Epigenetic Heterogeneity and Mitotic Heritability Prime Endothelial Cell Gene Induction

This information is current as Paul J. Turgeon, Gary C. Chan, Lucy Chen, Alisha N. Jamal, of October 2, 2021. Matthew S. Yan, J. J. David Ho, Lei Yuan, Neke Ibeh, Kyung Ha Ku, Myron I. Cybulsky, William C. Aird and Philip A. Marsden J Immunol published online 29 January 2020 http://www.jimmunol.org/content/early/2020/01/28/jimmun ol.1900744 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 29, 2020, doi:10.4049/jimmunol.1900744 The Journal of Immunology

Epigenetic Heterogeneity and Mitotic Heritability Prime Endothelial Cell Gene Induction

Paul J. Turgeon,*,†,‡ Gary C. Chan,* Lucy Chen,†,‡,x Alisha N. Jamal,* Matthew S. Yan,†,‡,x J. J. David Ho,x Lei Yuan,{ Neke Ibeh,‖ Kyung Ha Ku,*,†,‡ Myron I. Cybulsky,*,#,** William C. Aird,{ and Philip A. Marsden*,†,‡,x

Homogeneous populations of mature differentiated primary cell types can display variable responsiveness to extracellular stimuli, although little is known about the underlying mechanisms that govern such heterogeneity at the level of gene expression. In this article, we show that morphologically homogenous human endothelial cells exhibit heterogeneous expression of VCAM1 after TNF-a stimulation. Variability in VCAM1 expression was not due to stochasticity of intracellular signal transduction but rather to preexisting established heterogeneous states of DNA methylation that were generationally conserved through .

Variability in DNA methylation of the VCAM1 promoter resulted in graded RelA/p65 and RNA polymerase II binding that gave Downloaded from rise to a distribution of VCAM1 in the population after TNF-a stimulation. Microarray analysis and single-cell RNA sequencing revealed that a number of cytokine-inducible genes shared this heterogeneous response pattern. These results show that heritable epigenetic heterogeneity is fundamental in inflammatory signaling and highlight VCAM1 as a metastable epiallele. The Journal of Immunology, 2020, 204: 000–000.

issue inflammation requires the coordinated expression of inflammatory phenotype of atherosclerosis, VCAM1 is expressed within the vascular endothelial cells (EC) to fa- by EC at the periphery of early atherosclerotic lesions, in which http://www.jimmunol.org/ T cilitate a targeted and efficient immune response. VCAM1 it contributes to lesion initiation and progression (3). Despite is a membrane that is expressed by EC exposed to proin- its fundamental function in the vasculature, VCAM1 expression flammatory cytokines, such as TNF-a, in which VCAM1 partici- patterns are not well understood, as studies have shown that there pates in the integrin-dependent arrest of circulating leukocytes is variation in the relative inducibility of VCAM1 in EC across during the early phases of inflammation (1, 2). In the chronic individuals, vascular beds, and within a vascular bed (4–7). Recent work has recognized that seemingly homogeneous populations of mature cells can display substantial phenotypic

