Whole-genome DNA Methylation Profiles Research Articles

Reprogramming of DNA methylation and changes of gene expression in grafted Hevea brasiliensis.

Rubber tree (Hevea brasiliensis) is reproduced by bud grafting for commercial planting, but significant intraclonal variations exist in bud-grafted clones. DNA methylation changes related to grafting may be partly responsible for intraclonal variations. In the current study, whole-genome DNA methylation profiles of grafted rubber tree plants (GPs) and their donor plants (DPs) were evaluated by whole-genome bisulfite sequencing. Data showed that DNA methylation was downregulated and DNA methylations in CG, CHG, and CHH sequences were reprogrammed in GPs, suggesting that grafting induced the reprogramming of DNA methylation. A total of 5,939 differentially methylated genes (DMGs) were identified by comparing fractional methylation levels between GPs and DPs. Transcriptional analysis revealed that there were 9,798 differentially expressed genes (DEGs) in the DP and GP comparison. A total of 1,698 overlapping genes between DEGs and DMGs were identified. These overlapping genes were markedly enriched in the metabolic pathway and biosynthesis of secondary metabolites by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Global DNA methylation and transcriptional analyses revealed that reprogramming of DNA methylation is correlated with gene expression in grafted rubber trees. The study provides a whole-genome methylome of rubber trees and an insight into the molecular mechanisms underlying the intraclonal variations existing in the commercial planting of grafted rubber trees.

Whole-genome DNA methylation profiling revealed epigenetic regulation of NF-κB signaling pathway involved in response to Vibrio alginolyticus infection in the Pacific oyster, Crassostrea gigas

DNA methylation, an essential epigenetic alteration, is tightly linked to a variety of biological processes, such as immune response. To identify the epigenetic regulatory mechanism in Pacific oyster (Crassostrea gigas), whole-genome bisulfite sequencing (WGBS) was conducted on C. gigas at 0 h, 6 h, and 48 h after infection with Vibrio alginolyticus. At 6 h and 48 h, a total of 11,502 and 14,196 differentially methylated regions (DMRs) were identified (p<0.05, FDR<0.001) compared to 0 h, respectively. Gene ontology (GO) analysis showed that differentially methylated genes (DMGs) were significantly enriched in various biological pathways including immunity, cytoskeleton, epigenetic modification, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that transcription machinery (ko03021) is one of the most important pathways. Integrated transcriptome and methylome analyses allowed the identification of 167 and 379 DMG-related DEGs at 6 h and 48 h, respectively. These genes were significantly enriched in immune-related pathways, including nuclear factor kappa B (NF-κB) signaling pathway (ko04064) and tumor necrosis factor (TNF) signaling pathway (ko04668). Interestingly, it's observed that the NF-κB pathway could be activated jointly by TNF Receptor Associated Factor 2 (TRAF2) and Baculoviral IAP Repeat Containing 3 (BIRC3, the homolog of human BIRC2) which were regulated by DNA methylation in response to the challenge posed by V. alginolyticus infection. Through this study, we provided insightful information about the epigenetic regulation of immunity-related genes in the C. gigas, which will be valuable for the understanding of the innate immune system modulation and defense mechanism against bacterial infection in invertebrates.

Multi-omic integration reveals alterations in nasal mucosal biology that mediate air pollutant effects on allergic rhinitis.

Allergic rhinitis is a common inflammatory condition of the nasal mucosa that imposes a considerable health burden. Air pollution has been observed to increase the risk of developing allergic rhinitis. We addressed the hypotheses that early life exposure to air toxics is associated with developing allergic rhinitis, and that these effects are mediated by DNA methylation and gene expression in the nasal mucosa. In a case-control cohort of 505 participants, we geocoded participants' early life exposure to air toxics using data from the US Environmental Protection Agency, assessed physician diagnosis of allergic rhinitis by questionnaire, and collected nasal brushings for whole-genome DNA methylation and transcriptome profiling. We then performed a series of analyses including differential expression, Mendelian randomization, and causal mediation analyses to characterize relationships between early life air toxics, nasal DNA methylation, nasal gene expression, and allergic rhinitis. Among the 505 participants, 275 had allergic rhinitis. The mean age of the participants was 16.4 years (standard deviation = 9.5 years). Early life exposure to air toxics such as acrylic acid, phosphine, antimony compounds, and benzyl chloride was associated with developing allergic rhinitis. These air toxics exerted their effects by altering the nasal DNA methylation and nasal gene expression levels of genes involved in respiratory ciliary function, mast cell activation, pro-inflammatory TGF-β1 signaling, and the regulation of myeloid immune cell function. Our results expand the range of air pollutants implicated in allergic rhinitis and shed light on their underlying biological mechanisms in nasal mucosa.

