On correlative and causal links of replicative epimutations.

The mitotic inheritability of DNA methylation as an epigenetic marker in higher-order eukaryotes has been established for >40 years. The DNA methylome and mitotic division interplay is now considered bidirectional and highly intertwined. Various epigenetic writers, erasers, and modulators shape the perceived replicative methylation dynamics. This Review surveys the principles and complexity of mitotic transmission of DNA methylation, emphasizing the awareness of mitotic aging in analyzing DNA methylation dynamics in development and disease. We reviewed how DNA methylation changes alter mitotic proliferation capacity, implicating age-related diseases like cancer. We link replicative epimutation to stem cell dysfunction, inflammatory response, cancer risks, and epigenetic clocks, discussing the causative role of DNA methylation in health and disease.

The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

BACKGROUND: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell-type composition. RESULTS: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. CONCLUSIONS: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

mLiftOver: harmonizing data across Infinium DNA methylation platforms.

MOTIVATION: Infinium DNA methylation BeadChips are widely used for genome-wide DNA methylation profiling at the population scale. Recent updates to probe content and naming conventions in the EPIC version 2 (EPICv2) arrays have complicated integrating new data with previous Infinium array platforms, such as the MethylationEPIC (EPIC) and the HumanMethylation450 (HM450) BeadChip. RESULTS: We present mLiftOver, a user-friendly tool that harmonizes probe ID, methylation level, and signal intensity data across different Infinium platforms. It manages probe replicates, missing data imputation, and platform-specific bias for accurate data conversion. We validated the tool by applying HM450-based cancer classifiers to EPICv2 cancer data, achieving high accuracy. Additionally, we successfully integrated EPICv2 healthy tissue data with legacy HM450 data for tissue identity analysis and produced consistent copy number profiles in cancer cells. AVAILABILITY AND IMPLEMENTATION: mLiftOver is implemented R and available in the Bioconductor package SeSAMe (version 1.21.13+): https://bioconductor.org/packages/release/bioc/html/sesame.html. Analysis of EPIC and EPICv2 platform-specific bias and high-confidence mapping is available at https://github.com/zhou-lab/InfiniumAnnotationV1/raw/main/Anno/EPICv2/EPICv2ToEPIC_conversion.tsv.gz. The source code is available at https://github.com/zwdzwd/sesame/blob/devel/R/mLiftOver.R under the MIT license.

TET1 displays catalytic and non-catalytic functions in the adult mouse cortex.

TET1/2/3 dioxygenases iteratively demethylate 5-methylcytosine, beginning with the formation of 5-hydroxymethylcytosine (5hmC). The post-mitotic brain maintains higher levels of 5hmC than most peripheral tissues, and TET1 ablation studies have underscored the critical role of TET1 in brain physiology. However, deletion of Tet1 precludes the disentangling of the catalytic and non-catalytic functions of TET1. Here, we dissect these functions of TET1 by comparing adult cortex of Tet1 wildtype (Tet1 WT), a novel Tet1 catalytically dead mutant (Tet1 HxD), and Tet1 knockout (Tet1 KO) mice. Using DNA methylation array, we uncover that Tet1 HxD and KO mutations perturb the methylation status of distinct subsets of CpG sites. Gene ontology (GO) analysis on specific differential 5hmC regions indicates that TET1's catalytic activity is linked to neuronal-specific functions. RNA-Seq further shows that Tet1 mutations predominantly impact the genes that are associated with alternative splicing. Lastly, we performed High-performance Liquid Chromatography Mass-Spectrometry lipidomics on WT and mutant cortices and uncover accumulation of lysophospholipids lysophosphatidylethanolamine and lysophosphatidylcholine in Tet1 HxD cortex. In summary, we show that Tet1 HxD does not completely phenocopy Tet1 KO, providing evidence that TET1 modulates distinct cortical functions through its catalytic and non-catalytic roles.

