A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus

SUMMARYDysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in severe developmental defects and other diseases, but the molecular mechanisms that ensure imprint stability remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which serves as a non-canonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the DNA (de)methylation machinery.HIGHLIGHTSThe IG-DMR is a bipartite element with distinct allele-specific functionsA non-canonical enhancer within the IG-DMR prevents DNA methyltransferase activityTargeted epigenome editing allows induction of specific imprinting phenotypesCRISPRi reveals a functional hierarchy between DMRs that dictates imprint stability

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The long non-coding RNA Dali is an epigenetic regulator of neural differentiation

eLife ◽

10.7554/elife.04530 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 86

Author(s):

Vladislava Chalei ◽

Stephen N Sansom ◽

Lesheng Kong ◽

Sheena Lee ◽

Juan F Montiel ◽

...

Keyword(s):

Neural Differentiation ◽

Noncoding Rna ◽

Dna Methyltransferase ◽

Large Scale ◽

Cpg Island ◽

Methylation Status ◽

Neuroblastoma Cells ◽

Regulatory Elements ◽

Protein Coding ◽

Non Coding Rna

Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacent protein coding genes. Less clear is whether intergenic lncRNAs commonly regulate transcription by modulating chromatin at genomically distant loci. Here, we report both genomically local and distal RNA-dependent roles of Dali, a conserved central nervous system expressed intergenic lncRNA. Dali is transcribed downstream of the Pou3f3 transcription factor gene and its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali transcript regulates transcription of the Pou3f3 locus. Distally, it preferentially targets active promoters and regulates expression of neural differentiation genes, in part through physical association with the POU3F3 protein. Dali interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylation status of CpG island-associated promoters in trans. These results demonstrate, for the first time, that a single intergenic lncRNA controls the activity and methylation of genomically distal regulatory elements to modulate large-scale transcriptional programmes.

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RdDM pathway is required for Tobamovirus-induced symptomatology production

10.1101/2020.01.21.912923 ◽

2020 ◽

Author(s):

Melisa Leone ◽

Diego Zavallo ◽

Andrea Venturuzzi ◽

Sebastián Asurmendi

Keyword(s):

Dna Methylation ◽

Viral Infection ◽

Viral Infections ◽

Expression Patterns ◽

Initial Step ◽

List Type ◽

Direct Role ◽

Transcriptional Changes ◽

Virus Interactions

SummarySmall RNAs (sRNA) are important molecules for gene regulation in plants and play an essential role in plant-pathogen interactions. Researchers have evaluated the relationship between viral infections as well as the endogenous accumulation of sRNAs and the transcriptional changes associated with the production of symptoms, little is known about a possible direct role of epigenetics, mediated by 24-nt sRNAs, in the induction of these symptoms.With the use of different RNA directed DNA methylation pathway mutants and triple demethylase mutants, here we demonstrate that the disruption of RdDM pathway during viral infection produced alterations in the plant transcriptomic changes (because of the infection) and in symptomatology.This study represents the initial step in exposing that DNA methylation directed by endogenous sRNAs has an important role, uncoupled to defense, in the production of symptoms associated with plant-virus interactions.Significance statementThe crop yield losses induced by phytoviruses are mainly associated with the symptoms of the disease. DNA modifications as methylation, can modulate the information coded by the sequence, process named epigenetics. Viral infection can change the expression patterns of different genes linked to defenses and symptoms. This work represents the initial step to expose the role of epigenetic process, in the production of symptoms associated with plants-virus interactions.

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Blood‐Based Epigenetic Markers of FKBP5 Gene Methylation in Patients With Dilated Cardiomyopathy

Journal of the American Heart Association ◽

10.1161/jaha.121.021101 ◽

2021 ◽

Author(s):

Kento Wada ◽

Tomofumi Misaka ◽

Tetsuro Yokokawa ◽

Yusuke Kimishima ◽

Takashi Kaneshiro ◽

...

