
==== Front
Neoplasia
Neoplasia
Neoplasia (New York, N.Y.)
1522-8002
1476-5586
Neoplasia Press

S1476-5586(24)00084-8
10.1016/j.neo.2024.101042
101042
Original Research
PDGF-BB overexpression in p53 null oligodendrocyte progenitors increases H3K27me3 and induces transcriptional changes which favor proliferation
Huang Dennis abcd
Mela Angeliki e
Bhanu Natarajan V. f
Garcia Benjamin A. f
Canoll Peter e
Casaccia Patrizia pcasaccia@gc.cuny.edu
ac⁎
a Program in Molecular, Cellular and Developmental Biology at The Graduate Center of The City University of New, York 365 5th Ave, New York, NY 10016, United States
b Belfer Research Institute, City University of New York & Weill Cornell Medical College, 413 E 69th St, New York, NY 10021, United States
c Neuroscience Initiative, Advance Science Research Center, Graduate Center of The City University of New York, 85 St Nicholas Terrace, New York, NY 10031, United States
d Department of Biological Sciences, Hunter College, City University of New York, 695 Park Ave, New York, NY 10065, United States
e Department of Pathology and Cell Biology, Columbia University Irving Medical Center, 622 W 168th St, New York, NY 10032, United States
f Department Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S Euclid Ave, St. Louis, MO 63110, United States
⁎ Corresponding author at: Neuroscience Initiative at the Advanced Science Research Center, 85 St. Nicholas Terrace, New York, NY 10031, United States. pcasaccia@gc.cuny.edu
30 8 2024
11 2024
30 8 2024
57 10104228 3 2024
13 8 2024
22 8 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• OPCs lacking p53 form tumors when injected in mice only if they also express PDGF-BB.

• PDGF-BB overexpression in p53 null OPC results in more proliferation, less differentiation and altered growth characteristics.

• PDGF-BB overexpressing p53 null OPC display high levels of the repressive H3K27me3 histone mark and low levels of H4K20me3.

• Pharmacological inhibition of H3K27 methylation alone is not sufficient to revert the growth phenotype of the PDGF-BB overexpressing p53 null OPC.

Proneural gliomas are brain tumors characterized by enrichment of oligodendrocyte progenitor cell (OPC) transcripts and genetic alterations. In this study we sought to identify transcriptional and epigenetic differences between OPCs with Trp53 deletion and PDGF-BB overexpression (BB-p53n) and those carrying only p53 deletion (p53n). In culture, the BB-p53n OPCs display growth characteristics more similar to glioma cells than p53n OPCs. When injected in mouse brains, BB-p53n OPC form tumors, while the p53n OPCs do not. Unbiased histone proteomics and transcriptomic analysis on these OPC populations identified higher levels of the histone H3K27me3 mark and lower levels of the histone H4K20me3. The transcriptome of the BB-p53n OPCs was characterized by higher levels of transcripts related to proliferation and cell adhesion compared to p53n OPCs. Pharmacological inhibition of the enzyme responsible for histone H3K27 trimethylation (EZH2i) in BB-p53n OPCs, reduced cell cycle transcripts and increased the expression of differentiation markers, but was not sufficient to restore their growth characteristics. This suggests that PDGF-BB overexpression in p53n OPCs favors the early stages of transformation, by promoting proliferation and halting differentiation in a H3K27me3-dependent pathway, and favoring growth characteristics in a H3K27me3 independent manner

Keywords

Histone
Epigenetic
Brain tumor
Glioma
Polycomb
Abbreviations

OPC oligodendrocyte progenitor cells

T3 Triiodothyronine

PDGF platelet derived growth factor
==== Body
pmcIntroduction

Gliomas are the most common malignant tumor in the central nervous system (CNS). They are characterized by rapid disease course and by marked cellular [1], transcriptional and epigenetic [2,3] heterogeneity. This described complexity likely contributes to the difficulty of advancing treatment options and developing viable drug options, rendering these tumors incurable.

In an attempt to decipher glioma heterogeneity, previous studies on gene expression profiling [4] identified the presence of distinctive transcriptional signatures, defining four specific glioma subtypes, on the basis of the abundance of cell-specific patterns of gene expression [5]. One such subtype is the proneural glioma, characterized by the transcriptional signature of oligodendrocyte progenitor cells (OPCs) which are the precursors to myelinating oligodendrocytes and represent the most abundant population of proliferative cells in the adult brain [6]. Several studies of gliomagenesis, in animal models with distinct genetic alterations in diverse brain cell types [7] suggested that OPCs act as cells of origin for proneural gliomas [[8], [9], [10], [11]]. However, the early steps leading from OPCs to tumor formation remain elusive.

Integrated genomic analyses [[5], [12], [13]] revealed the prevalence of specific genetic mutations in distinct “transcriptional” subtypes, with the proneural gliomas being characterized by amplification of platelet derived growth factor (PDGF) signaling [5] and often accompanied by the loss of the tumor suppressor function of P53 during the progression of the disease [14]. Deregulation of the p53 pathway has been described in 85 % of the tumors and alterations in PDGF and p53 pathways have been reported to occur at the early stages of glioma progression [14], suggesting that these alterations can be early events in the transformation of oligodendrocyte progenitors.

In this study, we sought to define the growth characteristics, the histone marks and transcriptional changes induced in OPCs by loss of p53 function and increased PDGF signaling. While PDGF-AA is the primary mitogen for OPCs during brain development [15] and PDGF-BB has been related to neovascularization [16] both mitogen isoforms have been reported to be secreted by glioma cell lines [17], with PDGFR beta frequently detected in high-grade glioma [18]. However, overexpression of PDGF-BB alone was able to induce tumors in rats [8], but not in mice [10]. Here, we use unbiased histone proteomics and transcriptomic analysis to assess the intrinsic differences between OPCs with p53 loss of function, which do not form tumors when injected in mice, and OPCs with both p53 deletion and overexpression of PDGF-BB, which form tumors, in an attempt to decipher early events of transformation.