Whole-Genome DNA Methylation Profiling of Intrahepatic Cholangiocarcinoma Reveals Prognostic Subtypes with Distinct Biological Drivers.

Characterization of the DNA methylome of intrahepatic cholangiocarcinoma integrated with genomic, transcriptomic, and proteomic analyses uncovers molecular mechanisms affected by genome-wide DNA methylation alterations, providing a resource for identifying potential therapeutic targets.

Rare variant of large pediatric glioneuronal tumor with novel MYO5A::NTRK3 fusion: illustrative case.

Glioneuronal tumors (GNTs) comprise a rare class of central nervous system (CNS) neoplasms with varying degrees of neuronal and glial differentiation that predominately affect children and young adults. Within the current 2021 World Health Organization (WHO) classification of CNS tumors, GNTs encompass 14 distinct tumor types. Recently, the use of whole-genome DNA methylation profiling has allowed more precise classification of this tumor group. A 3-year-old male presented with a 3-month history of increasing head circumference, regression of developmental milestones, and speech delay. Magnetic resonance imaging of the brain was notable for a large left hemispheric multiseptated mass with significant mass effect and midline shift that was treated with near-total resection. Histological and molecular assessment demonstrated a glioneuronal tumor harboring an MYO5A::NTRK3 fusion. By DNA methylation profiling, this tumor matched to a provisional methylation class known as "glioneuronal tumor kinase-fused" (GNT kinase-fused). The patient was later started on targeted therapy with larotrectinib. This is the first report of an MYO5A::NTRK3 fusion in a pediatric GNT. GNT kinase-fused is a provisional methylation class not currently included in the WHO classification of CNS tumors. This case highlights the impact of thorough molecular characterization of CNS tumors, especially with the increasing availability of novel gene targeting therapies.

The prognostic value of whole-genome DNA methylation in response to Leflunomide in patients with Rheumatoid Arthritis.

Although Leflunomide (LEF) is effective in treating rheumatoid arthritis (RA), there are still a considerable number of patients who respond poorly to LEF treatment. Till date, few LEF efficacy-predicting biomarkers have been identified. Herein, we explored and developed a DNA methylation-based predictive model for LEF-treated RA patient prognosis. Two hundred forty-five RA patients were prospectively enrolled from four participating study centers. A whole-genome DNA methylation profiling was conducted to identify LEF-related response signatures via comparison of 40 samples using Illumina 850k methylation arrays. Furthermore, differentially methylated positions (DMPs) were validated in the 245 RA patients using a targeted bisulfite sequencing assay. Lastly, prognostic models were developed, which included clinical characteristics and DMPs scores, for the prediction of LEF treatment response using machine learning algorithms. We recognized a seven-DMP signature consisting of cg17330251, cg19814518, cg20124410, cg21109666, cg22572476, cg23403192, and cg24432675, which was effective in predicting RA patient's LEF response status. In the five machine learning algorithms, the support vector machine (SVM) algorithm provided the best predictive model, with the largest discriminative ability, accuracy, and stability. Lastly, the AUC of the complex model(the 7-DMP scores with the lymphocyte and the diagnostic age) was higher than the simple model (the seven-DMP signature, AUC:0.74 vs 0.73 in the test set). In conclusion, we constructed a prognostic model integrating a 7-DMP scores with the clinical patient profile to predict responses to LEF treatment. Our model will be able to effectively guide clinicians in determining whether a patient is LEF treatment sensitive or not.

