exposure to sodium arsenite, a well-established human carcinogen with documented effects on the epigenome. We found that the knockout of p53 alone was associated with extensive alterations in DNA methylation content, with predominant CpG hypermethylation concurrent with global demethylation, as determined by LINE-1 repetitive element pyrosequencing. While exposure to sodium arsenite induced little to no effects in parental cell lines, mutant cells, upon treatment with sodium arsenite, exhibited a markedly altered response in comparison to their wild-type counterparts. We further performed genome regional analyses and found that differentially methylated regions (DMRs) associated with exposure to sodium arsenite map to genes involved in chromatin remodeling and cancer development. Reconstitution of wild-type p53 only partially restored p53-mutant-specific differential methylation states in response to sodium arsenite exposure, which may be due to the insufficient reconstitution of p53 function, or suggestive of a potential exposure-specific epigenetic memory. Together, our results revealed wide-spread epigenetic alterations associated with p53 mutation that influence cellular response to sodium arsenite exposure, which may constate an important epigenetic mechanism by which tumour promoting agents synergize with driver mutations in cancer promotion.

Maternal e-cigarette exposure alters DNA methylome, site-specific CpG and CH methylation, and transcriptomic signatures in the neonatal brain

Maternal use of e-cigarette (e-cig) aerosols poses significant risks to fetal brain development, potentially increasing susceptibility to neurodevelopmental disorders in later life. However, the underlying mechanisms remain incompletely understood. This study aimed to understand the effects of fetal e-cig exposure on DNA methylome and transcriptomic changes in the neonatal brain. Pregnant rats were exposed to e-cig aerosols, and neonatal brains (5 males and 5 females/group) from both control and e-cig-exposed groups were used for experimental analysis. Results indicated that prenatal e-cig exposure altered site-specific DNA methylation patterns at both CpG and CH (non-CpG) sites, predominantly in intergenic and intronic regions, with sex dimorphism in methylation and gene expression changes. Gene ontology analysis revealed that e-cig exposure not only affected neuron projection development and axonogenesis but also altered pathways related to neurodegeneration and long-term depression. These findings provide novel insights into the dynamic changes of CpG and CH methylation induced by e-cig exposure, underscoring the susceptibility of the developing brain to maternal e-cig exposure and its potential implications for developmental disorders and neurodegenerative diseases later in life.

Long-term methylome changes after experimental seed demethylation and their interaction with recurrent water stress in Erodium cicutarium (Geraniaceae).

The frequencies and lengths of drought periods are increasing in subtropical and temperate regions worldwide. Epigenetic responses to water stress could be key for plant resilience to these largely unpredictable challenges. Experimental DNA demethylation, together with application of a stress factor is an appropriate strategy to reveal the contribution of epigenetics to plant responses to stress. We analysed leaf cytosine methylation changes in adult plants of the annual Mediterranean herb, Erodium cicutarium, in a greenhouse, after seed demethylation with 5-Azacytidine and/or recurrent water stress. We used bisulfite RADseq (BsRADseq) and a newly reported reference genome for E. cicutarium to characterize methylation changes in a 2 × 2 factorial design, controlling for plant relatedness. In the long term, 5-Azacytidine treatment alone caused both hypo- and hyper-methylation at individual cytosines, with substantial hypomethylation in CG contexts. In control conditions, drought resulted in a decrease in methylation in all but CHH contexts. In contrast, the genome of plants that experienced recurrent water stress and had been treated with 5-Azacytidine increased DNA methylation level by ca. 5%. Seed demethylation and recurrent drought produced a highly significant interaction in terms of global and context-specific cytosine methylation. Most methylation changes occurred around genic regions and within Transposable Elements. The annotation of these Differentially Methylated Regions associated with genes included several with a potential role in stress responses (e.g., PAL, CDKC, and ABCF), confirming an epigenetic contribution in response to stress at the molecular level.

Abstract We028: Decoding m6a RNA methylomes identifies Igf2bp1 as a common barrier to direct reprogramming

Direct reprogramming has revolutionized the fields of stem cell biology and regenerative medicine. Targeted transdifferentiation of cells in situ holds significant promise in treating a large swathe of illness and injury including cardiovascular disease. However, much remains unknown about the molecular mechanisms of direct reprogramming, particularly its post-transcriptional regulation, including the role of m6a RNA modification. Here, by characterizing early changes in the m6a RNA methylome during reprogramming of fibroblasts toward three distinct lineages—cardiac, hepatic and neuronal—we identify lineage-specific features as well as common regulatory patterns. Our data reveal extensive changes in m6a methylome of direct reprogramming with clear impacts on cell fate decisions. Through loss-of-function screening of m6a readers, we uncovered Igf2bp1(insulin-like growth factor 2 mRNA binding protein 1) as a shared barrier to direct reprogramming. Further mechanistic studies will show how Igf2bp1 influences the reprogramming process mediated via m6a modifications. Collectively, our results present a fascinating portrait of m6a RNA methylome during direct reprogramming and reveal a key role of Igf2bp1 in maintaining cell fate, enhancing our mechanistic understanding of the reprogramming process.