Materials and methods

OPC isolation, culture, infection

Primary mouse OPCs were isolated from the brain of Trp53fl/fl C57BL/6 (JAX:008462) mice at postnatal day 5-7 by with a rat anti-mouse CD140a antibody (Fisher Cat# 558774), recognizing PDGFRα, as previously described [19] and were cultured in SATO medium (Dulbecco's modified Eagle's medium, 10 mg/ml bovine serum albumin (BSA), 10 mg/ml apotransferrin, 1.6 mg/ml putrescine, 6 ng/ml progesterone, 4 µg/ml selenium, 5 mg/ml insulin, 1 mM sodium pyruvate, 2 mM l-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 5 mg/ml N-acetyl-cysteine, Trace Element B, 10 µg/ml biotin, 50 mM forskolin) supplemented with PDGF-AA (10 ng/ml) and basic fibroblast growth factor (bFGF) (20 ng/ml). Trp53fl/fl OPCs were then infected with an X-IRES-CRE or PDGFB-IRES-CRE retrovirus to obtain Trp53−/− (p53n) and Trp53-/- PDGFB expressing (BB-p53n) OPCs. Retrovirus production as described in Lei et al. [10]. In brief, plasmid expressing VSVG and viral plasmid PDGFB-IRES-CRE were mixed with CaCl2. 2X HBS (Hepes Buffered Saline) was added to the mix, which was overlaid onto the 293GP cells. After 48 h, the virus enriched media was filtered and concentrated. 500,000 primary mouse OPCs were plated into a 35 mm plate before virus infection. Virus infection was performed by adding the concentrated virus directly into the tissue culture medium. The virus-containing medium was replaced by fresh media 12 h after infection. Infected cells were harvested 6 days after infection for experimental analysis. The experiment was independently replicated by two investigators in the lab.

OPC Tazemetostat treatment and differentiation

OPCs were cultured on 10 cm Poly-D-Lysine (PDL) treated dishes in SATO growth media. Tazemetostat (EPZ-6438 Selleck cat#S7128) was dissolved in DMSO to a stock concentration of 50mM and diluted to 5uM for treatment. Vehicle controls were treated with DMSO at the same concentration. Tazemetostat and DMSO added media were replaced every 24 h for a total of 48 h of treatment. In differentiation assays, OPCs were first treated with Tazemetostat or DMSO for 48 h in 10 cm dishes and then plated on PDL coated coverslips (Electron Microscopy Sciences Cat#72294-12) at a density of 20,000 cells per coverslip. OPCs were kept in Tazemetostat or DMSO while on coverslips and stimulated with Triiodothyronine (T3 60 nM) (Sigma cat#T5516-1MG) in the absence of mitogens to induce differentiation [19] for an addition 48 h or PDGF-AA (10ng/mL) and bFGF (20ng/mL) as controls, as mitogenic stimulation prevents differentiation.

OPC H2O2 treatment for induction of p53 expression

OPCs grown to 90 % confluency, were washed twice with PBS and then 1mM H2O2 (Sigma Aldrich cat#88597) was added to normal SATO growth media for 5 min before aspiration followed by two additional washes with PBS. Cells were then allowed to recover for 1 h in normal SATO growth media in the incubator before being lysed with RIPA buffer for protein extraction and further analysis.

PDGF-BB ELISA Assay

Mutant OPC lines were cultured to 90 % confluency, as described above, after washing the cells with PBS, the medium was replaced with SATO chemically defined medium devoid of mitogens and after 24 h the conditioned media was harvested. After a 5-fold concentration of the collected medium, using Millipore Amicon centrifugal filters 10K MWCO (cat# UFC901024), the ELISA assay was run following company protocol for R&D Systems Quantikine ELISA Human PDGF-BB Immunoassay (cat# DBB00).

Mutant OPC in vivo injections

Cell implantation was performed by stereotactic intracranial injection of 50,000 OPCs in 1 µL of SATO, at a flow rate of 0.25 µL/min with a Hamilton syringe, as previously described [20]. C57BL/6J Mice (JAX:000664) were anesthetized with Ketamine/Xylazine (100 mg kg-1 and 10 mg kg-1, respectively) and assessed for lack of reflexes by toe pinch. A burr hole was made with a 17-gauge needle 2 mm lateral and 2 mm anterior to the bregma.

Immunocytochemistry

A list of antibodies and buffers used for immunocytochemistry and immunoblotting is provided in Key Resources Table within the Supplemental material. Cells for immunocytochemistry were seeded in 8-well chamber slides (Thermo Fisher Scientific, 154941PK) or on PDL coated coverslips (Electron Microscopy Sciences Cat#72294-12) and fixed with 4 % paraformaldehyde (PFA) for 15 min at room temperature. Membranes were permeabilized with 0.1 % (vol/vol) Triton X-100/PBS (Fisher Scientific cat#AAA16046AP). Incubation with blocking solution consisting of PGBA (Phosphate buffer with 0.1 % gelatin, 1 %BSA, 0.002 % Sodium Azide), and 5 % normal goat serum (Vector Laboratories cat#S-1000-20) was performed at room temperature for 60 min. Primary antibodies were applied overnight at 4 °C or 1hr at room temperature followed by incubation of appropriate secondary antibodies conjugated with fluorophores. DAPI (life technologies Cat#D21490) in PBS is applied for 10 min and washed before mounting. Confocal images were captured using the Zeiss LSM-800 fluorescent microscope and Zen Blue software. Blinded quantification of the immunofluorescent intensity was performed using Fiji/ImageJ . Barplots generated in Graphpad Prism (Graphpad version 10.0.0 for Windows).

EdU assay

Mutant OPC lines were plated on PDL coated coverslips (Electron Microscopy Sciences Cat#72294-12) at a density of 10,000 cells per slip and in low-growth media (SATO media supplemented with 1ng/mL PDGF-AA). The methods for this assay were adapted from the Click-iT® EdU Imaging Kit (Thermofisher cat#C10425). A day after cells were plated on coverslips, they were incubated with 10µM EdU for 18 h before being fixed with 4 % PFA for 15 min at room temperature. Membrane permeabilization was performed with 0.5 % Triton X-100 in PBS for 20 min at room temperature. EdU and Hoechst were identified following the kit's protocol and additional targets were labelled using specific antibodies following methods described above and provided in the Key resource file. Confocal images were captured using the Zeiss LSM-800 fluorescent microscope and Zen Blue software. Blinded quantification of the immunofluorescent intensity was performed using Fiji/ImageJ. Barplots generated in Graphpad Prism (Graphpad version 10.0.0 for Windows).

