Publications by authors named "Wanding Zhou"

46 Publications

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

Cell Genom 2022 Jul;2(7)

Department of Epigenetics, Van Andel Institute, Grand Rapids, MI 49503, USA.

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. 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.
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http://dx.doi.org/10.1016/j.xgen.2022.100144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9306256PMC
July 2022

Computational methods for single-cell DNA methylomes.

Genomics Proteomics Bioinformatics 2022 Jun 16. Epub 2022 Jun 16.

Center for Computational and Genomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, United States; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, 19104, United States. Electronic address:

Dissecting intercellular epigenetic differences is key to understanding tissue heterogeneity. Recent advances in single-cell DNA methylome profiling have presented opportunities to resolve this heterogeneity at the maximum resolution. While these advances enable us to explore frontiers of chromatin biology and better understand cell lineage relationships, they pose new challenges in data processing and interpretation. This review surveys the current state of computational tools developed for single-cell DNA methylome data. We discuss critical components of single-cell DNA methylome data analysis, including data preprocessing, quality control, imputation, dimensionality reduction, cell clustering, supervised cell annotation, cell lineage reconstruction, gene activity scoring, and integration with transcriptomics data. We will highlight unique aspects of single-cell DNA methylome data analysis and how techniques common to other single-cell omics data analyses can be adapted to analyzing DNA methylomes. Finally, we discuss existing challenges and opportunities for future development.
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http://dx.doi.org/10.1016/j.gpb.2022.05.007DOI Listing
June 2022

A mammalian methylation array for profiling methylation levels at conserved sequences.

Nat Commun 2022 02 10;13(1):783. Epub 2022 Feb 10.

Dept. of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, 90095, USA.

Infinium methylation arrays are not available for the vast majority of non-human mammals. Moreover, even if species-specific arrays were available, probe differences between them would confound cross-species comparisons. To address these challenges, we developed the mammalian methylation array, a single custom array that measures up to 36k CpGs per species that are well conserved across many mammalian species. We designed a set of probes that can tolerate specific cross-species mutations. We annotate the array in over 200 species and report CpG island status and chromatin states in select species. Calibration experiments demonstrate the high fidelity in humans, rats, and mice. The mammalian methylation array has several strengths: it applies to all mammalian species even those that have not yet been sequenced, it provides deep coverage of conserved cytosines facilitating the development of epigenetic biomarkers, and it increases the probability that biological insights gained in one species will translate to others.
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http://dx.doi.org/10.1038/s41467-022-28355-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8831611PMC
February 2022

Host methylation predicts SARS-CoV-2 infection and clinical outcome.

Commun Med (Lond) 2021 26;1(1):42. Epub 2021 Oct 26.

Illumina, Inc., San Diego, CA USA.

Background: Since the onset of the SARS-CoV-2 pandemic, most clinical testing has focused on RT-PCR. Host epigenome manipulation post coronavirus infection suggests that DNA methylation signatures may differentiate patients with SARS-CoV-2 infection from uninfected individuals, and help predict COVID-19 disease severity, even at initial presentation.

Methods: We customized Illumina's Infinium MethylationEPIC array to enhance immune response detection and profiled peripheral blood samples from 164 COVID-19 patients with longitudinal measurements of disease severity and 296 patient controls.

Results: Epigenome-wide association analysis revealed 13,033 genome-wide significant methylation sites for case-vs-control status. Genes and pathways involved in interferon signaling and viral response were significantly enriched among differentially methylated sites. We observe highly significant associations at genes previously reported in genetic association studies ( , ). Using machine learning techniques, models built using sparse regression yielded highly predictive findings: cross-validated best fit AUC was 93.6% for case-vs-control status, and 79.1%, 80.8%, and 84.4% for hospitalization, ICU admission, and progression to death, respectively.

Conclusions: In summary, the strong COVID-19-specific epigenetic signature in peripheral blood driven by key immune-related pathways related to infection status, disease severity, and clinical deterioration provides insights useful for diagnosis and prognosis of patients with viral infections.
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http://dx.doi.org/10.1038/s43856-021-00042-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8767772PMC
October 2021

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

PLoS One 2021 18;16(11):e0259197. Epub 2021 Nov 18.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0259197PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601427PMC
December 2021

EHMT2 suppresses the variation of transcriptional switches in the mouse embryo.