Inflammatory and tissue injury marker dynamics in pediatric acute respiratory distress syndrome.

BACKGROUNDThe molecular signature of pediatric acute respiratory distress syndrome (ARDS) is poorly described, and the degree to which hyperinflammation or specific tissue injury contributes to outcomes is unknown. Therefore, we profiled inflammation and tissue injury dynamics over the first 7 days of ARDS, and associated specific biomarkers with mortality, persistent ARDS, and persistent multiple organ dysfunction syndrome (MODS).METHODSIn a single-center prospective cohort of intubated pediatric patients with ARDS, we collected plasma on days 0, 3, and 7. Nineteen biomarkers reflecting inflammation, tissue injury, and damage-associated molecular patterns (DAMPs) were measured. We assessed the relationship between biomarkers and trajectories with mortality, persistent ARDS, or persistent MODS using multivariable mixed effect models.RESULTSIn 279 patients (64 [23%] nonsurvivors), hyperinflammatory cytokines, tissue injury markers, and DAMPs were higher in nonsurvivors. Survivors and nonsurvivors showed different biomarker trajectories. IL-1α, soluble tumor necrosis factor receptor 1, angiopoietin 2 (ANG2), and surfactant protein D increased in nonsurvivors, while DAMPs remained persistently elevated. ANG2 and procollagen type III N-terminal peptide were associated with persistent ARDS, whereas multiple cytokines, tissue injury markers, and DAMPs were associated with persistent MODS. Corticosteroid use did not impact the association of biomarker levels or trajectory with mortality.CONCLUSIONSPediatric ARDS survivors and nonsurvivors had distinct biomarker trajectories, with cytokines, endothelial and alveolar epithelial injury, and DAMPs elevated in nonsurvivors. Mortality markers overlapped with markers associated with persistent MODS, rather than persistent ARDS.FUNDINGNIH (K23HL-136688, R01-HL148054).

UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.

The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. Nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). Here, we profile contributions of UHRF1 and DNMT1 to genome-wide DNA methylation inheritance and dissect specific roles for ubiquitin signaling in this process. We reveal DNA methylation maintenance at low-density CpGs is vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMD), a methylation signature observed across human cancers. Furthermore, disrupting DNMT1 UIM2 function abolishes DNA methylation maintenance. Collectively, we show DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers.

Comprehensive Evaluation of The Infinium Human MethylationEPIC v2 BeadChip.

Infinium Methylation BeadChips are widely used to profile DNA cytosine modifications in large cohort studies for reasons of cost-effectiveness, accurate quantification, and user-friendly data analysis in characterizing these canonical epigenetic marks. In this work, we conducted a comprehensive evaluation of the updated Infinium MethylationEPIC v2 BeadChip (EPICv2). Our evaluation revealed that EPICv2 offers significant improvements over its predecessors, including expanded enhancer coverage, applicability to diverse ancestry groups, support for low-input DNA down to one nanogram, coverage of existing epigenetic clocks, cell type deconvolution panels, and human trait associations, while maintaining accuracy and reproducibility. Using EPICv2, we were able to identify epigenome and sequence signatures in cell line models of DNMT and SETD2 loss and/or hypomorphism. Furthermore, we provided probe-wise evaluation and annotation to facilitate the use of new features on this array for studying the interplay between somatic mutations and epigenetic landscape in cancer genomics. In conclusion, EPICv2 provides researchers with a valuable tool for studying epigenetic modifications and their role in development and disease.

IL-1ß expression driven by androgen receptor absence or inactivation promotes prostate cancer bone metastasis.