Keyword(s):

Dna Methylation ◽

Dilated Cardiomyopathy ◽

Dna Methyltransferase ◽

Cpg Island ◽

Epigenetic Modification ◽

Quantitative Polymerase Chain Reaction ◽

Pressure Overload ◽

Cpg Methylation ◽

Promoter Regions

Background Blood‐based DNA methylation patterns are linked to types of diseases. FKBP prolyl isomerase 5 (FKBP5), a protein cochaperone, is known to be associated with the inflammatory response, but the regulatory mechanisms by leukocyte FKBP5 DNA methylation in patients with dilated cardiomyopathy (DCM) remain unclear. Methods and Results The present study enrolled patients with DCM (n=31) and age‐matched and sex‐matched control participants (n=43). We assessed FKBP5 CpG (cytosine‐phosphate‐guanine) methylation of CpG islands at the 5′ side as well as putative promoter regions by methylation‐specific quantitative polymerase chain reaction using leukocyte DNA isolated from the peripheral blood. FKBP5 CpG methylation levels at the CpG island of the gene body and the promoter regions were significantly decreased in patients with DCM. Leukocyte FKBP5 and IL‐1β (interleukin 1β) mRNA expression levels were significantly higher in patients with DCM than in controls. The protein expressions of DNMT1 (DNA methyltransferase 1) and DNMT3A (DNA methyltransferase 3A) in leukocytes were significantly reduced in patients with DCM. In vitro methylation assay revealed that FKBP5 promoter activity was inhibited at the methylated conditions in response to immune stimulation, suggesting that the decreased FKBP5 CpG methylation was functionally associated with elevation of FKBP5 mRNA expressions. Histological analysis using a mouse model with pressure overload showed that FKBP5‐expressing cells were substantially infiltrated in the myocardial interstitium in the failing hearts, indicating a possible role of FKBP5 expressions of immune cells in the cardiac remodeling. Conclusions Our findings demonstrate a link between specific CpG hypomethylation of leukocyte FKBP5 and DCM. Blood‐based epigenetic modification in FKBP5 may be a novel molecular mechanism that contributes to the pathogenesis of DCM.

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AML1/ETO-Mediated Lysozyme Repression Is Independently Relieved by Inhibitors of DNA Methylation and Histone Deacetylation.

Blood ◽

10.1182/blood.v108.11.4310.4310 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4310-4310

Author(s):

Rainer Claus ◽

Manfred Fliegauf ◽

Michael Stock ◽

Jesus Duque ◽

Mateusz Kolanczyk ◽

...

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Dna Methyltransferase ◽

Cpg Island ◽

Trichostatin A ◽

Combined Treatment ◽

Marker Gene ◽

Dna Demethylation ◽

Histone Deacetylation ◽

Mrna Levels

Abstract The human lysozyme (LZM) gene, a marker gene for myeloid-specific development, is highly methylated in immature myeloid and in non-myeloid cells (all LZM-negative), and unmethylated in LZM-expressing mature phagocyte cells. Thus this gene provides an excellent model for investigating differentation-associated DNA methylation changes during myelopoiesis. There is now increasing evidence that LZM (containing five perfect consensus binding sites for AML1/RUNX1 in its 5′ region) is repressed by the AML1/ETO chimeric transcription factor (Fliegauf et al, Oncogene 23:9070–81, 2004), and this repression can be relieved by siRNA-mediated AML1/ETO depletion in AML1/ETO-positive Kasumi-1 cells (Dunne et al., Oncogene, 2006). Recently, AML1/ETO has also been implicated in gene-specific epigenetic repression of interleukin-3 (Liu et al, Cancer Res 65, 1277–84, 2005). By extensive methylation analyses of the LZM gene including bisulfite sequencing, we now demonstrate marked demethylation in both the CpG-poor 5′ region and the exonic CpG island after treatment of Kasumi-1 cells with non-cytotoxic concentrations of the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-azaCdR), which was not associated with cellular differentiation. By Northern blot analysis, LZM mRNA levels in Kasumi-1 cells but not in AML1/ETO-negative HL-60 and U-937 cell lines were specifically and independently upregulated upon treatment with 5-azaCdR and, to a lesser extent, with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Combined treatment with subliminal concentrations of 5-azaCdR and TSA applied in different schedules did not reveal synergistic effects on LZM transcription. Relative chromatin accessibility of the LZM 5′ region, as detected by “MspI protection” assay, and associated with partial demethylation in several myeloid cell lines, was increased in Kasumi-1 with 5-azaCdR-induced further DNA demethylation, but not by TSA. As shown by chromatin immunoprecitation, TSA increased the acetylation of histones H3 and H4 both in the 5′ flanking region and exonic CpG island. In a U-937 inducible model, antagonization of AML1/ETO-mediated repression of LZM was achieved by TSA, implying that the histone deacetylation in this region of the human LZM gene is mediated by AML1/ETO protein. In conclusion, we demonstrate functional interactions between DNA methylation and histone modifications in mediating LZM gene repression which implicate AML1/ETO as one component involved in local chromatin remodelling. Interestingly, inhibitors of DNA methylation and histone deacetylation independently relieve repression of this CpG-poor gene in AML1/ETO-positive cells.