Spheroid assay

For the spheroid assay we also used , BB-p53n glioma cells [21] as comparison. Those cells were thawed and cultured in chemically defined media (Dulbecco's modified Eagle's medium, 0.5 % Fetal Bovine Serum, N2 supplement, 10 ng/mL bFGF, 10 ng/mL PDGF-AA, Anti-bacterial/Anti-fungal cocktail). Spheroids growth assays for both glioma cells, BB-p53 and p53nOPCs were performed in low-attachment conditions and in spheroid growth medium (Dulbecco's modified Eagle's medium/F-12, N2 supplement, 20 ng/mL EGF, 10 ng/mL bFGF, 0.6 % Glucose, Anti-bacterial/Anti-fungal cocktail). Mutant OPCs and glioma cells were trypsinized and resuspended in spheroid growth medium in the absence or presence of the EZH2 inhibitor. Cell were plated at low density (1000 cells/cm2) in ultra-low-attachment 24-well plates (Sphera plates by Thermofisher cat#174930). Extreme care was taken to not disturb growing spheroids when refreshing medium every 2 days. 300µL were removed in each well and 300µL fresh medium slowly added. Phase contrast images were taken with the Zeiss Axio Vert.A1 microscope and Zen Blue software.

Western blotting

In vitro cells, cultured to 90 % confluency, were lysed for protein with RIPA buffer (Thermofisher cat#89900) supplemented with phosphatase (Sigma cat#P0044-5ML) and protease (Thermofisher cat#A32963) inhibitors. Histone extraction is described below. Concentrations measured by Biorad's DC protein assay (Biorad cat# 5000112) and 20ug of total protein or 1ug of acid extracted histones were loaded into the well of SurePAGE 4-20 % gradient gels (GenScript Cat#M00656) and run in accompanying running buffer (GenScript Cat#M00138). Transfers were performed with PVDF membrane and TG buffer 20 % methanol. Membranes were blocked and probed for targets before imaging on the Biorad chemidoc imaging system. Quantifications of western blot signals done in Fiji/ImageJ. Barplots generated in Graphpad Prism (Graphpad version 10.0.0 for Windows).

RNA isolation

Each RNA sample isolated from a 90 % confluent 10cm culture dish. In vitro cells are homogenized in 1mL of TRIZOL reagent (Fisher cat# 15596026) and incubated at room temperature for 5 min. Phase separation is initiated by 0.2mL chloroform: isoamyl alcohol (Invitrogen cat#15593-049) and disrupted by vortex and shaking. After centrifuging at 4C for 15 min at 12,000xg, the aqueous phase is removed and mixed with 0.5 mL isopropyl alcohol. Samples are incubated for 10 min at room temperature for the RNA to precipitate and then centrifuged at 12,000xg for 10 min at 4C. The collected RNA pellet is washed with 75 % cold ethanol before being centrifuged again and reconstituted in RNase free water. RNA quantification is initially done with Nanodrop One (ThermoScientific cat#ND-ONE-W). RNA used for sequencing is cleaned up with the RNeasy MinElute Cleanup Kit (Qiagen cat#74204).

RNA sequencing library preparation and read processing

100ng of total RNA was used for library prep using Kapa mRNA HyperPrep Kit (Roche cat#KK8580) and sequenced on a Novaseq 6000 sequencer configured to paired end 150. Raw reads were trimmed by trimmomatic, arguments set to ILLUMINACLIP:Illumina-adapt.fa:2:30:10, SLIDINGWINDOW:4:15, MINLEN:50. Trimmed reads aligned to mm39 reference genome (Mus Musculus mm39 Genome Assembly) with the subjunc tool of the subread package, arguments set to defaults. 27-46 million paired reads met quality checks and were used to count features with the featuresCounts tool of the subread package, annotation by Ensembl(Mus_musculus.GRCm39.108) with feature type set to exon.

RNA sequencing analysis

Gene count matrices were imported to R for all downstream analysis. Raw counts were normalized to TPM values and converted to scaled z-scores to plot heatmaps using the pheatmap (v1.0.12) package (Kolde, 2012). Differential expression analysis was done with limma in the edgeR (v4.0.16) package. GSEA was performed with the clusterProfiler (v4.10.0) package and Org.Mm.eg.db (v3.18.0) annotation database. Gene lists for GSEA were filtered by adjusted p-value less than 0.01, calculated by limma differential expression analysis. Curated G1/S and G2/M genesets obtained from Tirosh et al. [22] and other gene sets obtained from the Gene Ontology database . All enrichment plots are generated with the enrichplot (v1.22.0) package. All volcano plots generated by ggplot2 . R code is available at (https://github.com/dhuang-ASRC/BBp53n_Submission)

RT-qPCR

cDNA is synthesized with 500ng of RNA, 4uL of qScript XLT cDNA Supermix (Quantabio cat# 95161-500), and RNase free water to a volume of 20uL per sample. Thermocycler is set according to qScript instructions. Each PCR reaction contains 5uL of cDNA (1ng/uL), 6uL Perfecta Sybr Green FastMix (Quantbio cat# 95072-012), 0.5uL of RNase free water, 0.5uL 10uM Primer mix (primer sequences in supplementary material). Each reaction was pipetted into a 385 well plate and run on the QuantStudio 7 Flex with Perfecta Sybr Green FastMix thermocycling instructions. Log2 fold change was calculated by ΔΔCt method normalized first to the average Ct values of three house keeping genes (Ppia, Rpl13a, Pgk1) and then to p53n control values.