We report the inverse association between the expression of androgen receptor (AR) and interleukin-1beta (IL-1ß) in a cohort of patients with metastatic castration resistant prostate cancer (mCRPC). We also discovered that AR represses the IL-1ß gene by binding an androgen response element (ARE) half-site located within the promoter, which explains the IL-1ß expression in AR-negative (ARNEG) cancer cells. Consistently, androgen-depletion or AR-pathway inhibitors (ARIs) de-repressed IL-1ß in ARPOS cancer cells, both in vitro and in vivo. The AR transcriptional repression is sustained by histone de-acetylation at the H3K27 mark in the IL-1ß promoter. Notably, patients' data suggest that DNA methylation prevents IL-1ß expression, even if the AR-signaling axis is inactive. Our previous studies show that secreted IL-1ß supports metastatic progression in mice by altering the transcriptome of tumor-associated bone stroma. Thus, in prostate cancer patients harboring ARNEG tumor cells or treated with ADT/ARIs, and with the IL-1ß gene unmethylated, IL-1ß could condition the metastatic microenvironment to sustain disease progression.

Characterizing DNA methylation signatures of retinoblastoma using aqueous humor liquid biopsy.

Retinoblastoma (RB) is a cancer that forms in the developing retina of babies and toddlers. The goal of therapy is to cure the tumor, save the eye and maximize vision. However, it is difficult to predict which eyes are likely to respond to therapy. Predictive molecular biomarkers are needed to guide prognosis and optimize treatment decisions. Direct tumor biopsy is not an option for this cancer; however, the aqueous humor (AH) is an alternate source of tumor-derived cell-free DNA (cfDNA). Here we show that DNA methylation profiling of the AH is a valid method to identify the methylation status of RB tumors. We identify 294 genes directly regulated by methylation that are implicated in p53 tumor suppressor (RB1, p53, p21, and p16) and oncogenic (E2F) pathways. Finally, we use AH to characterize molecular subtypes that can potentially be used to predict the likelihood of treatment success for retinoblastoma patients.

DNA methylation dynamics and dysregulation delineated by high-throughput profiling in the mouse.

We have developed a mouse DNA methylation array that contains 296,070 probes representing the diversity of mouse DNA methylation biology. We present a mouse methylation atlas as a rich reference resource of 1,239 DNA samples encompassing distinct tissues, strains, ages, sexes, and pathologies. We describe applications for comparative epigenomics, genomic imprinting, epigenetic inhibitors, patient-derived xenograft assessment, backcross tracing, and epigenetic clocks. We dissect DNA methylation processes associated with differentiation, aging, and tumorigenesis. Notably, we find that tissue-specific methylation signatures localize to binding sites for transcription factors controlling the corresponding tissue development. Age-associated hypermethylation is enriched at regions of Polycomb repression, while hypomethylation is enhanced at regions bound by cohesin complex members. Apc Min/+ polyp-associated hypermethylation affects enhancers regulating intestinal differentiation, while hypomethylation targets AP-1 binding sites. This Infinium Mouse Methylation BeadChip (version MM285) is widely accessible to the research community and will accelerate high-sample-throughput studies in this important model organism.

A B-cell developmental gene regulatory network is activated in infant AML.

Infant Acute Myeloid Leukemia (AML) is a poorly-addressed, heterogeneous malignancy distinguished by surprisingly few mutations per patient but accompanied by myriad age-specific translocations. These characteristics make treatment of infant AML challenging. While infant AML is a relatively rare disease, it has enormous impact on families, and in terms of life-years-lost and life limiting morbidities. To better understand the mechanisms that drive infant AML, we performed integrative analyses of genome-wide mRNA, miRNA, and DNA-methylation data in diagnosis-stage patient samples. Here, we report the activation of an onco-fetal B-cell developmental gene regulatory network in infant AML. AML in infants is genomically distinct from AML in older children/adults in that it has more structural genomic aberrations and fewer mutations. Differential expression analysis of ~1500 pediatric AML samples revealed a large number of infant-specific genes, many of which are associated with B cell development and function. 18 of these genes form a well-studied B-cell gene regulatory network that includes the epigenetic regulators BRD4 and POU2AF1, and their onco-fetal targets LIN28B and IGF2BP3. All four genes are hypo-methylated in infant AML. Moreover, micro-RNA Let7a-2 is expressed in a mutually exclusive manner with its target and regulator LIN28B. These findings suggest infant AML may respond to bromodomain inhibitors and immune therapies targeting CD19, CD20, CD22, and CD79A.