Whole-genome CpG-resolution DNA Methylation Profiling of HNSCC Reveals Distinct Mechanisms of Carcinogenesis for Fine-scale HPV+ Cancer Subtypes.

This study revealed that the previously observed hypermethylation of HPV(+) HNSCC is due solely to the IMU subtype, illustrating the importance of fine-scale subtype analysis in such a heterogeneous disease. Particularly, IMU has significantly higher methylation of transposable elements, which can be tested as a prognosis biomarker in future translational studies.

Whole-genome DNA methylation and gene expression profiling in the livers of mice with nonalcoholic steatohepatitis

AimsNon-alcoholic fatty liver disease (NAFLD) has emerged as one of the major causes of liver-related morbidity and mortality. It ranges simple steatosis to non-alcoholic steatohepatitis (NASH). Previous studies have shown that epigenetic factors, such as DNA methylation, can contribute to the development and progression of simple steatosis. However, the profiling of whole-genome DNA methylation remains poorly characterized in NASH. Main methodsIn this study, we established a mouse model of diet-induced NASH, by maintaining male mice on a high-fructose-high-cholesterol diet (HFHC), to generate hepatic steatosis, inflammation and injury. We profiled hepatic gene expression by RNA-Sequencing and locus-specific 5-methylcytosine level, using Whole Genome Bisulfite Sequencing (WGBS). Key findingsWe identified >1000 differentially methylated regions in NASH versus control group, indicating that NASH diet could modulate the liver methylome. Furthermore, integrated analysis of methylome and transcriptome identified certain key methylated genes and pathways, which may be involved in steroid metabolism and inflammation response. The liver methylation levels of key genes especially Tgfb, Msn, Iqgap1, Cyba, Fcgr1 decreased, and their consequent increased expression may lead to NASH development. SignificanceWe found that HFHC diet-induced NASH could induces genome-wide differential DNA methylation changes. Thus, we proposed that DNA methylation profiles of genomes may be a useful signature of gene transcription and may play an important role in the development of NASH. We also screened and validated the changes of key genes, which may provide new perspectives for the mechanistic study of NASH in future.

Whole-genome DNA methylation profiling reveals epigenetic signatures in developing muscle in Tan and Hu sheep and their offspring.

The Tan sheep is a popular local breed in China because of its tenderness and flavor. The Hu sheep breed is also famous for its high litter size, and its muscle growth rate is faster than that of Tan sheep. However, the epigenetic mechanism behind these muscle-related phenotypes is unknown. In this study, the longissimus dorsi tissue from 18 6 month-old Tan sheep, Hu sheep, and Tan-Hu F2 generation (6 sheep per population) were collected. After genomic DNA extraction, whole-genome bisulfite sequencing (WGBS) and bioinformatics analysis were performed to construct genome-wide DNA methylome maps for the Tan sheep, Hu sheep and their Tan-Hu F2 generation. Distinct genome-wide DNA methylation patterns were observed between Tan sheep and Hu sheep. Moreover, DNA methylated regions were significantly increased in the skeletal muscle from Tan sheep vs. the F2 generation compared to the Hu sheep vs. F2 generation and the Tan sheep vs. Hu sheep. Compared with Hu sheep, the methylation levels of actin alpha 1 (ACTA1), myosin heavy chain 11 (MYH11), Wiskott-Aldrich syndrome protein (WAS), vav guanine nucleotide exchange factor 1 (VAV1), fibronectin 1 (FN1) and Rho-associated protein kinase 2 (ROCK2) genes were markedly distinct in the Tan sheep. Furthermore, Gene Ontology analysis indicated that these genes were involved in myotube differentiation, myotube cell development, smooth muscle cell differentiation and striated muscle cell differentiation. The findings from this study, in addition to data from previous research, demonstrated that the ACTA1, MYH11, WAS, VAV1, FN1, and ROCK2 genes may exert regulatory effects on muscle development.