PLoS Genet 2021 11 18;17(11):e1009908. Epub 2021 Nov 18.

Department of Epigenetics, Van Andel Institute, Grand Rapids, Michigan, United States of America.

EHMT2 is the main euchromatic H3K9 methyltransferase. Embryos with zygotic, or maternal mutation in the Ehmt2 gene exhibit variable developmental delay. To understand how EHMT2 prevents variable developmental delay we performed RNA sequencing of mutant and somite stage-matched normal embryos at 8.5-9.5 days of gestation. Using four-way comparisons between delayed and normal embryos we clarified what it takes to be normal and what it takes to develop. We identified differentially expressed genes, for example Hox genes that simply reflected the difference in developmental progression of wild type and the delayed mutant uterus-mate embryos. By comparing wild type and zygotic mutant embryos along the same developmental window we detected a role of EHMT2 in suppressing variation in the transcriptional switches. We identified transcription changes where precise switching during development occurred only in the normal but not in the mutant embryo. At the 6-somite stage, gastrulation-specific genes were not precisely switched off in the Ehmt2-/- zygotic mutant embryos, while genes involved in organ growth, connective tissue development, striated muscle development, muscle differentiation, and cartilage development were not precisely switched on. The Ehmt2mat-/+ maternal mutant embryos displayed high transcriptional variation consistent with their variable survival. Variable derepression of transcripts occurred dominantly in the maternally inherited allele. Transcription was normal in the parental haploinsufficient wild type embryos despite their delay, consistent with their good prospects. Global profiling of transposable elements revealed EHMT2 targeted DNA methylation and suppression at LTR repeats, mostly ERVKs. In Ehmt2-/- embryos, transcription over very long distances initiated from such misregulated 'driver' ERVK repeats, encompassing a multitude of misexpressed 'passenger' repeats. In summary, EHMT2 reduced transcriptional variation of developmental switch genes and developmentally switching repeat elements at the six-somite stage embryos. These findings establish EHMT2 as a suppressor of transcriptional and developmental variation at the transition between gastrulation and organ specification.
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http://dx.doi.org/10.1371/journal.pgen.1009908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601470PMC
November 2021

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

STAR Protoc 2021 12 20;2(4):100766. Epub 2021 Sep 20.

The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.

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).
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http://dx.doi.org/10.1016/j.xpro.2021.100766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8456058PMC
December 2021

Evaluation of whole-genome DNA methylation sequencing library preparation protocols.

Epigenetics Chromatin 2021 06 19;14(1):28. Epub 2021 Jun 19.

Department of Epigenetics, Van Andel Research Institute, 333 Bostwick Avenue NE, Grand Rapids, MI, 49503, USA.

Background: With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types.

Results: An assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor.

Results: The NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences's Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.
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http://dx.doi.org/10.1186/s13072-021-00401-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214260PMC
June 2021

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

STAR Protoc 2021 06 19;2(2):100483. Epub 2021 Apr 19.

Center for Computational and Genomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.

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).
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http://dx.doi.org/10.1016/j.xpro.2021.100483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082263PMC
June 2021

DNA methylation enables transposable element-driven genome expansion.

Proc Natl Acad Sci U S A 2020 08 27;117(32):19359-19366. Epub 2020 Jul 27.

Center for Epigenetics, Van Andel Institute, Grand Rapids, MI 49503

Multicellular eukaryotic genomes show enormous differences in size. A substantial part of this variation is due to the presence of transposable elements (TEs). They contribute significantly to a cell's mass of DNA and have the potential to become involved in host gene control. We argue that the suppression of their activities by methylation of the C-phosphate-G (CpG) dinucleotide in DNA is essential for their long-term accommodation in the host genome and, therefore, to its expansion. An inevitable consequence of cytosine methylation is an increase in C-to-T transition mutations via deamination, which causes CpG loss. Cytosine deamination is often needed for TEs to take on regulatory functions in the host genome. Our study of the whole-genome sequences of 53 organisms showed a positive correlation between the size of a genome and the percentage of TEs it contains, as well as a negative correlation between size and the CpG observed/expected (O/E) ratio in both TEs and the host DNA. TEs are seldom found at promoters and transcription start sites, but they are found more at enhancers, particularly after they have accumulated C-to-T and other mutations. Therefore, the methylation of TE DNA allows for genome expansion and also leads to new opportunities for gene control by TE-based regulatory sites.
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http://dx.doi.org/10.1073/pnas.1921719117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431005PMC
August 2020

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

Int J Nanomedicine 2020 25;15:4625-4637. Epub 2020 Jun 25.

Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.

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 β-cyclodextrins (Ac-β-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.
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http://dx.doi.org/10.2147/IJN.S252074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326693PMC
August 2020

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

Cancer Cell 2020 05;37(5):639-654.e6

The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, Boston, MA 02115, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ccell.2020.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328015PMC
May 2020

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

Mol Cell 2020 05;78(3):506-521.e6

Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2020.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357281PMC
May 2020

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

Cancer Res 2020 06 3;80(12):2441-2450. Epub 2020 Apr 3.

Department of Hematology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.

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.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507765PMC
June 2020

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

Cell Syst 2019 07;9(1):24-34.e10

National Cancer Institute, Bethesda, MD 20892, USA.

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.
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http://dx.doi.org/10.1016/j.cels.2019.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707074PMC
July 2019

Mother-child transmission of epigenetic information by tunable polymorphic imprinting.

Proc Natl Acad Sci U S A 2018 12 3;115(51):E11970-E11977. Epub 2018 Dec 3.

Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI 49503;

Genomic imprinting mediated by DNA methylation restricts gene expression to a single allele determined by parental origin and is not generally considered to be under genetic or environmental influence. Here, we focused on a differentially methylated region (DMR) of approximately 1.9 kb that includes a 101-bp noncoding RNA gene (/), which is maternally imprinted in ∼75% of humans. This is unlike other imprinted genes, which demonstrate monoallelic methylation in 100% of individuals. The DMR includes a CTCF binding site on the centromeric side defining the DMR boundary and is flanked by a CTCF binding site on the telomeric side. The centromeric CTCF binding site contains an A/C polymorphism (rs2346018); the C allele is associated with less imprinting. The frequency of imprinting of the DMR in infants was linked to at least two nongenetic factors, maternal age at delivery and season of conception. In a separate cohort, imprinting was associated with lower body mass index in children at 5 y of age. Thus, we propose that the imprinting status of the DMR is "tunable" in that it is associated with maternal haplotype and prenatal environment. This provides a potential mechanism for transmitting information, with phenotypic consequences, from mother to child.
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http://dx.doi.org/10.1073/pnas.1815005115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304996PMC
December 2018

The chromatin accessibility landscape of primary human cancers.

Science 2018 10;362(6413)

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.
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http://dx.doi.org/10.1126/science.aav1898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408149PMC
October 2018

Dual Inhibition of DNA and Histone Methyltransferases Increases Viral Mimicry in Ovarian Cancer Cells.

Cancer Res 2018 10 5;78(20):5754-5766. Epub 2018 Sep 5.

Van Andel Research Institute, Grand Rapids, Michigan.

Ovarian cancer ranks as the most deadly gynecologic cancer, and there is an urgent need to develop more effective therapies. Previous studies have shown that G9A, a histone methyltransferase that catalyzes mono- and dimethylation of histone H3 lysine9, is highly expressed in ovarian cancer tumors, and its overexpression is associated with poor prognosis. Here we report that pharmacologic inhibition of G9A in ovarian cancer cell lines with high levels of expression induces synergistic antitumor effects when combined with the DNA methylation inhibitor (DNMTi) 5-aza-2'-deoxycytidine (5-aza-CdR). These antitumor effects included upregulation of endogenous retroviruses (ERV), activation of the viral defense response, and induction of cell death, which have been termed "viral mimicry" effects induced by DNMTi. G9Ai treatment further reduced H3K9me2 levels within the long terminal repeat regions of ERV, resulting in further increases of ERV expression and enhancing "viral mimicry" effects. In contrast, G9Ai and 5-aza-CdR were not synergistic in cell lines with low basal levels. Taken together, our results suggest that the synergistic effects of combination treatment with DNMTi and G9Ai may serve as a novel therapeutic strategy for patients with ovarian cancer with high levels of G9A expression. Dual inhibition of DNA methylation and histone H3 lysine 9 dimethylation by 5-aza-CdR and G9Ai results in synergistic upregulation of ERV and induces an antiviral response, serving as a basis for exploring this novel combination treatment in patients with ovarian cancer. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191339PMC
October 2018

SeSAMe: reducing artifactual detection of DNA methylation by Infinium BeadChips in genomic deletions.