Methylome-proteome integration after late-life voluntary exercise training reveals regulation and target information for improved skeletal muscle health.

Exercise is a potent stimulus for combatting skeletal muscle ageing. To study the effects of exercise on muscle in a preclinical setting, we developed a combined endurance-resistance training stimulus for mice called progressive weighted wheel running (PoWeR). PoWeR improves molecular, biochemical, cellular and functional characteristics of skeletal muscle and promotes aspects of partial epigenetic reprogramming when performed late in life (22-24months of age). In this investigation, we leveraged pan-mammalian DNA methylome arrays and tandem mass-spectrometry proteomics in skeletal muscle to provide detailed information on late-life PoWeR adaptations in female mice relative to age-matched sedentary controls (n=7-10 per group). Differential CpG methylation at conserved promoter sites was related to transcriptional regulation genes as well as Nr4a3, Hes1 and Hox genes after PoWeR. Using a holistic method of -omics integration called binding and expression target analysis (BETA), methylome changes were associated with upregulated proteins related to global and mitochondrial translation after PoWeR (P=0.03). Specifically, BETA implicated methylation control of ribosomal, mitoribosomal, and mitochondrial complex I protein abundance after training. DNA methylation may also influence LACTB, MIB1 and UBR4 protein induction with exercise - all are mechanistically linked to muscle health. Computational cistrome analysis predicted several transcription factors including MYC as regulators of the exercise trained methylome-proteome landscape, corroborating prior late-life PoWeR transcriptome data. Correlating the proteome to muscle mass and fatigue resistance revealed positive relationships with VPS13A and NPL levels, respectively. Our findings expose differential epigenetic and proteomic adaptations associated with translational regulation after PoWeR that could influence skeletal muscle mass and function in aged mice. KEY POINTS: Late-life combined endurance-resistance exercise training from 22-24months of age in mice is shown to improve molecular, biochemical, cellular and in vivo functional characteristics of skeletal muscle and promote aspects of partial epigenetic reprogramming and epigenetic age mitigation. Integration of DNA CpG 36k methylation arrays using conserved sites (which also contain methylation ageing clock sites) with exploratory proteomics in skeletal muscle extends our prior work and reveals coordinated and widespread regulation of ribosomal, translation initiation, mitochondrial ribosomal (mitoribosomal) and complex I proteins after combined voluntary exercise training in a sizeable cohort of female mice (n=7-10 per group and analysis). Multi-omics integration predicted epigenetic regulation of serine β-lactamase-like protein (LACTB - linked to tumour resistance in muscle), mind bomb 1 (MIB1 - linked to satellite cell and type 2 fibre maintenance) and ubiquitin protein ligase E3 component N-recognin 4 (UBR4 - linked to muscle protein quality control) after training. Computational cistrome analysis identified MYC as a regulator of the late-life training proteome, in agreement with prior transcriptional analyses. Vacuolar protein sorting 13homolog A (VPS13A) was positively correlated to muscle mass, and the glycoprotein/glycolipid associated sialylation enzyme N-acetylneuraminate pyruvate lyase (NPL) was associated to in vivo muscle fatigue resistance.

Blood DNA methylation in post-acute sequelae of COVID-19 (PASC): a prospective cohort study

DNA methylation integrates environmental signals with transcriptional programs. COVID-19 infection induces changes in the host methylome. While post-acute sequelae of COVID-19 (PASC) is a long-term complication of acute illness, its association with DNA methylation is unknown. No universal blood marker of PASC, superseding single organ dysfunctions, has yet been identified. In this single centre prospective cohort study, PASC, post-COVID without PASC, and healthy participants were enrolled to investigate their symptoms association with peripheral blood DNA methylation data generated with state-of-the-art whole genome sequencing. PASC-induced quality-of-life deterioration was scored with a validated instrument, SF-36. Analyses were conducted to identify potential functional roles of differentially methylated loci, and machine learning algorithms were used to resolve PASC severity. 103 patients with PASC (22.3% male, 77.7% female), 15 patients with previous COVID-19 infection but no PASC (40.0% male, 60.0% female), and 27 healthy volunteers (48.1% male, 51.9% female) were enrolled. Whole genome methylation sequencing revealed 39 differentially methylated regions (DMRs) specific to PASC, each harbouring an average of 15 consecutive positions, that differentiate patients with PASC from the two control groups. Motif analyses of PASC-regulated DMRs identify binding domains for transcription factors regulating circadian rhythm and others. Some DMRs annotated to protein coding genes were associated with changes of RNA expression. Machine learning support vector algorithm and random forest hierarchical clustering reveal 28unique differentially methylated positions (DMPs) in the genome discriminating patients with better and worse quality of life. Blood DNA methylation levels identify PASC, stratify PASC severity, and suggest that DNA motifs are targeted by circadian rhythm-regulating pathways in PASC. This project has been funded by the following agencies: NIH-AI173035 (A. Jaitovich and R. Alisch); and NIH-AG066179 (R. Alisch).