Histone extraction and LC-MS/MS analysis

BB-p53n and p53n OPCs were cultured in mitogen supplemented SATO media to a 90 % confluency on 15cm dishes, The histones were extracted and prepared for chemical derivatization and digestion as described previously [23]. In brief, cells were lysed in nuclear isolation buffer supplemented with protease and histone deacetylase inhibitors. Histones were precipitated with 25 % trichloroacetic acid overnight and washed with acetic acid before reconstituted with RNase free water. The lysine residues from histones were derivatized with the propionylation reagent (1:2 reagent:sample ratio) containing acetonitrile and propionic anhydride (3:1), and the solution pH was adjusted to 8.0 using ammonium hydroxide. The propionylation was performed twice and the samples were dried on speed vac. The derivatized histones were then digested with trypsin at a 1:50 ratio (wt/wt) in 50 mM ammonium bicarbonate buffer at room temperature overnight. The N-termini of histone peptides were derivatized with the propionylation reagent twice and dried on speed vac. The peptides were desalted with the self-packed C18 stage tip. The purified peptides were then dried and reconstituted in 0.1 % formic acid. Histone peptides were separated with Vanquish Neo UHPLC system fitted with 75 µm i.d. x 15 cm fused silica columns (Polymicro Tech) packed with ReproSil-Pur 120 C18-AQ (3 µm, Dr. Maisch GmbH) and connected in line with a mass spectrometer (Thermo QE). The chromatography conditions generally consisted of a linear gradient from 2 to 45 % solvent B (0.1 % formic acid in 80 % acetonitrile) in solvent A (0.1 % formic acid in water) over 55 mins and then 45 to 99 % solvent B over 5 mins at a flow rate of 300 nL/min. The mass spectrometer was programmed for data-independent acquisition (DIA). One acquisition cycle consisted of a full MS scan, 35 DIA MS/MS scans of 24 m/z isolation width starting from 295 m/z to reach 1100 m/z. Typically, full MS scans were acquired in the Orbitrap mass analyzer across 295–1100 m/z at a resolution of 70,000 in positive profile mode with a maximum injection time of 50 ms and an AGC target of 1e6. MS/MS data from HCD fragmentation was collected in the ion trap (when available) or the Orbitrap. These scans typically used an NCE of 30, an AGC target of 2e5, and a maximum injection time of 60 ms. Histone MS data were analyzed with EpiProfile [24].

Histone ratios analysis

Ratio values were converted to scaled z-scores for selected histone modifications in R and heatmaps were plotted in R with the ComplexHeatmap (v2.18.0) package.

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Patrizia Casaccia (pcasaccia@gc.cuny.edu).

Materials availability

Mouse lines used in this study are subject to MTA from the original investigator.

Data and code availability

RNA-Seq data have been deposited at NCBI GEO Repository and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. All data reported in this study and any additional information required to reanalyze the data reported in this study are available from the lead contact upon request.

Results

OPCs lacking p53 expression and over-expressing PDGF-BB form tumors when injected in mice

The experimental model in this study relies on the use of retroviral vectors and of a Trp53 flox/flox transgenic mouse line (JAX stock #008462). OPCs with p53 deletion alone (p53n) or also overexpressing PDGF-BB (BB-p53n) (Fig. 1A-C) were generated from the infection of Trp53 flox/flox OPCs with either a retroviral vector expressing only the recombinase cre or with a bicistronic vector expressing also PDGF-BB [10,21]. These cells were then subcortically injected into recipient mice, and only those receiving the BB-p53n OPCs formed tumors by 35 days post injection (35dpi), while those injected with p53n OPCs did not reveal any sign of tumor formation, even after 4 months of observations (Fig. 1D). Accordingly, survival experiments revealed that mice injected with BB-p53n OPCs succumbed to tumor morbidity by 60 dpi, while those injected with p53n OPCs survived past 120 dpi (Fig. 1E). Histopathology of BB-p53n injected brain tissue at end stage, revealed tumors with features of high-grade glioma, including dense neoplastic growth with pseudo-palisading necrosis and vascular proliferation. Together, these experiments suggested that analysis of transcriptional and epigenetic differences between the more tumorigenic BB-p53n OPCs and the non-tumorigenic p53n OPCs, may shed light on the early stages of tumor formationFig. 1 Cultured p53 null OPC over-expressing PDGF-BB form tumors when injected in mice. (A). Schematics of mutant OPCs generation. OPCs cultured from Trp53 floxed transgenic mice were infected either with Cre alone retrovirus (X-IRES-Cre) to generate p53n OPCs or with a bicistronic virus expressing both Cre and PDGF-BB (PDGF-BB IRES Cre) to generate BB-p53n OPCs. (B) Western blot analysis of protein lysates from H2O2 treated wildtype (WT), p53n, and BB-p53n OPCs. probed with antibodies for P53 and total histone H3 as loading control. (C) Bar graphs of PDGF-BB levels measured by ELISA in the conditioned medium collected from cultured p53n and BB-p53n OPCs. Dots represent average +/- SEM of three biological replicates, each averaged from three technical replicates. (D) Schematic of the experimental protocol of injection of the mutant OPCs in recipient mice followed byhematoxylin-eosin staining of brain sections 35 days after injection (35dpi). Only BB-p53null OPC induced histological signs of glioma, as assessed by histopathology . Histology image viewed at 200X magnification, scale bar = 100 µm. (E) Survival plot of mice injected with the BB-p53n (red) or p53n (blue) OPCs (n = 5 mice per group). Mantel-Cox test used to calculate the p-value (p-value = 0.0018).

Fig 1

Cultured OPCs lacking p53 expression and over-expressing PDGF-BB are characterized by increased proliferation and growth patterns similar to glioma cells

The proliferative rate in the two populations of p53n and of BB-p53n OPCs were assessed using KI67 immunocytochemistry, to identify proliferating cells at any stage of the cell cycle, and the 5-ethynyl-2′-deoxyuridine (EdU) Assay (Thermofisher cat# C10337) to identify OPCs in the S phase. KI67 immunocytochemistry was performed on p53n and BB-p53n OPCs, using antibodies specific for this proliferation marker [25] and those for OLIG2, a pan-oligodendrocyte lineage cell marker [26], which revealed a greater percentage of KI67+/OLIG2+ cells in BB-p53n OPC cultures compared to p53n OPCs (Fig. 2A, B). Both mutant populations of OPCs were also treated with EdU while in low growth conditions (1ng/mL PDGF-AA) for 18 h and then processed for immunocytochemistry, using antibody specific for OLIG2 and Edu, to label only cells engaged in DNA synthesis. Also in this case, the percentage of Edu+/OLIG2+ cells in BB-p53n OPCs was significantly higher compared to p53n OPCs (Fig. 2C, D). Together these data indicate that over-expression of PDGF-BB grants a proliferative advantage to the BB-p53n OPCs over the proliferation capacity of the p53n OPCs.Fig. 2 Cultured p53 null OPC over-expressing PDGF-BB are highly proliferative and tend to aggregate in low-adhesion conditions. (A). Confocal images of cultured p53n and BB-p53n OPCs, stained for OLIG2 (red), Ki67 (gray), and DAPI as nuclear counterstain (blue). Scale bar = 10 µm. (B) Bar graphs representing the percentage of OLIG2+ proliferating p53n (white bar) and BBp53n (gray bar) OPCs characterized by high KI67 mean fluorescence intensity (calculated as the value above the median value of all samples). Data represent the average +/- SEM obtained from 2 biological replicates with 3 experimental replicates each and 30 total cells counted from each determinations. Two-tailed Student t-test (p value = 0.001). (C) Confocal images of p53n and BB-p53n OPCs, cultured in low growth conditions, incubated for 18 h with EdU and stained for OLIG2 (red), EdU (gray), and Hoechst as nuclear counterstain (blue). Scale bar = 10µm. (D) Bar graphs represent the percentage of EdU+/OLIG2+ p53n (white bar) and BBp53n (gray bar) OPCs. Data represent average +/- SEM from 3 biological replicates. 50-100 cells counted for each biological replicate. Significance two-tailed Student t-test (p-value = 0.0066). (E) Phase contrast images of spheroid formation Assay for p53n, BB-p53n OPCs, and BB-p53n glioma primary cell line. Images captured from day of plating at day 0 to day 4 in culture. Scale bar = 100µm.