Histone H3-wild type diffuse midline gliomas with H3K27me3 loss are a distinct entity with exclusive EGFR or ACVR1 mutation and differential methylation of homeobox genes

Diffuse midline gliomas (DMG) harbouring H3K27M mutation are paediatric tumours with a dismal outcome. Recently, a new subtype of midline gliomas has been described with similar features to DMG, including loss of H3K27 trimethylation, but lacking the canonical H3K27M mutation (H3-WT). Here, we report a cohort of five H3-WT tumours profiled by whole-genome sequencing, RNA sequencing and DNA methylation profiling and combine their analysis with previously published cases. We show that these tumours have recurrent and mutually exclusive mutations in either ACVR1 or EGFR and are characterised by high expression of EZHIP associated to its promoter hypomethylation. Affected patients share a similar poor prognosis as patients with H3K27M DMG. Global molecular analysis of H3-WT and H3K27M DMG reveal distinct transcriptome and methylome profiles including differential methylation of homeobox genes involved in development and cellular differentiation. Patients have distinct clinical features, with a trend demonstrating ACVR1 mutations occurring in H3-WT tumours at an older age. This in-depth exploration of H3-WT tumours further characterises this novel DMG, H3K27-altered sub-group, characterised by a specific immunohistochemistry profile with H3K27me3 loss, wild-type H3K27M and positive EZHIP. It also gives new insights into the possible mechanism and pathway regulation in these tumours, potentially opening new therapeutic avenues for these tumours which have no known effective treatment.This study has been retrospectively registered on clinicaltrial.gov on 8 November 2017 under the registration number NCT03336931 (https://clinicaltrials.gov/ct2/show/NCT03336931).

Purified regenerating retinal neurons reveal regulatory role of DNA methylation-mediated Na+/K+-ATPase in murine axon regeneration

While embryonic mammalian central nervous system (CNS) axons readily grow and differentiate, only a minority of fully differentiated mature CNS neurons are able to regenerate injured axons, leading to stunted functional recovery after injury and disease. To delineate DNA methylation changes specifically associated with axon regeneration, we used a Fluorescent-Activated Cell Sorting (FACS)-based methodology in a rat optic nerve transection model to segregate the injured retinal ganglion cells (RGCs) into regenerating and non-regenerating cell populations. Whole-genome DNA methylation profiling of these purified neurons revealed genes and pathways linked to mammalian RGC regeneration. Moreover, whole-methylome sequencing of purified uninjured adult and embryonic RGCs identified embryonic molecular profiles reactivated after injury in mature neurons, and others that correlate specifically with embryonic or adult axon growth, but not both. The results highlight the contribution to both embryonic growth and adult axon regeneration of subunits encoding the Na+/K+-ATPase. In turn, both biochemical and genetic inhibition of the Na+/K+-ATPase pump significantly reduced RGC axon regeneration. These data provide critical molecular insights into mammalian CNS axon regeneration, pinpoint the Na+/K+-ATPase as a key regulator of regeneration of injured mature CNS axons, and suggest that successful regeneration requires, in part, reactivation of embryonic signals.

A comparison of DNA methylation profiles of blood mononuclear cells in patients with multiple sclerosis in remission and relapse

To study the whole-genome DNA methylation profiles of peripheral blood mononuclear blood cells (PBMCs) of patients with relapsing-remitting multiple sclerosis (RRMS) in remission and relapse in order to assess the contribution of this epigenetic mechanism of gene expression regulation to the activity of the pathological process. Eight patients with RRMS in remission and 6 patients in relapse were included in the study. Methylation levels of DNA CpG sites in PBMCs were analyzed using Infinium HumanMethylation450 BeadChip DNA microarrays. Seven differentially methylated positions (DMPs) were identified, of which 3 were hypermethylated (cg02981003, cg18486102, cg19533582) and 4 were hypomethylated (cg16814680, cg1964802, cg18584440, cg08291996) during RRMS relapse. Five DMPs are located in protein-coding genes (GPR123, FAIM2, BTNL2, ZNF8, ASAP2), one in microRNA gene (MIR548N), and one in an intergenic region. For all identified DMPs, we observed a change in DNA methylation levels of more than 20% (range 20.2-57.5%). Hierarchical clustering of DNA samples on the heatmap shows their clear aggregation into separate clusters corresponding to RRMS patients in the stages of relapse and remission. For the first time it was shown that during relapse and remission of RRMS there are differences in the DNA methylation profile that allow discrimination between these clinical stages. These data indicate the involvement of the epigenetic mechanism of DNA methylation in the activation of the pathological process in RRMS.