O-306 Sperm DNA methylome changes in testicular cancer patients following chemotherapy treatment

Abstract Study question Does chemotherapy (CT) treatment for testicular cancer induce sperm methylome changes? Summary answer The analysis of 27 pre- and post-CT samples from testicular cancer survivors identified 65 differentially methylated regions. What is known already Research has shown that cancer treatment can cause DNA damage and aneuploidy in sperm, even years after the treatment. However, there is limited information available on the impact of CT on the sperm epigenome, and most of the information has been obtained from rodent models. Two recent studies, one involving one patient and the other involving three, have investigated this topic in humans. Both studies have suggested that cancer survivors may experience changes in sperm’s DNA methylation following oncological treatment. Study design, size, duration This is a multicenter prospective study. We included 27 patients [31.23 years (CI 95% 29.29-33.17)] diagnosed with testicular cancer who cryopreserved semen before CT (2009-2020). After receiving oncological treatment, these patients recovered their spermatogenesis. Patients who agreed to participate in this IRB-approved study, donated a second semen sample after their recovery, which was also frozen. Participants/materials, setting, methods Semen samples were thawed; sperm DNA was isolated and subsequently hybridized using the Human Infinitum DNA Methylation 850K EPIC BeadChip array platform (Illumina). A paired differential methylation analysis was performed between pre-CT and post-CT samples from the same patient, fitting a linear model of the methylated positions and regions. Minfi and DMRcate R packages were used to find differentially methylated positions (DMPs) and regions (DMRs). Subsequently, the functional impact of involved genes was evaluated. Main results and the role of chance DNA methylation data were available for the 54 sperm samples (27 collected before CT and 27 after CT treatment). Results were controlled by age and time elapsed between the cryopreservation of pre-CT and post-CT samples, which was 4.01 years (CI 95%: 3.42-5.64). After filtering for initial QC, the assessment was done on 445.000 CpGs per sample. The analysis revealed that a total of 228 CpG positions showed a tendency towards statistical significance. DMRcate identified 65 DMRs (40 hypomethylated and 25 hypermethylated) that reached a smoothed FDR p-value < 0.05 when compared post-CT samples to pre-CT within the same patient. We observed that 61 genes were located within DMRs, being TBC1D20, EN1, HOXB1 and SCNN1A the ones within the most significant DMRs (min-smoothed-FDR < 0.001). The functional enrichment analysis of putatively affected genes revealed that the biological processes more affected were: Regulation of macromolecule biosynthetic process, hormone receptor binding, signaling pathways regulating pluripotency of stem cells and pathways in cancer. Limitations, reasons for caution Epigenetics may be affected during cryopreservation and thawing. To ensure comparability, we also froze the samples after undergoing the CT process. Due to the high-throughput nature of the study and the limited access to samples, we only achieved marginal significance. Therefore, our results should be further validated. Wider implications of the findings This is the largest study suggesting distinct sperm DNA methylation signatures in testicular cancer survivors. Our findings emphasize the importance of counseling cancer patients in pretreatment sample collection and may explain the reported increase in birth defects after treatment. Trial registration number NOT APPLICABLE

Human TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, the most severe symptoms of TSC are often neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes, we observed aberrant early neurodevelopment in vitro, including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions, including loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued.

NFKBIA deletions reshape the epigenome antithetical to the IDH mutation and indication of prognosis in diffuse gliomas.

2083 Background: Genetic alterations help predict the clinical behavior of diffuse gliomas, but some variability remains uncorrelated. The NFKBIA gene at 14q13.2 encodes IkBa, a protein that regulates the activity of NF-kB in the cytoplasm. Evidence that, in chromatin, nuclear NFKBIA dynamically interacts with histones H2A and H4 to regulate polycomb-dependent transcriptional repression and, thus, stem cell maturation and lineage specification and our characterization of NFKBIA as a tumor suppressor in glioblastoma prompted our investigation of the relationship of NFKBIA deletions to other genetic markers, alterations in the methylome, and the clinical course of gliomas. Methods: We analyzed the genetic profiles of 2,343 WHO grade 2 to 4 diffuse gliomas in multiple clinically well-characterized patient populations, including national randomized phase III consortium trial RTOG 9802, to determine whether incorporation of NFKBIA deletions could enhance the prognostic value of current molecular descriptions. Results: We demonstrate that haploinsufficient deletions of NFKBIA display distinct patterns of occurrence in relation to other genetic markers, including driver mutations IDH, TERT, ATRX, and PTEN, 1p19q codeletion, and CDKN2A/B deletion, and are disproportionally present at recurrence. NFKBIA haploinsufficiency is associated with unfavorable patient outcomes, independent of genetic and clinicopathologic predictors. NFKBIA deletions reshape the DNA and histone methylome antipodal to the IDH mutation. The methylome changes facilitated through NFKBIA deletions engender a transcriptome landscape partly reminiscent of H3K27M mutant pediatric gliomas and are highly enriched for neural stem cell and neurogenesis genes bearing repressive H3K27 marks. Our findings imply that the NFKBIA deletion in adult gliomas and the H3K27M mutation in pediatric diffuse midline gliomas define aggressive disease subtypes that may share an epigenetic state reflecting a common PRC2 loss-of-function phenotype antipodal to the quasi PRC2 gain-of-function phenotype of IDH mutant gliomas and their favorable clinical course. Lower-grade gliomas harboring NFKBIA deletions behave much like high-grade gliomas. In IDH mutant gliomas, NFKBIA deletions are common in tumors with a clinical course similar to that of IDH wildtype tumors. A sparse, easily interpretable, and externally validated nomogram model for estimating individual patient survival in IDH mutant gliomas, incorporating NFKBIA deletion and current best-established genetic and clinicopathologic factors, confirms that NFKBIA deletions predict comparatively brief survival. Conclusions: NFKBIA haploinsufficiency aligns with distinct epigenome changes, portends a poor prognosis, and should be incorporated into clinical models predicting the disease fate of diffuse gliomas.