Fig 2

To further characterize these cells in terms of growth patterns, we cultured p53n OPCs, BB-p53n OPCs and a mouse glioma cell line [21] in low-attachment conditions and very low density in order to assess their ability to form spheroids. This property is a known characteristic of gliomas [27]. P53n, BB-p53n OPCs and glioma cells were all plated at low density (1000 cells/cm2) in low-attachment plates (Thermofisher cat#174930) and spheroid-growth media, and their growth pattern was assessed by acquiring daily images over a four day period (Fig. 2E). All three cell types were able to form spheroids. However, while p53n cells started to form spheroids after 3-4 days in culture, the glioma cells formed large spheroid aggregates as soon as day 1 in culture and the BB-p53n OPCs displayed spheroid aggregates as early as day 2 in culture (Fig. 2E). This revealed that the growth characteristics of BB-p53n OPCs were more similar to those of glioma cells than those of p53n OPCs.

The transcriptome of BB-p53n compared to p53n OPCs reveals increased cell cycle transcripts, decreased markers of OPC differentiation and differential expression of proneural markers, cell adhesion genes, and histone modifying enzymes

To further investigate potential mechanisms underlying the pro-transformation effect of PDGF-BB overexpression in p53n OPCs, we performed RNA-seq. Differential expression analysis between BB-p53n and p53n OPCs revealed over 4000 significantly differentially expressed genes (Supp. Table 1). Among the transcripts with higher expression in the BB-p53n OPCs, we detected genes encoding for positive cell cycle regulation (e.g. Cdc6, Cdc45, Ccne2) (Fig. 3A). GSEA analysis of the upregulated genes confirmed a significant enrichment in gene sets related to proliferation, including those regulating the G1-S and the G2-M transition [22], as well as ontologies related to DNA replication and cell division (Fig. 3B, C). Among the downregulated transcripts, we detected several genes related to OPC differentiation (e.g. Cnp, Myrf, Mbp), fatty acid metabolism and central nervous system myelination, a major feature of mature oligodendrocytes (Fig. 3D). By filtering for highly significant genes enriched in proneural glioma, we noted the upregulation of several cell cycle controlling genes (Cdc7,Cdc25a, Atad5), in BB-p53n OPCs and the down-regulation of OPC markers (Nkx2-2, Gpr17) (Fig. 3E). Consistent with the growth pattern of BB-p53n OPCs, compared to p53n OPCs, it was not surprising to detect differential expression of genes regulating cell adhesion and glioma growth (Fig. 3F). For instance, Cd9 [28,29], Cd93 [30], known to be expressed in gliomas, were upregulated, together with oligodendrocyte specific connexins, such as Gjb1, while the astrocytic connexin Gja1, previously reported in astrocytomas [31] was downregulated. This is of great interest as differential connexin expression was previously reported to play an important pro-tumorigenic role and enhance self-renewal [32].Fig. 3 Altered transcriptome in BB-p53n OPCs compared to p53n reveals increased cell cycle transcripts, decreased markers of OPC differentiation and differential expression of proneural markers, cell adhesion genes, and histone modifying enzymes. (A). Volcano plot of differentially expressed genes (adjusted p-value cutoff of 0.05 and log2 fold change of 1) between BB-p53n and p53n OPCs. Of the 2149 upregulated genes (red), the ones positively regulating cell cycle are indicated in text boxes (red text). Of the 2312 downregulated genes (blue), those related to differentiation are indicated (blue text). (B) Dot plot of GSEA data visualizing multiple GO gene sets, obtained by running the differential expression data against the full GO database. Red to blue color gradient represents adjusted p-value. Size of dots represents the number of genes matching between gene set and input gene list. Normalized enrichment score (NES) is plotted on the x-axis. See supplemental Table for full enrichment data table. (C) GSEA of the differential expression data shown in Fig. 3A identifying positive regulation of G1-S and G2-M transition. Curated gene sets obtained from Tirosh et al. [22,]. Adjusted p-values < 1e-10. (D) GSEA of the differentially expressed genes shown in Fig. 3A. Note decreased transcripts relative to OPC differentiation (GO:0048709), adjusted p-values = 0.07; central nervous system myelination (GO:0022010), adjusted p-value = 0.06; and fatty acid metabolic process (GO:0006631) , adjusted p-value < 0.001. (E–G) Heatmap of gene expression data filtered for highly significant (adj p-value < 1e-8) proneural markers [5] (E), cell adhesion genes, including members of the connexin (top) and of the tetraspanin families (bottom) (F) and selected histone lysine modifying enzymes (G) in p53n (teal bar on top) and BBp53 (orange bar on top). Red indicates relatively higher gene expression and blue indicates relatively lower represented by z-scores. Z-score calculated with tpm normalized gene counts.

Fig 3

In addition, BB-p53n OPCs were characterized by differential expression of enzymes responsible for post-translational modifications of histone tails compared to p53n OPCs (Fig. 3G). For instance, transcripts for the enzymes responsible for repressive methylation of histone H3K27 (e.g. Ezh2, Eed, Suz12) were upregulated, while those responsible for the removal of this mark (e.g. Kdm6a, Kdm6b), were significantly downregulated. These data support the interpretation that overexpression of PDGF-BB in p53n OPCs alters their transcriptome providing them with a proliferative advantage and growth characteristic that enable them to form tumors when injected into the brain of healthy recipient mice.