Nucleic Acids Res 2018 11;46(20):e123

Center for Epigenetics, Van Andel Research Institute, 333 Bostwick Ave., N.E., Grand Rapids, MI 49503 USA.

We report a new class of artifacts in DNA methylation measurements from Illumina HumanMethylation450 and MethylationEPIC arrays. These artifacts reflect failed hybridization to target DNA, often due to germline or somatic deletions and manifest as incorrectly reported intermediate methylation. The artifacts often survive existing preprocessing pipelines, masquerade as epigenetic alterations and can confound discoveries in epigenome-wide association studies and studies of methylation-quantitative trait loci. We implement a solution, P-value with out-of-band (OOB) array hybridization (pOOBAH), in the R package SeSAMe. Our method effectively masks deleted and hyperpolymorphic regions, reducing or eliminating spurious reports of epigenetic silencing at oft-deleted tumor suppressor genes such as CDKN2A and RB1 in cases with somatic deletions. Furthermore, our method substantially decreases technical variation whilst retaining biological variation, both within and across HM450 and EPIC platform measurements. SeSAMe provides a light-weight, modular DNA methylation data analysis suite, with a performant implementation suitable for efficient analysis of thousands of samples.
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http://dx.doi.org/10.1093/nar/gky691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237738PMC
November 2018

Switching roles for DNA and histone methylation depend on evolutionary ages of human endogenous retroviruses.

Genome Res 2018 08 3;28(8):1147-1157. Epub 2018 Jul 3.

Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.

We provide a comprehensive genomic and epigenomic map of the more than 500,000 endogenous retroviruses (ERVs) and fragments that populate the intergenic regions of the human genome. The repressive epigenetic marks associated with the ERVs, particularly long terminal repeats (LTRs), show a remarkable switch in silencing mechanisms, depending on the evolutionary age of the LTRs. Young LTRs tend to be CpG rich and are mainly suppressed by DNA methylation, whereas intermediate age LTRs are associated predominantly with histone modifications, particularly histone H3 lysine 9 (H3K9) methylation. Young LTRs can be reactivated by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) alone, but their level of expression is much increased by 5-aza-CdR treatment plus knockdown of one of several H3K9 methyltransferases or of the H3K27 methyltransferase EZH2. The removal of cytosine methylation led to rapid, widespread increases in H3K9me3 in the LTRs. Intermediate age LTRs had lower CpG densities and were not up-regulated by 5-aza-CdR treatment, but they were sensitive to knockdown of H3K9 methyltransferases. Unlike the situation in embryonic stem cells, the polycomb repressive complex (PRC2) has a minor role in LTR suppression by itself and is only a player after removal of cytosine methylation in the analyzed cancer cell line. Up-regulation of LTRs and induction of "viral mimicry" is rapidly becoming of interest for predicting cancer patient response to epigenetic therapies. Understanding the mechanism for LTR suppression is of major importance in order to improve patient treatment strategies.
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http://dx.doi.org/10.1101/gr.234229.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071641PMC
August 2018

Integrated Molecular Characterization of Testicular Germ Cell Tumors.

Cell Rep 2018 06;23(11):3392-3406

The Eli and Edythe L. Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02142, USA.

We studied 137 primary testicular germ cell tumors (TGCTs) using high-dimensional assays of genomic, epigenomic, transcriptomic, and proteomic features. These tumors exhibited high aneuploidy and a paucity of somatic mutations. Somatic mutation of only three genes achieved significance-KIT, KRAS, and NRAS-exclusively in samples with seminoma components. Integrated analyses identified distinct molecular patterns that characterized the major recognized histologic subtypes of TGCT: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma. Striking differences in global DNA methylation and microRNA expression between histology subtypes highlight a likely role of epigenomic processes in determining histologic fates in TGCTs. We also identified a subset of pure seminomas defined by KIT mutations, increased immune infiltration, globally demethylated DNA, and decreased KRAS copy number. We report potential biomarkers for risk stratification, such as miRNA specifically expressed in teratoma, and others with molecular diagnostic potential, such as CpH (CpA/CpC/CpT) methylation identifying embryonal carcinomas.
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http://dx.doi.org/10.1016/j.celrep.2018.05.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075738PMC
June 2018

Syntenin-targeted peptide blocker inhibits progression of cancer cells.