Clinical utility of whole-genome DNA methylation profiling as a primary molecular diagnostic assay for central nervous system tumors-A prospective study and guidelines for clinical testing.

Central nervous system (CNS) cancer is the 10th leading cause of cancer-associated deaths for adults, but the leading cause in pediatric patients and young adults. The variety and complexity of histologic subtypes can lead to diagnostic errors. DNA methylation is an epigenetic modification that provides a tumor type-specific signature that can be used for diagnosis. We performed a prospective study using DNA methylation analysis as a primary diagnostic method for 1921 brain tumors. All tumors received a pathology diagnosis and profiling by whole genome DNA methylation, followed by next-generation DNA and RNA sequencing. Results were stratified by concordance between DNA methylation and histopathology, establishing diagnostic utility. Of the 1602 cases with a World Health Organization histologic diagnosis, DNA methylation identified a diagnostic mismatch in 225 cases (14%), 78 cases (5%) did not classify with any class, and in an additional 110 (7%) cases DNA methylation confirmed the diagnosis and provided prognostic information. Of 319 cases carrying 195 different descriptive histologic diagnoses, DNA methylation provided a definitive diagnosis in 273 (86%) cases, separated them into 55 methylation classes, and changed the grading in 58 (18%) cases. DNA methylation analysis is a robust method to diagnose primary CNS tumors, improving diagnostic accuracy, decreasing diagnostic errors and inconclusive diagnoses, and providing prognostic subclassification. This study provides a framework for inclusion of DNA methylation profiling as a primary molecular diagnostic test into professional guidelines for CNS tumors. The benefits include increased diagnostic accuracy, improved patient management, and refinements in clinical trial design.

Integrative Methylome and Transcriptome Characterization Identifies SERINC2 as a Tumor-Driven Gene for Papillary Thyroid Carcinoma

Most papillary thyroid carcinomas (PTCs) can be diagnosed preoperatively by routine evaluation, such as thyroid ultrasonography and fine-needle aspiration biopsy. Nevertheless, understanding how to differentiate indolent thyroid tumors from aggressive thyroid cancers remains a challenge, which may cause overtreatment. This study aimed to identify papillary thyroid cancer-specific indicators with whole-genome DNA methylation and gene expression profiles utilizing Infinium Methylation EPIC BeadChip (850k) and RNA arrays. In this paper, we report SERINC2 as a potential tumor-driven indicator in PTC. The up-regulated expression levels of SERINC2 were verified in PTC cell lines via qPCR. Then, cell counting kit 8 (CCK-8), wound healing, and flow cytometric assays were performed to confirm the influence of SERINC2 on proliferation and apoptosis in PTC cell lines after intervention or overexpression. Moreover, the investigation of data from the Cancer Dependency Map (DepMap) provided a potential pathway targeted by SERINC2. The activation of the tryptophan metabolic pathway may reduce the dependency of SERINC2 in thyroid cancers. In conclusion, our results demonstrate the whole-genome DNA methylation and gene expression profiles of papillary thyroid carcinoma, identify SERINC2 as a potential tumor-driven biomarker, and preliminarily verify its function in PTC.

Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling

Background: DNA methylation, the most common epigenetic modification, is defined as the removal or addition of methyl groups to cytosine bases. Studying DNA methylation provides insight into the regulation of gene expression, transposon mobility, genomic stability, and genomic imprinting. Whole-genome DNA methylation profiling (WGDM) is a powerful tool to find DNA methylation. This technique combines standard whole-genome sequencing methodology (e.g., Illumina high-throughput sequencing) with additional steps where unmethylated cytosine is converted to uracil. However, factors such as low cytosine conversion efficiency and inadequate DNA recovery during sample preparation oftentimes render poor-quality data. It is therefore imperative to benchmark sample preparation protocols to increase sequencing data quality and reduce false positives in methylation detection. Methods: A survey analysis was performed to investigate the efficiency of the following commercially available cytosine conversion kits when coupled with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB): Zymo Research EZ DNA Methylation™ kit (hereafter known as Zymo Conversion kit), QIAGEN EpiTect Bisulfite kit (hereafter known as QIAGEN Conversion kit), and NEBNext® Enzymatic Methyl-seq Conversion Module (hereafter known as NEB EM-seq kit). Input DNA was derived from soybean (Glycine max [L.] Merrill) leaf tissue. Results: Of those tested, the QIAGEN Conversion kit provided the best sample recovery and the highest number of sequencing reads, whereas the Zymo Conversion kit had the best cytosine conversion efficiency and the least duplication. The sequence library obtained with the NEB EM-seq kit had the highest mapping efficiency (percentage of reads mapped to the genome). The data quality (defined by Phred score) and methylated cytosine call were similar between kits. Conclusions: This study offers the groundwork for selecting an effective DNA methylation detection kit for crop genome research.

Single-base methylome analysis reveals dynamic changes of genome-wide DNA methylation associated with rapid stem growth of woody bamboos.

CG and CHG methylation levels in the rapid shoot growth stages (ST2-ST4) of woody bamboos were obviously decreased, which might regulate the internode elongation during rapid shoot growth, while CHH methylation was strongly associated with shoot developmental time or age. DNA methylation plays a critical role in the regulation of plant growth and development. Woody bamboos have a unique trait of rapid stem growth resulted from internode elongation at the shooting period. However, it is still unclear whether DNA methylation significantly controls the bamboo rapid stem growth. Here we present whole-genome DNA methylation profiles of the paleotropical woody bamboo Bonia amplexicaulis at five newly defined stages of shoot growth, named ST1-ST5. We found that CG and CHG methylation levels in the rapid shoot growth stages (ST2-ST4) were significantly lower than in the incubation (ST1) and plateau stages (ST5). The changes in methylation levels mainly occurred in flanking regions of genes and gene body regions, and 23647 differentially methylated regions (DMRs) were identified between ST1 and rapid shoot growth stages (ST2-ST4). Combined with transcriptome analysis, we found that DMR-related genes enriched in the auxin and jasmonic acid (JA) signal transduction, and other pathways closely related to plant growth. Intriguingly, CHH methylation was not involved in the rapid shoot growth, but strongly associated with shoot developmental time by gradually accumulating in transposable elements (TEs) regions. Overall, our results reveal the importance of DNA methylation in regulating the bamboo rapid shoot growth and suggest a role of DNA methylation associated with development time or age in woody bamboos.

Upfront Biology-Guided Therapy in Diffuse Intrinsic Pontine Glioma: Therapeutic, Molecular, and Biomarker Outcomes from PNOC003.

PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Patients (3-25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN). Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2-18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7-14; TP53wt 13.3 mo; 95% CI, 11.8-NA; P = 3.4e-2), genome instability (P = 3.1e-3), and RT resistance (P = 6.4e-4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome. Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs.

EPEN-18. Oncogenic 3D genome conformations identify novel therapeutic targets in ependymoma

Ependymoma (EPN) is an aggressive pediatric tumor that occurs throughout the central nervous system. The two most aggressive molecular subgroups of EPN are the supratentorial ZFTA-fusion associated group (ST-EPN-ZFTA) and the posterior fossa group A (PF-EPN-A). Although the molecular characteristics underlying the tumorigenesis of these subgroups have been extensively studied, these tumors remain difficult to treat. Hence, innovative therapeutic approaches are urgently needed. Here, we used genome-wide chromosome conformation capture (Hi-C), complemented with CTCF (insulators) and H3K27ac (active enhancers) ChIP-seq, as well as gene expression and whole-genome DNA methylation profiling in primary and relapsed EPN tumors and cell lines, to identify chromosomal rearrangements and regulatory mechanisms underlying aberrant expression of genes that are essential for EPN tumorigenesis. By integrating these heterogenous data types, we have observed the formation of new topologically associated domains (‘neo-TADs’) caused by intra- and inter-chromosomal structural variants in both tumors. In addition, we observed 3D chromatin complexes of regulatory elements, and the replacement of CTCF insulators by DNA hyper-methylation in PF-EPN-A tumors. These tumor-specific 3D genome conformations can be associated with the transcriptional upregulation of nearby genes. Through inhibition experiments we validated that these newly identified genes, including RCOR2, ITGA6, LAMC1, and ARL4C, are highly essential for the survival of patient-derived EPN cell lines in a disease subgroup-specific manner. Thus, our study identifies novel potential therapeutic vulnerabilities in EPN and extends our ability to reveal tumor-dependency genes and pathways by oncogenic 3D genome conformations even in tumors that lack known genetic alterations.