BB-p53n OPCs are characterized by higher levels of H3K27me3 than p53n OPCs

The detection of altered transcript levels of histone modifying enzymes led us to hypothesize the presence of higher levels of repressive histone post-translational modifications (e.g. H3K27me3), which could be responsible for the transcriptional changes. We therefore performed unbiased histone proteomics on histones extracted from cultured p53n and BB-p53n OPCs, using mass spectrometry. Although the differences in histone modifications were mild and did not meet statistical significance, the unbiased histone proteomic analysis suggested differing levels of several histone marks in BB-p53n OPC compared to p53n OPCs, including increased trimethylation of K9 and K27 on histone H3 and decreased trimethylation of K20 on histone H4 (Fig. 4A). These results were then validated using western blot of acid-extracted histones from the populations of OPCs, carrying one or two mutations (Fig. 4B). Of the tested histone marks, we found only H3K27me3 to be significantly higher in BB-p53n OPCs compared to p53n OPCs, while the H4K20me3 mark was significantly lower (Fig. 4C). Since H3K27me3 has been previously reported to play an important role on the oligodendrocyte lineage [[33], [34], [35]], while the H4K20me3 is only beginning to be studied [36], in agreement also with the significant differential expression of components of the PRC2 complex in BB-p53n OPCs, we opted to focus on this mark for further analysis. The levels of H3K27me3 were further evaluated using immunocytochemistry (ICC) and antibodies highly specific for the histone marks. The nuclear mean fluorescent intensity of H3K27me3 immunoreactivity (Fig. 4D) was quantified and revealed higher values in BB-p53n OPCs than p53n OPCs (Fig. 4E). These data suggest that the increased levels of H3K27me3 may play a significant role in the transcriptional changes induced by PDGF-BB overexpression in p53 null OPCs and possibly contribute to facilitating their transformation.Fig. 4 BB-p53n OPCs are characterized by higher levels of H3K27me3 than p53n OPCs. (A) Heatmap of histone modification ratios generated from acid extracted histones and mass spectroscopy. Ratios were converted into z-scores between p53n and BB-p53n samples. Rows organized into unique combinations of lysine modifications in H3 and H4 histone tails. Red indicates relatively higher and blue relatively lower levels in OPC of the indicated genotype. (B) Western blot analysis of acid-extracted histones from cultured p53n and BB-p53n OPCs. Antibodies specific for the post-translational modifications indicated on the left side of the blots and molecular weights on the right. Total histone H3 was used as control for loading and reference for H3K4me3, H3K9me3 and H3K27me3, while total histone H4 was used as control for loading for H4K20me3. (C) Bar graphs representing the ratio of the indicated histone modifications over total histone levels in p53n (white bars) and BBp53n (gray bars) OPCs. Data represent the average values +/- SEM obtained from 3 biological replicates. H3K27me3 significance calculated by two-tailed student t-test (p-value < 0.0001). H4K20me3 significance calculated by two-tailed student t-test (p-value = 0.047). (D) Confocal images of cultured p53n and BB-p53n OPCs stained for OLIG2 (red), H3K27me3 (green), and DAPI as nuclear counterstain (blue). Scale bar = 20 mm. (E) Bar graphs representing the quantification of the nuclear mean fluorescence intensity of H3K27me3 in OLIG2+ OPCs visualized in Fig. 4D. Data represent the average +/- SEM obtained from 3 experimental replicates with 30 cells counted in each replicate. Significance calculated by two-tailed student t-test. p-value = 0.025.

Fig 4

Pharmacological inhibition of EZH2 in BB-p53n OPC reduces proliferation while increasing differentiation but does not change their overall growth pattern

Considering the significant increase of H3K27me3 in BB-p53n OPCs compared to p53n OPCs, we asked whether the inhibition of the enzyme responsible for trimethylation of H3K27 could reverse the differential gene expression in the BB-p53n OPCs and return it to that of p53n OPC, which do not form tumors. Since the enzyme EZH2 is responsible for the deposition of the H3K27me3 mark, we treated BB-p53n OPCs with the pharmacological inhibitor of EZH2, Tazemetostat (EZH2i) for 48 h and validated the effectiveness of the treatment by detecting lower H3K27me3 levels in the EZH2i treated cultures compared to BB-p53n cultures treated with DMSO, as vehicle control (Fig. 5 A, B). Treated cells (BB-p53n-EZH2i) and their controls (BB-p53n DMSO) were also processed for bulk RNA sequencing. Full transcriptome analysis revealed that the majority of genes in the treated BB-p53n OPCs were not reversed by EZH2 inhibition (Supp Table 3; Supp. Fig. S1A). However, a proportion of transcripts was affected by the treatment and returned to levels similar to the ones detected in p53n OPCs. Visualizing those genes as a heatmap, we could identify three clusters, based on the response to EZH2 inhibition (Fig. 5C). Clusters 1 and 2 included genes regulating cell cycle, mitosis and chromosome segregation, which were expressed at higher levels in the BB-p53 OPCs compared to p53n OPCs, and whose expression was lowered by treatment with the EZH2i (Fig. 5C, D). Cluster 3 included genes related to lipid metabolism, which were expressed at lower levels in the BB-p53n OPCs compared to p53n and upregulated by treatment with the EZH2i (Fig. 5C, D). As validation of these results, we selected transcripts from those clusters for further validation by rt-qPCR. Those transcripts included Ccne2, the gene encoding for cyclin E2 and Atad2, encoding an ATPase known to induce the expression of other cell cycle regulators [37], and positively regulate the G1-S transition. The results confirmed the higher transcript levels in BB-p53n OPCs compared to p53n and a statistically significant reduction after 48 h of treatment with the EZH2 inhibitor (Fig. 5E). In addition, even though the GSEA analysis of OPC differentiation gene set showed insignificant enrichment (data not shown), the transcript levels of genes involved in OPC differentiation such as: Prmt5, an arginine methyl transferase important for OPC differentiation [19] and Erbb2, a tyrosine kinase involved in OPC differentiation [38], showed a trend towards increased expression after 48 h treatment with EZH2i (Supp. Fig. S2A). These data suggest that EZH2 inhibition of BB-p53n OPCs significantly decreased the expression of genes regulating proliferation, while only partially affected the increase of genes associated with differentiation.Fig. 5 Reduction of H3K27me3 in BB-p53n OPC using pharmacological inhibitors of EZH2 reduced the levels of cell cycle-related transcripts while increasing markers of differentiation. (A). Western blot analysis of acid extracted histones from cultured BB-p53nOPCs treated with either EZH2i or DMSO, as vehicle control. Histones were probed with antibodies specific for H3K27me3 and for total H3. (B) Bar graph represents the ratio of H3K27me3 immunoreactivity over total H3 calculated by dividing the intensity of the H3K27me3 bands by the intensity of total H3 bands. Data represent the average +/- SEM from 3 biological replicates.. Significance calculated by one-tail student t-test (p-value = 0.015). (C) Heatmap of gene expression data of selected differentially expressed genes comparing p53n in blue columns, vehicle treated (DMSO) BB-p53n in orange columns, and EZH2 inhibitor treated BB-p53n OPCs in purple columns. All samples done in triplicate. Genes represented in rows and high relative gene expression is red while low relative gene expression is blue. Clusters identified by k-means implemented on the full transcriptome. Z-scores calculated with normalized tpm values. (D) Dot plots of overrepresentation analysis on genes from the three clusters labelled in panel C. Analysis run against the gene ontology database. Gene ratio on the x-axis represents the number of genes matching between input gene list and ontology gene set divided by the total number of genes in the input list. Blue to red color gradient per dot represents the adjusted p-value of overrepresentation analysis, blue is higher and red is lower p-value. The size of the dots represents number of genes matching between input gene list and ontology gene set. (E) Bar graphs represent the levels of Atad2 and Ccne2 transcripts. The Y-axis represents log2 fold change of ΔΔCt values normalized to housekeeping genes in experiments performed in triplicate. Significance calculated by one-way ANOVA. * = p-value < 0.05, ** = p-value < 0.005.