Integrative analysis of the methylome and transcriptome of tomato fruit (Solanum lycopersicum L.) induced by postharvest handling.

Tomato fruit ripening is triggered by the demethylation of key genes, which alters their transcriptional levels thereby initiating and propagating a cascade of physiological events. What is unknown is how these processes are altered when fruit are ripened using postharvest practices to extend shelf-life, as these practices often reduce fruit quality. To address this, postharvest handling-induced changes in the fruit DNA methylome and transcriptome, and how they correlate with ripening speed, and ripening indicators such as ethylene, abscisic acid, and carotenoids, were assessed. This study comprehensively connected changes in physiological events with dynamic molecular changes. Ripening fruit that reached 'Turning' (T) after dark storage at 20°C, 12.5°C, or 5°C chilling (followed by 20°C rewarming) were compared to fresh-harvest fruit 'FHT'. Fruit stored at 12.5°C had the biggest epigenetic marks and alterations in gene expression, exceeding changes induced by postharvest chilling. Fruit physiological and chronological age were uncoupled at 12.5°C, as the time-to-ripening was the longest. Fruit ripening to Turning at 12.5°C was not climacteric; there was no respiratory or ethylene burst, rather, fruit were high in abscisic acid. Clear differentiation between postharvest-ripened and 'FHT' was evident in the methylome and transcriptome. Higher expression of photosynthetic genes and chlorophyll levels in 'FHT' fruit pointed to light as influencing the molecular changes in fruit ripening. Finally, correlative analyses of the -omics data putatively identified genes regulated by DNA methylation. Collectively, these data improve our interpretation of how tomato fruit ripening patterns are altered by postharvest practices, and long-term are expected to help improve fruit quality.

Abstract 3454: Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk

Abstract Cell-free DNA (cfDNA) epigenetic and fragmentomic profiling has emerged as prominent non-invasive approaches for early cancer detection and subtyping. However, owing to difficulties in observing the early development of human malignancies, most cancer biomarker and evolution studies to date have primarily examined biologics following a diagnosis. Utilizing cfDNA as a screening tool for early disease detection requires assessment of blood plasma samples collected from asymptomatic individuals prior to the diagnosis of cancers. Here, we leverage the Ontario Health Study (OHS), a prospective longitudinal population cohort, to assess cfDNA profiles in blood plasma collected from participants that developed breast (n=476), prostate (n=342) and pancreatic (n=32) cancers throughout the study follow up time between 2009 and 2019. We performed cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) on baseline plasma samples from incident cancer cases up to eight years prior to diagnosis, in addition to matched controls (n=404) with no history of cancer through follow-up. Genome-wide plasma cfDNA differential methylation analysis revealed significantly hypermethylated regions, particularly among CpG island and shore regions, were predictive of early breast cancers, and overlapped hypermethylated regions in solid cancer tissues relative to adjacent normal tissues and peripheral blood leukocytes. Using a repeated cross-validation strategy we found that as few as 200 hypermethylated regions can identify individuals with breast cancer in blood up to seven years prior to diagnosis among discovery set samples (AUC=0.725) and are highly generalizable to samples processed in separate batches (AUC = 0.650). Remarkably, integrating biomarker hypermethylated regions with fragmentomic features including fragment length and 5’ tetranucleotide motif proportions further increased accuracy for detecting individuals with early prostate cancers (AUC=0.804). In addition, we observed shared genome-wide cfDNA methylome changes associated with aging and heavy alcohol consumption in cfDNA. We identified increased shedding of tissue-specific methylation markers from liver and pancreatic tissue among individuals consuming more than three drinks a week relative to non-drinkers, reflective of potential signatures of tissue damage. Citation Format: Nicholas Cheng, Kimberly Skead, Tom Oullette, Scott Bratman, Daniel De Carvalho, David Soave, Philip Awadalla. Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk [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 3454.