CD19-CAR T Cells Develop Exhaustion Epigenetic Programs During a Clinical Response

Abstract The goal of this study was to determine the epigenetic landscape of CD19-CAR T cells pre and post infusion in leukemia patients as an initial step to elucidate intrinsic mechanisms that limit CAR T-cell effector functions in humans. A longitudinal analysis of CD8+ CD19-CAR T cell epigenetic changes was performed by whole-genome DNA methylation profiling of CAR T cells during manufacturing and from peripheral blood mononuclear cells (PBMCs) of 15 patients enrolled on our institutional, autologous CD19-CAR T cell therapy study (NCT03573700). CAR T cell expansion and persistence were determined by measuring vector copy numbers in the PBMCs of treated patients. We had previously established novel exhaustion DNA methylation datasets that delineate between progenitor and fully exhausted T cells. These datasets served as a guide for stratifying our post-infusion CAR T cells along the exhaustion developmental trajectory. Our data show that CD19-CAR T cells lose repressive DNA methylation at effector loci (e.g. PRF1, TBET) while gaining methylation at genes associated with memory potential (e.g. LEF1, TCF7). We confirmed these epigenetic changes are coupled to endogenous human T cell effector and memory differentiation by cross-referencing our epigenetic data with publicly available transcriptional profiles for antigen-specific effector and long-lived memory CD8 T cells from individuals vaccinated for yellow fever. Furthermore, we show that CAR T cells were unable to mount an in vivo recall response after relapse of antigen-positive disease or recovery of endogenous B cells. These observations support the conclusion that CD19-CAR T cells acquire stable epigenetic exhaustion programs that limit their protective capacity. This work was supported by the National Institutes of Health (1R01AI114442 to BY and LRP to CCZ), the National Comprehensive Cancer Network Young Investigator Award (to CZ), Alex’s Lemonade Stand Foundation Young Investigator Grant (to CZ), Stand Up to Cancer- SU2C (to BY), the American Lebanese Syrian Associated Charities (ALSAC) to BY, and Assisi foundation to BY.

Inheritance of social dominance is associated with global sperm DNA methylation in inbred male mice.

Dominance relationships between males and their associated traits are usually heritable and have implications for sexual selection in animals. In particular, social dominance and its related male pheromones are heritable in inbred mice; thus, we wondered whether epigenetic changes due to altered levels of DNA methylation determine inheritance. Here, we used C57BL/6 male mice to establish a social dominance-subordination relationship through chronic dyadic encounters, and this relationship and pheromone covariation occurred in their offspring, indicative of heritability. Through transcriptome sequencing and whole-genome DNA methylation profiling of the sperm of both generations, we found that differential methylation of many genes was induced by social dominance-subordination in sires and could be passed on to the offspring. These methylated genes were mainly related to growth and development processes, neurodevelopment, and cellular transportation. The expression of the genes with similar functions in whole-genome methylation/bisulfite sequencing was also differentiated by social dominance-subordination, as revealed by RNA-seq. In particular, the gene Dennd1a, which regulates neural signaling, was differentially methylated and expressed in the sperm and medial prefrontal cortex in paired males before and after dominance-subordination establishment, suggesting the potential epigenetic control and inheritance of social dominance-related aggression. We suggest that social dominance might be passed on to male offspring through sperm DNA methylation and that the differences could potentially affect male competition in offspring by affecting the development of the nervous system.