Analytical protocol to identify local ancestry-associated molecular features in cancer.

People of different ancestries vary in cancer risk and outcome, and their molecular differences may indicate sources of these variations. Determining the "local" ancestry composition at each genetic locus across ancestry-admixed populations can suggest causal associations. We present a protocol to identify local ancestry and detect the associated molecular changes, using data from the Cancer Genome Atlas. This workflow can be applied to cancer cohorts with matched tumor and normal data from admixed patients to examine germline contributions to cancer. For complete details on the use and execution of this protocol, please refer to Carrot-Zhang et al. (2020).

Integrative modeling identifies genetic ancestry-associated molecular correlates in human cancer.

Cellular and molecular aberrations contribute to the disparity of human cancer incidence and etiology between ancestry groups. Multiomics profiling in The Cancer Genome Atlas (TCGA) allows for querying of the molecular underpinnings of ancestry-specific discrepancies in human cancer. Here, we provide a protocol for integrative associative analysis of ancestry with molecular correlates, including somatic mutations, DNA methylation, mRNA transcription, miRNA transcription, and pathway activity, using TCGA data. This protocol can be generalized to analyze other cancer cohorts and human diseases. For complete details on the use and execution of this protocol, please refer to Carrot-Zhang et al. (2020).

Mimicking the Endometrial Cancer Tumor Microenvironment to Reprogram Tumor-Associated Macrophages in Disintegrable Supramolecular Gelatin Hydrogel.

PURPOSE: Besides the tumor cells themselves, solid tumors are comprised of numerous cell types including infiltrating immune cells such as tumor-associated macrophages (TAMs). TAMs are vital stromal components of host immune system and play a critical role in the development of cancer. TAMs can be divided into two subtypes: M1 tumor-suppressive macrophage and M2 tumor-supportive macrophage. To better address the observations of TAMs functional performance, we describe an in vitro system that mimics the populations of TAMs infiltrated into the tumor mass by using our disintegrable supramolecular gelatin (DSG) hydrogels, which are physically crosslinked by host-guest complexations. MATERIALS AND METHODS: The host-guest interaction was adopted between the aromatic groups of gelatin and the photocrosslinkable acrylated ß-cyclodextrins (Ac-ß-CDs) to form the DSG hydrogels. The convenient macrophage/endometrial cancer cells heterospheroid 3D model was set up by DSG hydrogels. RT-PCR and Western blot assays were developed to evaluate the efficiencies of inducers on the macrophages. The ELISA and oxygen saturation assays were performed to measure the secretion of VEGF and consumption of oxygen of tumor and/or macrophages, respectively. To determine the antitumor effects of M2 reprogrammed macrophages in vitro and in vivo, migration assay and tumor xenograft model were used, respectively. RESULTS: The host-guest complexations of DSG hydrogels were controllably broken efficiently by soaking into the solution of competitive guest monomers 1-adamantanamine hydrochloride. The DSG hydrogels help IFN-γ reprogram the M2 to M1 and then decrease the tumor/M2 reprogrammed macrophage cells heterospheroid secretion of VEGF and increase the relative oxygen saturation. Significantly, the co-cultural tumor/M2 reprogrammed group from the disintegrated DSG hydrogels reduced the migration of cancer cells in vitro and the tumor growth in vivo. CONCLUSION: We obtain a TAMs/tumor microenvironment-responsive 3D model based on the novel DSG hydrogels, and will be of utility in cancer therapy and drug discovery.

Large DNA Methylation Nadirs Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity.

Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of ∼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.

Comprehensive Analysis of Genetic Ancestry and Its Molecular Correlates in Cancer.