Methylome-wide and meQTL analysis helps to distinguish treatment response from non-response and pathogenesis markers in schizophrenia.

Schizophrenia is a complex condition with entwined genetic and epigenetic risk factors, posing a challenge to disentangle the intermixed pathological and therapeutic epigenetic signatures. To resolve this, we performed 850K methylome-wide and 700K genome-wide studies on the same set of schizophrenia patients by stratifying them into responders, non-responders, and drug-naïve patients. The key genes that signified the response were followed up using real-time gene expression studies to understand the effect of antipsychotics at the gene transcription level. The study primarily implicates hypermethylation in therapeutic response and hypomethylation in the drug-non-responsive state. Several differentially methylated sites and regions colocalized with the schizophrenia genome-wide association study (GWAS) risk genes and variants, supporting the convoluted gene-environment association. Gene ontology and protein-protein interaction (PPI) network analyses revealed distinct patterns that differentiated the treatment response from drug resistance. The study highlights the strong involvement of several processes related to nervous system development, cell adhesion, and signaling in the antipsychotic response. The ability of antipsychotic medications to alter the pathology by modulating gene expression or methylation patterns is evident from the general increase in the gene expression of response markers and histone modifiers and the decrease in class II human leukocyte antigen (HLA) genes following treatment with varying concentrations of medications like clozapine, olanzapine, risperidone, and haloperidol. The study indicates a directional overlap of methylation markers between pathogenesis and therapeutic response, thereby suggesting a careful distinction of methylation markers of pathogenesis from treatment response. In addition, there is a need to understand the trade-off between genetic and epigenetic observations. It is suggested that methylomic changes brought about by drugs need careful evaluation for their positive effects on pathogenesis, course of disease progression, symptom severity, side effects, and refractoriness.

Long-term effects of myo-inositol on traumatic brain injury: Epigenomic and transcriptomic studies

Background and purposeTraumatic brain injury (TBI) and its consequences remain great challenges for neurology. Consequences of TBI are associated with various alterations in the brain but little is known about long-term changes of epigenetic DNA methylation patterns. Moreover, nothing is known about potential treatments that can alter these epigenetic changes in beneficial ways. Therefore, we have examined myo-inositol (MI), which has positive effects on several pathological conditions. MethodsTBI was induced in mice by controlled cortical impact (CCI). One group of CCI animals received saline injections for two months (TBI+SAL), another CCI group received MI treatment (TBI+MI) for the same period and one group served as a sham-operated control. Mice were sacrificed 4 months after CCI and changes in DNA methylome and transcriptomes were examined. ResultsFor the first time we: (i) provide comprehensive map of long-term DNA methylation changes after CCI in the hippocampus; (ii) identify differences by methylation sites between the groups; (iii) characterize transcriptome changes; (iv) provide association between DNA methylation sites and gene expression. MI treatment is linked with upregulation of genes covering 33 biological processes, involved in immune response and inflammation. In support of these findings, we have shown that expression of BATF2, a transcription factor involved in immune-regulatory networks, is upregulated in the hippocampus of the TBI+MI group where the BATF2 gene is demethylated. ConclusionTBI is followed by long-term epigenetic and transcriptomic changes in hippocampus. MI treatment has a significant effect on these processes by modulation of immune response and biological pathways of inflammation.

Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomato

BackgroundGeminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV).ResultsWe showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes.ConclusionWe have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.

Amyloid‐beta immunotherapy induces novel methylomic changes in Alzheimer’s disease brain

AbstractBackgroundPost‐mortem neuropathological examinations following the first active immunotherapy strategy (AN‐1792, Elan Pharmaceuticals, 2000) for Alzheimer’s disease (AD) have evidenced amyloid‐β (Aβ) plaque clearance and increased microglial phagocytic activity in immunised individuals. This study characterises the epigenetic profiles of individuals who underwent Aβ immunotherapy with the aim of discovering novel therapeutic targets and biomarkers.MethodDNA was isolated from post‐mortem prefrontal cortex tissue of 16 immunised AD cases who received varying doses (ug) and number of doses during the trial period, including placebos. DNA methylation was quantified using methylation arrays and the raw intensity values processed and normalised for subsequent statistical analysis to identify differentially methylated positions (DMPs) across the genome associated with Aβ immunotherapy. Additional analyses included regions of enrichment analysis for identification of differentially methylated regions (DMRs) and weighted gene co‐expression network analysis to identify gene clusters with highly correlated methylation levels.ResultAfter correcting for known (age, sex, cell type composition) and unknown variables, a DMP located within Chromosome 19 Open Reading Frame 12 (C19Orf12) at the genome‐wide significance level (P < 9.00E−08) was associated with Aβ immunisation. 10 DMRs were further associated with immunisation, with three regions consisting of ≥ 3 CpG sites and having a Sidak‐corrected P < 0.05. These regions were located within the CRISP2 (chr6; 49681178:49681391 [8 probes], Sidak‐corrected P = 8.29E‐05), CTTNA1 (chr5; 138210906:138211184 [8 probes], Sidak‐corrected P = 5.96E‐06), and RNF39 (chr6; 30038859:30039025 [12 probes], Sidak‐Corrected P = 2.60E‐05) genes.ConclusionIndicating better inflammatory markers can help inform of effective preventative care strategies for elders. This study provides evidence for altered epigenetic processes in the pathophysiology of AD and identifies novel processes specific to Aβ immunotherapy. The next step for this project includes analysing RNA sequencing data, to determine if observed methylomic changes correlate with gene expression, and genotyping array data.