Fig 5

Based on the transcriptional effects of EZH2i on BB-p53n OPCs, we asked whether also the functional properties of the cells were affected. We therefore assessed proliferation using immunocytochemistry and quantified the proportion of OLIG2+ cells that were also KI67+. After 48 hour of EZH2i treatment, the percentage of proliferating BB-p53n OPCs significantly decreased from 83 % +/-4.99 % to 32 % +/-9.72 % (Fig. 6A, B). Similar results were noted for the EdU assay of cells in S phase, which decreased from 49.71 % +/-3.82 % in DMSO treated BB-p53n OPCs to 31.25 % +/-5.17 % in EZH2i treated cells (Fig. 6C, D). To further assess whether the same treatment also affected the ability of BB-p53n OPCs to differentiate, we removed mitogens and cultured cells in differentiation medium in the presence of either EZH2i or DMSO for an additional 48 h. Differentiation was monitored using immunofluorescence with antibodies specific for the myelin protein for 2′,3′-Cyclic Nucleotide 3′ Phosphodiesterase (CNP) [39], a marker for differentiated oligodendrocytes (Supp. Fig. S2B). The quantification of the CNP+ cells in EZH2i treated BB-p53n OPCs revealed an average of 28 % +/-3.05 % compared to the 15 % +/-3.21 % CNP+ cells in DMSO controls (Supp. Fig. S2C). We asked whether reduction of H3K27me3 would affect the tendency BB-p53n OPCs had to form large spheroid aggregates when grown in low-density and low-attachment conditions in the presence or absence of the EZH2 inhibitor. While the cells grown in the presence of the inhibitors appeared to form smaller clusters on days 1-2 of culture, the effect was no longer visible starting at day 3 (Fig. 6E). Lastly, since spheroid growth is considered a hallmark of stemness, we also asked whether the EZH2i would decrease the levels of well-known stem cell markers (Klf4, Sox2, Myc), expressed at higher levels in the BB-p53n than in p53n OPCs (Fig. 6F). Interestingly, EZH2 inhibition reduced the levels of Sox2 and Myc, while the levels of Klf4 remained elevated. Taken together, reduction of the H3K27me3 mark reduced the proliferation advantage induced by PDGF-BB in BB-p53n OPCs while also increasing their ability to differentiate when stimulated by T3 but only partially affected the stemness properties of these cells.Fig. 6 Reduction of H3K27me3 levels in BB-p53null OPCs by pharmacological inhibition of EZH2, reduces their proliferation but does not affect their growth pattern in low-attachment conditions. (A) Confocal images of BB-p53n OPCs treated for 48 h with either vehicle (DMSO) or the EZH2 inhibitor Tazemetostat (EZH2i) and then stained for OLIG2 (red), Ki67 (gray), and DAPI as a nuclear counterstain (blue). Scalebar = 20 mm. (B) Bar graph representing the percentage of proliferating Ki67+/OLIG2+ cells in cells treated with DMSO (CTRL, gray bar) or Tazemetostat (EZH2i, black bar). Values represent the average +/- SEM obtained from 3 biological replicates with 3 experimental replicates each and 30 cells counted for each experiment. High Ki67 was determined by nuclear mean fluorescent value above the median value of all samples. Significance two-way Student t-test (p value = 0.0003). (C) Confocal images of BB-p53n OPCs treated for 48 h with either vehicle (DMSO) or Tazemetostat (EZH2i) and incubated for 18 h with EdU and then stained for OLIG2 (red), EdU (gray), and Hoechst as nuclear counterstain (blue). Scale bar = 10 um. (D) Bar graph represents the percentage of EdU+ in OPC treated as described in 6C. Values represent the average +/- SEM in BB-p53n OPCs treated with DMSO (CTRL, gray bar) or Tazemetostat (EZH2i, black bar). Data obtained from 3 biological replicates and 50–100 cells counted for each biological replicate. Significance two-tailed Student t-test (p-value = 0.045). (E) Phase contrast images of spheroid formation assay in BB-p53n OPC untreated or treated with Tazemetostat for 48 h. Images were captured daily, starting from the day of plating. Scale bar = 100 um. (F) Heatmap of gene expression data filtered for stem markers. Red indicates relatively higher gene expression and blue indicates relatively lower represented by z-scores. Z-score calculated with tpm normalized gene counts.