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Up-Regulation of miR-195 Expression Leads to Decreased Expression of Basic Fibroblast Growth Factor in CLL Patients Treated with DNA Methylation Inhibitors.

Blood ◽

10.1182/blood.v110.11.3183.3183 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3183-3183

Author(s):

John D. Phillips ◽

Ioana Pop ◽

Ken Boucher ◽

Margaret K. Yu

Keyword(s):

Dna Methylation ◽

Growth Factor ◽

Tumor Suppressor ◽

Fibroblast Growth Factor ◽

Basic Fibroblast Growth Factor ◽

Dna Methyltransferase ◽

Cpg Island ◽

Fibroblast Growth ◽

Substrate Inhibitor ◽

Dna Methylation Inhibitors

Abstract A higher than age-expected DNA methylation index is predictive for early disease progression in patients with CLL (Yu et al. Leukemia Research 2006). We hypothesized that miRNA expression can also be silenced by promoter hypermethylation in CLL. Thus, methyl pool or DNA methyltransferase inhibitors can upregulate microRNAs with tumor suppressor characteristics by restoring the “normal” pattern of methylation. Results of patients treated with cladribine, a methyl substrate inhibitor, or 5-azacitidine, a DNA methyltransferase inhibitor were compared in 2 separate clinical protocols. The global DNA methylation decreased after treatment with cladribine or 5-azacitidine in 60% of the patients. At the 2006 ASH meeting, we reported on the consistent upregulation of miR-17-3p, miR-21, miR-29a, miR-29b, miR-29c, miR-30e, miR-104, miR-126, miR-128a, miR-130a, miR-141, miR-142-3p, miR-148a, miR-151, miR-199a, miR-199a*, and miR-301 by real-time PCR using the Early Access Human Panel from Applied Biosystems. Data from two patients on the cladribine protocol and 10 patients on the 5-azacitidine protocol were used for statistical analyses. Non-parametric Wilcoxon tests as well as t-tests were performed. Comparisons were made of the responders versus non-responders, and of cladribine versus 5-azacytidine. Since many of the miRNAs showed differences in the cladribine versus 5-azacytidine comparison, a second responder vs non-responder analysis was performed in which the two cladribine subjects were removed. MiR-195 was statistically more upregulated in the cladribine treated responder whereas miR-29c was statistically most upregulated in the 5-azacitidine treated patients (p=0.02). In the patients with global demethylation after treatment, upregulation of microRNA-195 was observed and directly correlated with regional demethylation of the CpG island, confirmed by bisulfite sequencing. Some of the predicted targets of miR-195 include bcl-2, CNOT6L, USP15, PADAH1B1, and ESRRG. MiR-195 may also have tumor suppressor characteristics as it also targets basic fibroblast growth factor (FGF-2), a gene important in CLL angiogenesis. There has been some evidence suggesting FGF-2 is an oncogene. For example, overexpression of FGF-2 isoforms facilitates growth of NIH 3T3 cells in low serum media and also mediates radioresistance of HeLa cells. FGF-2 is also protective against irradiation activation of p53 in the leukemia cells derived from patients with CLL. Although cladribine has been reported to downregulate FGF-2 by inhibiting adenosine deaminase, downregulation of FGF-2 at the transcript and protein levels was also observed in before and after treatment samples from patients treated with 5-azacytidine. We propose an alternative mechanism by which the FGF-2 transcript is degraded after binding to excess miR-195. In patients responsive to treatment with DNA methylation inhibitors, a regional decrease in the methylation status of the CpG island 5′ to miR-195 may lead to increased expression.