Epigenetic Age Acceleration in Hematological Malignancies: Beyond Chronological Age, Clinical Implications, and Therapeutic Perspectives

Background: Age is important in prognosticating hematological malignancies. Biological based on DNA methylome changes offers new insights into the complex interplay between epigenetics, genetics, environment, and cancer. DNA methylation levels at specific CpG sites evolve in a predictable fashion with aging and can be used to develop biomarkers that estimate the epigenetic age (EA) of a tissue. Such biomarkers are called epigenetic clocks (Fig 1-B). Based on the EA, we can determine Epigenetic Age Acceleration (EAA), a tool to identify patients aging faster biologically than their chronological age. We sought to build a blood-based clock to predict epigenetic age in healthy young and old adults and subsequently assess the degree of EAA in AML patients. Finally, among multiple planned downstream applications, we propose to use EAA as a biomarker for monitoring treatment responses in targeted therapies (Fig 1-A). Methods: We used ClockBase and NCBI's Gene Expression Omnibus (GEO) repositories to collect methylation data based on Illumina's 450k array for healthy adults and patients with AML/MDS. Pre-processing was done using the minifi BioConductor package. We used a cohort of healthy young adults (YA, aged 18-30 ys.) to train our epigenetic clock and calibrate it with three well-studied epigenetic clocks: Horvath clock (HC), Hannum clock (HAC), and PhenoAge (PA). We hypothesized that EA and chronological ages (CA) should approximate each other in younger patients as they do not generally carry comorbidities or have a higher CHIP prevalence which could influence EA. We compared our clock's performance to known epigenetic clocks, and after fine-tuning, all 4 models were validated in a cohort of healthy older adults (OA, aged 70-85 ys.) with a higher risk of CHIP. Then, we tested our clock in an AML patient cohort and illustrated how EAA can predict therapy responses in AML patients treated with IDH1/2 inhibitors (Published methylation data from Wang et al 2021, Nat Comm.) Results: We designed the clock using an Elastic-Net regression scheme. We screened 130,000 patient samples from GEO and 1,813 eligible samples were divided into 3 cohorts: 800 healthy younger adults (aged 18-30 ys.), 800 healthy elderly adults (aged 70-85 ys.), and 213 patients with AML. To train and test our clock, we used the YA cohort (n=800) with a median age of 20 ys. (SD 3.168 ys) and our epigenetic clock predicted a median EA (MEA) of 19.1 ys. (SD 3.4 ys). HC, HAC and PA predicted MEA of 21.6 ys. (SD 3.9 ys.), 19.4 ys. (SD 4.1 ys.) and 21.5 ys. (SD 3.9 ys.). After fine-tuning of weights, we tested our clock in OA cohort (n=800) with a median age of 78 ys. (SD 4.8 ys.). Our clock predicted a MEA of 82.3 ys. (SD 4.9 ys.). HC, HAC and PA predicted a MEA of 83.7 ys. (SD 5.7 ys.), 85.2 ys. (SD 6.0 ys.) and 84.9 ys. (SD of 5.1 ys.) resp. The AML cohort (n=213) had a median age of 80.9 ys. (SD 2.5 ys.). We expected a significantly higher EA due to the mutation burden along with genomic and epigenomic changes induced by the disease, our clock predicted a MEA of 84.7 ys. (SD 3.0 ys.). EAA was calculated from the difference between EA and CA. YA cohort had a median EAA of 0.9 ys. (SD 1.8 ys.) and our clock predicted a median EAA of 1.1 ys. (SD 2.0 ys.). HC, HAC, and PA predicted a median EAA of 1.4 ys. (SD 2.3 ys.), 1.4 ys. (SD 2.9 ys.), and 1.5 ys. (SD 2.4 ys.). In the OA cohort, we expected higher EAA due to age-associated cardiometabolic disease and CHIP; our clock predicted a median EAA of 4.2 (SD 2.1). HC, HAC, and PA predicted a median EAA of 6.0 ys. (SD 2.7), 5.6 ys. (SD 3.0 ys.), and 5.1 ys. (SD 2.2 ys.). For the AML cohort, our clock predicted a median EAA of 8.0 ys. (SD 5.6). We then applied our clock to a cohort of 60 AML patients treated with IDH inhibitors to predict whether a patient cleared the IDH clone after therapy. We discovered that patients who failed to clear the IDH-clone (n=14) had a higher EAA (3.23 vs 7.91 ys., p=0.012) and shorter RFS compared to patients who cleared the IDH-clone (n=7) [RFS 20.6 vs 6.1 mo., p=0.0086]. Conclusion: We built an epigenetic clock that outperforms three standard clocks in predicting epigenetic age and applied it to AML patients demonstrating a significant increase in epigenetic age. We also illustrated how EAA can be used as a biomarker for targeted therapies. Ongoing work at Karmanos Cancer Center is focused on improving the underlying regression model for more sensitive epigenetic age predictions and implementing novel pace-of-aging algorithms for EAA calculations.