Eur J Med Chem 2018 Jun 19;154:354-366. Epub 2018 May 19.

Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China; Institute for Advanced Study, Tongji University, Shanghai, China. Electronic address:

The multidomain adaptor protein syntenin is known to mediate cancer cell metastasis and invasion through its tandem PDZ1 and PDZ2 domains, leading to the postulation that the PDZ tandem may serve as a potential drug target for cancer treatment. Here we report the development of high-affinity peptide blockers to target the syntenin tandem PDZ domain, and elucidate that blocking syntenin correlates with the inhibition of cell migration and spreading. Two strategies are employed to derive high-affinity blockers from the low-affinity natural binding peptides: first, dimerization of the C termini of natural syntenin-binding peptides confers dimer peptides with much higher affinity than the monomers; second, unnatural amino acid substitution at P-1 and P-2 positions of the PDZ-binding sequence increases the binding affinity. Through several rounds of optimization, we discovered a dimeric peptide that binds tightly to syntenin tandem PDZ domain, with a dissociation constant of 0.21 μM based on fluorescence polarization measurement. The peptide dimer inhibits the migration and invasion of syntenin high-expression human cancer cells through attenuating the ERK phosphorylation of the MAPK kinase pathway. This work showcases an effective strategy to derive high-affinity blocker of multidomain adaptor proteins, which resulted in a syntenin-targeted antagonist with potential pharmaceutical values for the treatment of syntenin over-expressing cancers.
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http://dx.doi.org/10.1016/j.ejmech.2018.05.015DOI Listing
June 2018

Integrative Epigenetic Analysis Reveals Therapeutic Targets to the DNA Methyltransferase Inhibitor Guadecitabine (SGI-110) in Hepatocellular Carcinoma.

Hepatology 2018 10;68(4):1412-1428

Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, CA.

There is an urgent need to develop more effective therapies for hepatocellular carcinoma (HCC) because of its aggressiveness. Guadecitabine (SGI-110) is a second-generation DNA methyltransferase inhibitor (DNMTi), which is currently in clinical trials for HCC and shows greater stability and performance over first-generation DNMTis. In order to identify potential therapeutic targets of SGI-110 for clinical trials, HCC cell lines (SNU398, HepG2, and SNU475) were used to evaluate the effects of transient SGI-110 treatment by an integrative analysis of DNA methylation, nucleosome accessibility, gene expression profiles, and its clinical relevance by comparison to The Cancer Genome Atlas (TCGA) HCC clinical data. Each HCC cell line represents a different DNA methylation subtype of primary HCC tumors based on TCGA data. After SGI-110 treatment, all cell lines were sensitive to SGI-110 with prolonged antiproliferation effects. Expression of up-regulated genes, including tumor suppressors, was positively correlated with nucleosome accessibility and negatively correlated with gene promoter DNA methylation. Alternatively, expression of down-regulated genes, such as oncogenes, was negatively correlated with nucleosome accessibility and positively correlated with gene body DNA methylation. SGI-110 can also act as a dual inhibitor to down-regulate polycomb repressive complex 2 (PRC2) genes by demethylating their gene bodies, resulting in reactivation of PRC2 repressed genes without involvement of DNA methylation. Furthermore, it can up-regulate endogenous retroviruses to reactivate immune pathways. Finally, about 48% of frequently altered genes in primary HCC tumors can be reversed by SGI-110 treatment.

Conclusion: Our integrative analysis has successfully linked the antitumor effects of SGI-110 to detailed epigenetic alterations in HCC cells, identified potential therapeutic targets, and provided a rationale for combination treatments of SGI-110 with immune checkpoint therapies.
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http://dx.doi.org/10.1002/hep.30091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173644PMC
October 2018

The Immune Landscape of Cancer.

Immunity 2018 04 5;48(4):812-830.e14. Epub 2018 Apr 5.