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The novel DNA methylation inhibitor zebularine is effective against the development of murine T-cell lymphoma

Blood ◽

10.1182/blood-2005-05-2033 ◽

2006 ◽

Vol 107 (3) ◽

pp. 1174-1177 ◽

Cited By ~ 43

Author(s):

Michel Herranz ◽

Juan Martín-Caballero ◽

Mario F. Fraga ◽

Jesús Ruiz-Cabello ◽

Juana Maria Flores ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cpg Island ◽

Promoter Hypermethylation ◽

Dna Hypomethylation ◽

Chemotherapy Drugs ◽

Positron Emission ◽

Methylation Inhibitor ◽

Dna Methylation Inhibitor ◽

T Lymphomas

AbstractGene silencing by CpG island promoter hypermethylation has awakened the interest for DNA demethylating agents as chemotherapy drugs. Zebularine (1-[β-D-ribofuranosil]-1,2-dihydropyrimidin-2-1) has been recently described as a new DNA methylation inhibitor. Here we have studied its effects in a mouse model of radiation-induced lymphomagenesis using nuclear magnetic resonance (NMR) and positron emission tomography (PET). All control animals presented large thymic T lymphomas and died between 4 and 5.5 months. In contrast, 40% (12 of 30) of zebularine-treated animals were still alive after 1 year (Kaplan-Meier P < .001). NMR and PET imaging showed that surviving animals presented a thymus structure/volume similar to normal mice of the same age. Most important, zebularine demonstrated a complete lack of toxicity in nonirradiated control mice. DNA hypomethylation induced by zebularine occurred in association with depletion in extractable DNA methyltransferase 1 protein. Thus, our data support the role of zebularine as a DNA demethylating agent with antitumor activity and little toxicity.

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Single cell multi-omics profiling reveals a hierarchical epigenetic landscape during mammalian germ layer specification

10.1101/519207 ◽

2019 ◽

Cited By ~ 5

Author(s):

Ricard Argelaguet ◽

Hisham Mohammed ◽

Stephen J Clark ◽

L Carine Stapel ◽

Christel Krueger ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Cell Fate ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

List Type ◽

Germ Layer ◽

Epigenetic Landscape ◽

Germ Layers ◽

Cell Fate Decisions

AbstractFormation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan. Recent studies employing single cell RNA-sequencing have identified major transcriptional changes associated with germ layer specification. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different epigenetic layers is unclear. Here we describe the first single cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during the exit from pluripotency and the onset of gastrulation in mouse embryos. We find dynamic dependencies between the different molecular layers, with evidence for distinct modes of epigenetic regulation. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of local lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements, driven by loss of methylation in enhancer marks and a concomitant increase of chromatin accessibility. In striking contrast, the epigenetic landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or epigenetically remodelled prior to overt cell fate decisions during gastrulation, providing the molecular logic for a hierarchical emergence of the primary germ layers.HighlightsFirst map of mouse gastrulation using comprehensive single cell triple-omic analysis.Exit from pluripotency is associated with a global repressive epigenetic landscape, driven by a sharp gain of DNA methylation and a gradual decrease of chromatin accessibility.DNA methylation and chromatin accessibility changes in enhancers, but not in promoters, are associated with germ layer formation.Mesoderm and endoderm enhancers become open and demethylated upon lineage commitment.Ectoderm enhancers are primed in the early epiblast and protected from the global repressive dynamics, supporting a default model of ectoderm commitment in vivo.

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Stable DNMT3L Overexpression in SH-SY5Y Neurons Recreates a Facet of the Genome-Wide Down Syndrome DNA Methylation Signature

10.1101/2020.11.09.374793 ◽

2020 ◽

Author(s):

Benjamin I. Laufer ◽

J. Antonio Gomez ◽

Julia M. Jianu ◽

Janine M. LaSalle

Keyword(s):

Dna Methylation ◽

Down Syndrome ◽

Dna Methyltransferase ◽

Molecular Mechanisms ◽

Cpg Island ◽

Neuroblastoma Cell ◽

Chromosome 21 ◽

Pairwise Comparisons ◽

Genome Wide ◽

A Genome

AbstractDown syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing at three different developmental phases (undifferentiated, differentiating, and differentiated). DNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Merging the DMRs into a consensus profile where the cell lines clustered by genotype and then phase demonstrated that different regions of common genes are affected. The hypermethylated DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated CpGs from previous DS studies of diverse tissues. In contrast, the hypomethylated DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development. Taken together, we propose a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during development are targeted by excess DNMT3L and become hypermethylated, while excess DNMT3L also evicts DNMT3A from heterochromatin, resulting in hypomethylation. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the DS DNA methylation signature.