We evaluated ancestry effects on mutation rates, DNA methylation, and mRNA and miRNA expression among 10,678 patients across 33 cancer types from The Cancer Genome Atlas. We demonstrated that cancer subtypes and ancestry-related technical artifacts are important confounders that have been insufficiently accounted for. Once accounted for, ancestry-associated differences spanned all molecular features and hundreds of genes. Biologically significant differences were usually tissue specific but not specific to cancer. However, admixture and pathway analyses suggested some of these differences are causally related to cancer. Specific findings included increased FBXW7 mutations in patients of African origin, decreased VHL and PBRM1 mutations in renal cancer patients of African origin, and decreased immune activity in bladder cancer patients of East Asian origin.

Activation of a Subset of Evolutionarily Young Transposable Elements and Innate Immunity Are Linked to Clinical Responses to 5-Azacytidine.

The DNA methyltransferase inhibitors (DNMTi) 5-azacytidine and 5-aza-2-deoxycytidine have been approved for the treatment of different types of hematologic malignancies. However, only about 50% of patients respond to treatment. Therefore, a more comprehensive understanding of the molecular changes in patients treated with DNMTi is needed. Here, we examined gene expression profiles in a total of 150 RNA samples from two adult cohorts and one pediatric cohort with hematologic cancers taken before, during, and after treatment with 5-azacytidine (40 patients; 15 nonresponders, 25 responders). Using each patient as their own control, malignant cells showed preferential activation of a subset of evolutionarily young transposable elements (TE), including endogenous retroviral long terminal repeats (LTR), short and long interspersed nuclear elements (SINE and LINE), and the type I IFN pathway in responders, all independent of disease classification. Transfection of eight upregulated LTRs into recipient human cells in culture showed robust and heterogenous activation of six genes in the type I IFN pathway. These results, obtained in diverse hematologic disease entities, show that common targets (TE) activated by the same drug (5-azacytidine) elicit an immune response, which may be important for patient's responses to DNMTi. SIGNIFICANCE: Activation of specific classes of evolutionarily young transposable elements can lead to activation of the innate immune system.

Before and After: Comparison of Legacy and Harmonized TCGA Genomic Data Commons' Data.

We present a systematic analysis of the effects of synchronizing a large-scale, deeply characterized, multi-omic dataset to the current human reference genome, using updated software, pipelines, and annotations. For each of 5 molecular data platforms in The Cancer Genome Atlas (TCGA)-mRNA and miRNA expression, single nucleotide variants, DNA methylation and copy number alterations-comprehensive sample, gene, and probe-level studies were performed, towards quantifying the degree of similarity between the 'legacy' GRCh37 (hg19) TCGA data and its GRCh38 (hg38) version as 'harmonized' by the Genomic Data Commons. We offer gene lists to elucidate differences that remained after controlling for confounders, and strategies to mitigate their impact on biological interpretation. Our results demonstrate that the hg19 and hg38 TCGA datasets are very highly concordant, promote informed use of either legacy or harmonized omics data, and provide a rubric that encourages similar comparisons as new data emerge and reference data evolve.

The chromatin accessibility landscape of primary human cancers.

We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709 transposase-accessible DNA elements that substantially extend the compendium of known cis-regulatory elements. Integration of ATAC-seq (the assay for transposase-accessible chromatin using sequencing) with TCGA multi-omic data identifies a large number of putative distal enhancers that distinguish molecular subtypes of cancers, uncovers specific driving transcription factors via protein-DNA footprints, and nominates long-range gene-regulatory interactions in cancer. These data reveal genetic risk loci of cancer predisposition as active DNA regulatory elements in cancer, identify gene-regulatory interactions underlying cancer immune evasion, and pinpoint noncoding mutations that drive enhancer activation and may affect patient survival. These results suggest a systematic approach to understanding the noncoding genome in cancer to advance diagnosis and therapy.