THU082 Longitudinal Multiomics Characterization Of Paired Primary And Recurrent Aggressive Pituitary Tumors From The Same Patient Reveals Genomic Stability And Transcriptomic And Epigenetic Heterogeneity With Metabolic Pathways Alterations

Abstract Disclosure: K. Taniguchi-Ponciano: None. A. Chavez-Santoscoy: None. S. Hinojosa-Alvarez: None. J. Hernandez-Perez: None. A. Valenzuela-Perez: None. S. Vela-Patiño: None. S. Andonegui-Elguera: None. A. Cano-Zaragoza: None. F. Martinez-Mendoza: None. J. Kerbel: None. Z. Zhang: None. G. Smith: None. A. Rubenstein: None. W. Cheng: None. N. Mendelev: None. M. Amper: None. M. Zamojski: None. E. Zaslavsky: None. F.M. Ruf-Zamojski: None. S.C. Sealfon: None. M. Mercado: None. D. Marrero-Rodríguez: None. Pituitary tumors (PT) represent 20% of all intracranial neoplasms. Some behave aggressively, growing rapidly and invading surrounding tissues, with high recurrence rates and are frequently resistant to conventional therapies. We performed exome and transcriptome sequencing, as well as methylation profiling of primary and recurrent PT of the same patient. The cohort consisted of patients with GH- and gonadotropin-producing PT and a patient with an ACTH carcinoma. We sought genome evolution as well as transcriptomic and methylomic changes trough time, trying to identify molecular markers of recurrence. We performed scRNAseq to identify tumor cell populations. Compared to the primary PT, recurrent lesions showed less than 5% genomic changes in the SNV profile. The gonadotrophin-PT did not show common variants between patients or between primary and recurrent lesions. The ACTH-carcinoma showed USP8, TP53 and EGFR variants in both tumor samples, but no genomic changes between tumors despite a Ki67 of 80% in the recurrent lesion. A GH-tumor showed an AIP variant with high genetic heterogeneity. No CNV between the primary and recurrent tumors were observed. Regardless of their lineage, all PT showed different degrees of transcriptomic and methylomic heterogeneity between the primary and the recurrent lesions. Primary and recurrent tumors clustered separately from each other but together among themselves. Interestingly the differentially expressed and methylated genes in gonadotropin-producing PT are related to fatty acid biosynthesis and metabolism, GPI-anchor biosynthesis, and other metabolic pathways. ACTH-carcinoma differs significantly from the silent corticotrope in gene expression and methylation patterns. The ACTH-carcinoma showed alteration in N-Glycan biosynthesis, glycerophospholipid metabolism, purine and pyrimidine metabolism, whereas the silent corticotrope showed one carbon pool by folate, inositol phosphate metabolism, carbohydrate digestion and absorption and biosynthesis of unsaturated fatty acids. The GH-tumor showed alteration in pyruvate metabolism, propanoate metabolism, insulin secretion, and aldosterone synthesis and secretion. Our results suggest that there are several clones conforming a PT and some of them remain after surgical resection and can re-grow, this was confirmed by our scRNAseq analysis and immunofluorescence assays. We conclude that PT are genomically stable trough time, indicating that mechanisms other than somatic mutations are involved in pituitary tumorigenesis and that their biology could be driven by transcriptomically and epigenetically heterogeneous clones within the tumor itself. Presentation: Thursday, June 15, 2023

Epigenomic response to albuterol treatment in asthma-relevant airway epithelial cells