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DNA methylation and histone H1 cooperatively repress transposable elements and aberrant intragenic transcripts

10.1101/527523 ◽

2019 ◽

Cited By ~ 3

Author(s):

Jaemyung Choi ◽

David B. Lyons ◽

M. Yvonne Kim ◽

Jonathan D. Moore ◽

Daniel Zilberman

Keyword(s):

Dna Methylation ◽

Transposable Elements ◽

Dna Methyltransferase ◽

Housekeeping Genes ◽

Histone H1 ◽

List Type ◽

Antisense Transcripts ◽

Methylated Dna ◽

Nucleosome Organization ◽

Genomic Methylation

SummaryDNA methylation and histone H1 mediate transcriptional silencing of genes and transposable elements, but how they interact is unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. Here we show that H1 is enriched in methylated sequences, including genes, of Arabidopsis thaliana, yet this enrichment is independent of DNA methylation. Loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. Loss of H1 also activates antisense transcripts within demethylated genes. Our results demonstrate that H1 and DNA methylation cooperatively maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes.HighlightsHistone H1 is enriched in methylated DNA independently of methylationLoss of H1 activates genes, alters nucleosome organization and disperses heterochromatinDNA methylation and H1 jointly silence transposonsDNA methylation and H1 cooperatively suppress intragenic antisense transcripts

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SAT-LB34 Repressive Epigenetic Programs Reinforce Steroidogenic Differentiation and Wnt/β-Catenin Signaling in Aggressive Adrenocortical Carcinoma

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.2265 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Dipika R Mohan ◽

Isabella Finco ◽

Christopher Ryan LaPensee ◽

Juilee Rege ◽

Tobias Else ◽

...

Keyword(s):

Cell Cycle ◽

Dna Methylation ◽

Adrenocortical Carcinoma ◽

Dna Methyltransferase ◽

Cpg Island ◽

Cpg Islands ◽

Embryonic Stem ◽

High Status ◽

Tissue Specific ◽

Genome Wide

Abstract Adrenocortical carcinoma (ACC) is a rare, aggressive cancer. Up to 75% of patients develop incurable metastatic disease, highlighting an urgent need for novel medical therapies. We recently identified a rapidly progressive ACC subtype characterized by CpG island hypermethylation (CIMP-high), sustained Wnt/β-catenin signaling, steroidogenic differentiation, and cell cycle activation. CIMP-high status alone accounts for 40% of ACC, but predicts 70% of recurrences and >50% of deaths. Intriguingly, hypermethylated CpG islands in CIMP-high ACC are unmethylated in fetal and adult adrenal cortex, suggesting DNA methylation is supported by cancer-specific mechanisms. We therefore sought to investigate how aberrant epigenetic programming contributes to ACC biology. In embryonic stem cells, the Polycomb repressive complex 2 (PRC2) represses differentiation programs through EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) deposition in promoter CpG islands free of DNA methylation. Gain or loss of EZH2/PRC2 function prevails in a variety of human cancers, enabling proliferation in a tissue-specific manner. Here, we identify that CIMP-high ACC exhibit high expression of EZH2/H3K27me3, but paradoxically bear DNA hypermethylation in annotated PRC2 target regions. To determine if DNA methylation of PRC2 targets disrupts or is controlled by EZH2, we characterized EZH2’s role in CIMP-high ACC cell line NCI-H295R at baseline and in response to EZH2 inhibition (EZH2i). EZH2-directed IP-MS revealed EZH2 interacts with PRC2 members and DNA methylation-sensitive accessory proteins, but no DNA methyltransferase machinery. ChIP-seq revealed EZH2 and H3K27me3 colocalize in repressive domains genome-wide, but DNA methylation and H3K27me3 are mutually exclusive. EZH2i induced H3K27 demethylation and loss of viability, but with no effect on CIMP-high DNA methylation. These data suggest PRC2 target DNA methylation in CIMP-high ACC is maintained independently of EZH2, enabling EZH2/PRC2 to coordinate alternative programs required for cell survival. We then measured the consequences of EZH2i on the NCI-H295R transcriptome (RNA-seq), EZH2/H3K27me3 deposition genome-wide (ChIP-seq), and chromatin accessibility landscape (ATAC-seq). EZH2i led to global downregulation of cell cycle, Wnt/β-catenin transcriptional programming, and steroidogenic differentiation, partially explained by EZH2i-induced offloading of EZH2 from H3K27me3 domains to accessible promoters genome-wide. Taken together, our studies illustrate how aberrant CpG island hypermethylation in CIMP-high ACC participates in a targetable repressive epigenetic cascade that reinforces oncogenic adrenocortical transcriptional programs. Ultimately, we hope to illuminate novel strategies for tissue-specific disruption of the aberrant epigenetic wiring that defines CIMP-high ACC.

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