Department of Systems Biology and Department of Electrical Engineering, Columbia University, New York, NY 10027, USA.

We performed an extensive immunogenomic analysis of more than 10,000 tumors comprising 33 diverse cancer types by utilizing data compiled by TCGA. Across cancer types, we identified six immune subtypes-wound healing, IFN-γ dominant, inflammatory, lymphocyte depleted, immunologically quiet, and TGF-β dominant-characterized by differences in macrophage or lymphocyte signatures, Th1:Th2 cell ratio, extent of intratumoral heterogeneity, aneuploidy, extent of neoantigen load, overall cell proliferation, expression of immunomodulatory genes, and prognosis. Specific driver mutations correlated with lower (CTNNB1, NRAS, or IDH1) or higher (BRAF, TP53, or CASP8) leukocyte levels across all cancers. Multiple control modalities of the intracellular and extracellular networks (transcription, microRNAs, copy number, and epigenetic processes) were involved in tumor-immune cell interactions, both across and within immune subtypes. Our immunogenomics pipeline to characterize these heterogeneous tumors and the resulting data are intended to serve as a resource for future targeted studies to further advance the field.
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http://dx.doi.org/10.1016/j.immuni.2018.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5982584PMC
April 2018

Oncogenic Signaling Pathways in The Cancer Genome Atlas.

Cell 2018 04;173(2):321-337.e10

Van Andel Research Institute, 333 Bostwick Ave NE, Grand Rapids Michigan, 49503, USA.

Genetic alterations in signaling pathways that control cell-cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations, copy-number changes, mRNA expression, gene fusions and DNA methylation in 9,125 tumors profiled by The Cancer Genome Atlas (TCGA), we analyzed the mechanisms and patterns of somatic alterations in ten canonical pathways: cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGFβ signaling, p53 and β-catenin/Wnt. We charted the detailed landscape of pathway alterations in 33 cancer types, stratified into 64 subtypes, and identified patterns of co-occurrence and mutual exclusivity. Eighty-nine percent of tumors had at least one driver alteration in these pathways, and 57% percent of tumors had at least one alteration potentially targetable by currently available drugs. Thirty percent of tumors had multiple targetable alterations, indicating opportunities for combination therapy.
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http://dx.doi.org/10.1016/j.cell.2018.03.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070353PMC
April 2018

DNA methylation loss in late-replicating domains is linked to mitotic cell division.

Nat Genet 2018 04 2;50(4):591-602. Epub 2018 Apr 2.

Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, late-replicating regions termed partially methylated domains (PMDs). We profiled 39 diverse primary tumors and 8 matched adjacent tissues using whole-genome bisulfite sequencing (WGBS) and analyzed them alongside 343 additional human and 206 mouse WGBS datasets. We identified a local CpG sequence context associated with preferential hypomethylation in PMDs. Analysis of CpGs in this context ('solo-WCGWs') identified previously undetected PMD hypomethylation in almost all healthy tissue types. PMD hypomethylation increased with age, beginning during fetal development, and appeared to track the accumulation of cell divisions. In cancer, PMD hypomethylation depth correlated with somatic mutation density and cell cycle gene expression, consistent with its reflection of mitotic history and suggesting its application as a mitotic clock. We propose that late replication leads to lifelong progressive methylation loss, which acts as a biomarker for cellular aging and which may contribute to oncogenesis.
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http://dx.doi.org/10.1038/s41588-018-0073-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893360PMC
April 2018

The genomic landscape of tuberous sclerosis complex.

Nat Commun 2017 06 15;8:15816. Epub 2017 Jun 15.

Center for Cancer and Cell Biology, Van Andel Research Institute, 333 Bostwick Avenue NE, Grand Rapids, Michigan 49503, USA.