BackgroundAlbuterol is the first-line asthma medication used in diverse populations. Although DNA methylation (DNAm) is an epigenetic mechanism involved in asthma and bronchodilator drug response (BDR), no study has assessed whether albuterol could induce changes in the airway epithelial methylome. We aimed to characterize albuterol-induced DNAm changes in airway epithelial cells, and assess potential functional consequences and the influence of genetic variation and asthma-related clinical variables.ResultsWe followed a discovery and validation study design to characterize albuterol-induced DNAm changes in paired airway epithelial cultures stimulated in vitro with albuterol. In the discovery phase, an epigenome-wide association study using paired nasal epithelial cultures from Puerto Rican children (n = 97) identified 22 CpGs genome-wide associated with repeated-use albuterol treatment (p < 9 × 10–8). Albuterol predominantly induced a hypomethylation effect on CpGs captured by the EPIC array across the genome (probability of hypomethylation: 76%, p value = 3.3 × 10–5). DNAm changes on the CpGs cg23032799 (CREB3L1), cg00483640 (MYLK4-LINC01600), and cg05673431 (KSR1) were validated in nasal epithelia from 10 independent donors (false discovery rate [FDR] < 0.05). The effect on the CpG cg23032799 (CREB3L1) was cross-tissue validated in bronchial epithelial cells at nominal level (p = 0.030). DNAm changes in these three CpGs were shown to be influenced by three independent genetic variants (FDR < 0.05). In silico analyses showed these polymorphisms regulated gene expression of nearby genes in lungs and/or fibroblasts including KSR1 and LINC01600 (6.30 × 10–14 ≤ p ≤ 6.60 × 10–5). Additionally, hypomethylation at the CpGs cg10290200 (FLNC) and cg05673431 (KSR1) was associated with increased gene expression of the genes where they are located (FDR < 0.05). Furthermore, while the epigenetic effect of albuterol was independent of the asthma status, severity, and use of medication, BDR was nominally associated with the effect on the CpG cg23032799 (CREB3L1) (p = 0.004). Gene-set enrichment analyses revealed that epigenomic modifications of albuterol could participate in asthma-relevant processes (e.g., IL-2, TNF-α, and NF-κB signaling pathways). Finally, nine differentially methylated regions were associated with albuterol treatment, including CREB3L1, MYLK4, and KSR1 (adjusted p value < 0.05).ConclusionsThis study revealed evidence of epigenetic modifications induced by albuterol in the mucociliary airway epithelium. The epigenomic response induced by albuterol might have potential clinical implications by affecting biological pathways relevant to asthma.

Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37.

Epichloë spp. often form mutualistic interactions with cool-season grasses, such as Lolium perenne. However, the molecular mechanisms underlying this interaction remain poorly understood. In this study, we employed reduced representation bisulfite sequencing method (epiGBS) to investigate the impact of the Epichloë sp. LpTG-3 strain AR37 on the methylome of L. perenne across multiple grass generations and under drought stress conditions. Our results showed that the presence of the endophyte leads to a decrease in DNA methylation across genomic features, with differentially methylated regions primarily located in intergenic regions and CHH contexts. The presence of the endophyte was consistently associated with hypomethylation in plants across generations. This research sheds new light on the molecular mechanisms governing the mutualistic interaction between Epichloë sp. LpTG-3 strain AR37 and L. perenne. It underscores the role of methylation changes associated with endophyte infection and suggests that the observed global DNA hypomethylation in L. perenne may be influenced by factors such as the duration of the endophyte-plant association and the accumulation of genetic and epigenetic changes over time.

Sex differences in muscle protein expression and DNA methylation in response to exercise training

BackgroundExercise training elicits changes in muscle physiology, epigenomics, transcriptomics, and proteomics, with males and females exhibiting differing physiological responses to exercise training. However, the molecular mechanisms contributing to the differing adaptations between the sexes are poorly understood.MethodsWe performed a meta-analysis for sex differences in skeletal muscle DNA methylation following an endurance training intervention (Gene SMART cohort and E-MTAB-11282 cohort). We investigated for sex differences in the skeletal muscle proteome following an endurance training intervention (Gene SMART cohort). Lastly, we investigated whether the methylome and proteome are associated with baseline cardiorespiratory fitness (maximal oxygen consumption; VO2max) in a sex-specific manner.ResultsHere, we investigated for the first time, DNA methylome and proteome sex differences in response to exercise training in human skeletal muscle (n = 78; 50 males, 28 females). We identified 92 DNA methylation sites (CpGs) associated with exercise training; however, no CpGs changed in a sex-dependent manner. In contrast, we identified 189 proteins that are differentially expressed between the sexes following training, with 82 proteins differentially expressed between the sexes at baseline. Proteins showing the most robust sex-specific response to exercise include SIRT3, MRPL41, and MBP. Irrespective of sex, cardiorespiratory fitness was associated with robust methylome changes (19,257 CpGs) and no proteomic changes. We did not observe sex differences in the association between cardiorespiratory fitness and the DNA methylome. Integrative multi-omic analysis identified sex-specific mitochondrial metabolism pathways associated with exercise responses. Lastly, exercise training and cardiorespiratory fitness shifted the DNA methylomes to be more similar between the sexes.ConclusionsWe identified sex differences in protein expression changes, but not DNA methylation changes, following an endurance exercise training intervention; whereas we identified no sex differences in the DNA methylome or proteome response to lifelong training. Given the delicate interaction between sex and training as well as the limitations of the current study, more studies are required to elucidate whether there is a sex-specific training effect on the DNA methylome. We found that genes involved in mitochondrial metabolism pathways are differentially modulated between the sexes following endurance exercise training. These results shed light on sex differences in molecular adaptations to exercise training in skeletal muscle.