Fig 6

Discussion

This study was designed to address the early steps of gliomagenesis in oligodendrocyte progenitors, as these cells have been previously identified as the cell of origin of proneural gliomas [9,10], a subtype of tumors characterized by the prevalence of OPC transcriptional signature [5,10]. Since specific genetic alterations have been identified for each subtype, with deletion of P53, NF1 or PTEN, IDH1 mutations and amplification of PDGF signaling reported in proneural gliomas, several groups adopted different strategies to create distinct animal models. Those included the use of the avian RCAS system [7], the injection of retroviral vectors [8], the combination of viral vector injection in transgenic mice [[9], [10], [11]]. Our results show that primary cultures of OPCs carrying specific genetic alterations, begin to show transcriptional and epigenetic changes in vitro, and give rise to gliomas when injected into the brain of recipient mice, providing a well-controlled experimental platform to interrogate the molecular events characterizing the early stages of tumor formation.

An open question that remains unanswered is whether and how specific genetic mutations occurring in OPCs lead to transformation. This study addresses this question by investigating the effect of p53 deletion alone or in combination with PDGF-BB on the OPC transcriptome and on histone post-translational modifications. We show here that OPCs carrying PDGF-BB overexpression and p53 deletion (BB-p53n OPC), are intrinsically distinct from OPCs carrying p53 deletion alone (p53n OPC) and are capable to induce the formation of gliomas 35 days after injection in the brain of recipient adult mice. Primary cultures of OPC carrying p53 deletion and PDGF overexpression were characterized by higher proliferative rates and decreased ability to differentiate than OPCs with p53 deletion alone. These findings are consistent with previous reports in mice with deletions of both Trp53 and Nf1 in adult OPC, where the premalignant phase of OPCs was characterized by increased proliferation and decreased differentiation [11], thereby suggesting that proliferative advantage and inability to differentiate are common steps in gliomagenesis. In addition, to begin deciphering the molecular mechanisms underlying these early changes occurring in premalignant cells, we conducted an unbiased histone proteomics analysis, followed by validation using confocal imaging and western blot analysis of histone extracts. Collectively, these experiments detected higher levels of the repressive H3K27me3 histone mark in the BB-p53n OPCs compared to p53n OPCs, a finding which was also consistent with the higher expression levels of enzymes responsible for the deposition of this mark (e.g. EZH2), and lower transcript levels of enzymes responsible for erasing the repressive H3K27me3 histone mark. At a transcriptional level, BB-p53n OPCs were characterized by higher expression levels of genes encoding for positive regulation of the cell cycle and lower levels of transcripts related to oligodendrocyte differentiation.

These data are consistent with reports in adult gliomas, where higher levels of EZH2 [40] and higher levels of H3K27me3 [41,42] have been associated with worse prognosis. In our study, the reduction of H3K27me3 levels in BB-p53n OPCs, by treatment with a well-recognized EZH2 inhibitor (Tazemetostat), significantly reduced the expression of genes regulating cell division, while increasing the expression of genes related to differentiation. These data are consistent with the previously suggested prognostic value of H3K27me3 levels in human gliomas [41,42]. These data also validate the previously reported beneficial effect of reduced EZH2 activity in decreasing proliferation and migration of adult human glioma cells [43].

Importantly, Tazemetostat treatment of BBp53n OPCS, despite reducing proliferation and the expression levels of some stemness genes, it did not entirely revert the growth characteristics of BBp53n OPCs in low attachment conditions. This partial effect raised important concerns towards the suggestion of monotherapy with EZH2i as a potential therapeutic option. In agreement with this consideration, previous work in animal models showed the beneficial synergistic effect of treatment with inhibitors of both, EZH2 (e.g. Tazemetostat) and HDAC (e.g. Panobistat) in reducing tumor growth [44,].

Our results also differ from those reported for pediatric diffuse pontine intrinsic gliomas, characterized by the H3K27M mutation, where lysine residue K27 in histone H3 is replaced by a methionine, thereby resulting in overall decreased H3K27me3 mark [45,46]. In agreement with the cell specificity of the effect of EZH2 inhibition, and previous reports on the importance of EZH2 in OPC cell fate [34], it is important to note that short term inhibition was beneficial for survival, while long-term depletion resulted in cell fate switch and tumor progression [47]. Collectively, these data suggest that the effect of EZH2 inhibition is time-dependent and cell type specific, and suitable to target early pre-malignant states of transformation.

These data also highlight the limitations of treating cells with a single inhibitor, as the epigenomic landscape of cells cannot be singly ascribed to specific histone modifications. Accordingly, the overall effect of Tazemetostat in reversing the transcriptional differences between the BB-p53n and the p53n OPCs was only partial and affected genes regulating proliferation, resulting in elimination of the proliferative advantage and favoring differentiation. The partial reversal of the BB-p53n OPC transcriptome also suggests the possible existence of additional epigenetic differences with p53n OPC, such as the reduction of histone H4K20me3, as identified in this study, and possibly DNA and RNA methylation or hydroxymethylation and microRNAs, which could not be captured by the histone proteomic analysis.

Conclusions

This study identifies important histone post-transcriptional modifications and transcriptional changes induced by PDGF-BB overexpression in p53 null OPCs. However, a potential limitation of this study is that we did not characterize the entire extent of the epigenetic landscape, but focused on one histone mark (e.g.H3K27me3) as more abundant in the tumor-inducing BBp53n cells compared to the p53n OPCs. Since these cells were also characterized by decreased levels of the heterochromatic mark H4K20me3, future studies will be needed to further characterize the functional relevance of this mark and possibly inform on novel combination therapies.

Inclusion and diversity

We worked to ensure sex balance selection in the selection of non-human subjects. While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list.

Funding

This work was performed thanks to grants from the 10.13039/100000002 National Institute of Health to Patrizia Casaccia (R35- NS111604 ), to Peter Canoll and Patrizia Casaccia (5R01NS052738 ) and to Benjamin Garcia (P01CA196539 ; R01HD106051 ).

CRediT authorship contribution statement

Dennis Huang: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Angeliki Mela: Writing – review & editing, Investigation, Data curation. Natarajan V. Bhanu: Writing – review & editing, Investigation. Benjamin A. Garcia: Writing – review & editing, Methodology, Investigation, Funding acquisition. Peter Canoll: Writing – review & editing, Funding acquisition, Conceptualization. Patrizia Casaccia: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Image, application 2

Acknowledgments

The authors thank members of the Casaccia Lab for helpful discussions, the Epigenetic Core at the ASRC for help with the preparation of samples for RNA sequencing.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neo.2024.101042.
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