Tuberous sclerosis complex (TSC) is a rare genetic disease causing multisystem growth of benign tumours and other hamartomatous lesions, which leads to diverse and debilitating clinical symptoms. Patients are born with TSC1 or TSC2 mutations, and somatic inactivation of wild-type alleles drives MTOR activation; however, second hits to TSC1/TSC2 are not always observed. Here, we present the genomic landscape of TSC hamartomas. We determine that TSC lesions contain a low somatic mutational burden relative to carcinomas, a subset feature large-scale chromosomal aberrations, and highly conserved molecular signatures for each type exist. Analysis of the molecular signatures coupled with computational approaches reveals unique aspects of cellular heterogeneity and cell origin. Using immune data sets, we identify significant neuroinflammation in TSC-associated brain tumours. Taken together, this molecular catalogue of TSC serves as a resource into the origin of these hamartomas and provides a framework that unifies genomic and transcriptomic dimensions for complex tumours.
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http://dx.doi.org/10.1038/ncomms15816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481739PMC
June 2017

Comprehensive evaluation of extracellular small RNA isolation methods from serum in high throughput sequencing.

BMC Genomics 2017 01 7;18(1):50. Epub 2017 Jan 7.

Department of Surgery, Vanderbilt University, MRBIV 8465A Langford Hall, 2213 Garland Ave, Nashville, TN, 37232, USA.

Background: DNA and RNA fractions from whole blood, serum and plasma are increasingly popular analytes that are currently under investigation for their utility in the diagnosis and staging of disease. Small non-coding ribonucleic acids (sRNAs), specifically microRNAs (miRNAs) and their variant isoforms (isomiRs), and transfer RNA (tRNA)-derived small RNAs (tDRs) comprise a repertoire of molecules particularly promising in this regard.

Results: In this designed study, we compared the performance of various methods and kits for isolating circulating extracellular sRNAs (ex-sRNAs). ex-sRNAs from one healthy individual were isolated using five different isolation kits: Qiagen Circulating Nucleic Acid Kit, ThermoFisher Scientific Ambion TRIzol LS Reagent, Qiagen miRNEasy, QiaSymphony RNA extraction kit and the Exiqon MiRCURY RNA Isolation Kit. Each isolation method was repeated four times. A total of 20 small RNA sequencing (sRNAseq) libraries were constructed, sequenced and compared using a rigorous bioinformatics approach. The Circulating Nucleic Acid Kit had the greatest miRNA isolation variability, but had the lowest isolation variability for other RNA classes (isomiRs, tDRs, and other miscellaneous sRNAs (osRNA). However, the Circulating Nucleic Acid Kit consistently generated the fewest number of reads mapped to the genome, as compared to the best-performing method, Ambion TRIzol, which mapped 10% of the miRNAs, 7.2% of the tDRs and 23.1% of the osRNAs. The other methods performed intermediary, with QiaSymphony mapping 14% of the osRNAs, and miRNEasy mapping 4.6% of the tDRs and 2.9% of the miRNAs, achieving the second best kit performance rating overall.

Conclusions: In summary, each isolation kit displayed different performance characteristics that could be construed as biased or advantageous, depending upon the downstream application and number of samples that require processing.
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http://dx.doi.org/10.1186/s12864-016-3470-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5219650PMC
January 2017

Comprehensive characterization, annotation and innovative use of Infinium DNA methylation BeadChip probes.

Nucleic Acids Res 2017 02;45(4):e22

Center for Epigenetics, Van Andel Research Institute, 333 Bostwick Ave NE, Grand Rapids, MI 49503, USA.

Illumina Infinium DNA Methylation BeadChips represent the most widely used genome-scale DNA methylation assays. Existing strategies for masking Infinium probes overlapping repeats or single nucleotide polymorphisms (SNPs) are based largely on ad hoc assumptions and subjective criteria. In addition, the recently introduced MethylationEPIC (EPIC) array expands on the utility of this platform, but has not yet been well characterized. We present in this paper an extensive characterization of probes on the EPIC and HM450 microarrays, including mappability to the latest genome build, genomic copy number of the 3΄ nested subsequence and influence of polymorphisms including a previously unrecognized color channel switch for Type I probes. We show empirical evidence for exclusion criteria for underperforming probes, providing a sounder basis than current ad hoc criteria for exclusion. In addition, we describe novel probe uses, exemplified by the addition of a total of 1052 SNP probes to the existing 59 explicit SNP probes on the EPIC array and the use of these probes to predict ethnicity. Finally, we present an innovative out-of-band color channel application for the dual use of 62 371 probes as internal bisulfite conversion controls.
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http://dx.doi.org/10.1093/nar/gkw967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389466PMC
February 2017
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