*Department of Laboratory and Pathobiology, University of Toronto, heterogeneity at the single-cell level, both in vitro and in vivo. by guest on October 2, 2021 Toronto, Ontario M5S 1A8, Canada; †Keenan Research Centre, Li Ka Shing Knowl- Although often classified as stochastic noise, it is becoming clear edge Institute, St. Michael’s Hospital, Toronto, Ontario M5B 1T8, Canada; ‡Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, that fundamental cellular behaviors govern phenotypic distribu- Canada; xDepartment of Medical Biophysics, University of Toronto, Toronto, tions. Single-cell variability has been traced to alterations in protein { Ontario M5G 1L7, Canada; Center for Vascular Biology Research at Beth Israel allocation during mitosis, differential paracrine signaling in cul- Deaconess Medical Center, Harvard Medical School, Harvard University, Boston, MA 02215; ‖Princess Margaret Cancer Center, University Health Network, Toronto, ture, and variable receptor–ligand interactions (8–10). Recent Ontario M5G 2C1, Canada; #Department of Immunology, University of Toronto, work on the endothelial restricted gene von Willebrand factor Toronto, Ontario M5S 1A8, Canada; and **Toronto General Hospital Research In- (vWF) has shown dynamic mosaic heterogeneity that was sto- stitute, University Health Network, Toronto, Ontario M5G 2C4, Canada chastic in nature and related to alterations in DNA methylation ORCIDs: 0000-0001-9063-3933 (P.J.T.); 0000-0003-2027-6162 (L.C.); 0000-0002- 1696-2867 (M.I.C.); 0000-0003-4877-3523 (P.A.M.). at the promoter region (11). Moreover, differential expression of Received for publication September 26, 2019. Accepted for publication December VCAM1 has become prominent in literature, but there is no 23, 2019. mechanism has been identified (12, 13). Heterogeneity in gene This work was supported by Canadian Institutes of Health Grant MOP 142307 to expression extends many possible advantages for cells, namely in P.A.M. buffering population responses (10). We observed heterogeneous P.J.T. performed and analyzed experiments and wrote the manuscript. G.C.C. per- VCAM1 expression in vascular EC and sought to explore the formed and analyzed experiments and edited the manuscript. L.C., A.N.J., M.S.Y., underlying mechanisms governing VCAM1 heterogeneity. J.J.D.H., L.Y., N.I., and K.H.K. performed and analyzed experiments. W.C.A., M.I.C., and P.A.M. conceived experiments and edited the manuscript. The microarray data presented in this article have been submitted to the Gene Ex- Materials and Methods pression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number , cloning GSE141374. Primary HUVEC were isolated and maintained as previously described Address correspondence and reprint requests to Dr. Philip A. Marsden, University of (14). For HUVEC cloning experiments, single cells were counted using the Toronto, St. Michael’s Hospital, 209 Victoria Street, Room 521, Toronto, ON M5B 1C6, Canada. E-mail address: [email protected] Beckman-Coulter Vi-CELL counter and either serially diluted to a con- centration of one cell/100 ml or sorted using FACS and seeded on gelatin- The online version of this article contains supplemental material. coated 96-well plates. Cells were grown to confluence 24-well, six-well, Abbreviations used in this article: ChIP, chromatin immunoprecipitation; ChIP-BA, and finally 10-cm gelatin-coated plates. Cells were maintained according ChIP bisulfite analysis; EC, endothelial cell; hnRNA, heterogeneous nuclear RNA; to the suppliers’ instructions, as previously described (15). IP, immunoprecipitated; LUMA, Luminometric Methylation Assay; Pol II, polymer- ase II; qPCR, quantitative PCR; qRT-PCR, quantitative real-time PCR; scRNA, Chromatin immunoprecipitation assay single-cell RNA sequencing; UMI, unique molecular index. Chromatin immunoprecipitation (ChIP) assay was performed on postconfluent Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 passage three HUVEC using the ChIP Assay Kit (Upstate Biotechnology), as

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900744 2 EPIGENETIC HETEROGENEITY IN ENDOTHELIAL VCAM1 EXPRESSION described (15). Approximately 3 3 106 cells were used per ChIP assay. for 1 h. Five microliters of the reaction mixture was spotted onto Whatman Briefly, formaldehyde was added to culture medium to a final concentra- DE81 ion-exchange filter paper in triplicate, washed twice in 20 ml of tion of 1% and incubated for 10 min at 37˚C. Sonication achieved soluble 0.5 M sodium phosphate wash buffer (pH 7) with shaking for 20 min, chromatin fragments containing DNA ranging in size from 200 to 400 bp. dipped in 2 ml of 70% ethanol and then in 2 ml of absolute ethanol, and air Sonicated cell lysates were centrifuged for 10 min at 14,000 rpm at 4˚C. dried, and radioactivity was measured by scintillation counting. Raw The supernatant was diluted to 2000 ml with ChIP dilution buffer. A 20-ml values were corrected for background radioactivity by subtracting value aliquot was removed, the cross-links were reversed by heating at 65˚C for from control reactions that were performed using nonradioactive SAM and 4 h, and then 2 ml was used to quantitate the amount of input DNA. The reactions that excluded SssI methylase. remaining diluted supernatant was precleared with salmon sperm DNA/ protein A agarose and then incubated overnight at 4˚C with either 5 mlof Cytosine extension assay antiserum, 5 mg of specific Ab, 5 mg of control rabbit IgG, or no Ab (Santa Five hundred nanograms of genomic DNA was digested to completion with Cruz: RNA polymerase II [Pol II] [sc-899]; Upstate Biotechnology: H2A HpaII and MspI for 16–18 h. Digested DNA was then subjected to a single- [07-146], H2B [07-371], H4 [05-858], acetyl H3 [06-599], acetyl H4 [06- nucleotide extension reaction by incubating in a 25-ml reaction mixture 866], H3K4me2 [07-030], and MeCP2 [07-013]; Abcam: H3 [ab1791], containing 13 PCR buffer II, 1 mM MgCl2, 0.25 U of Taq polymerase, H3K9me2 [ab7312], and H3K9me3 [ab8898]). To collect the immuno- and0.25Uof3[H]-dCTP (sp. act. 64 Ci/mmol; Amersham) for 1 h at precipitated (IP) complexes, salmon sperm DNA/protein A agarose was 56˚C, as described (18). Duplicate 10-ml aliquots from each reaction added and incubated for 1 h at 4˚C. After washing, the immune complexes were applied to Whatman DE-81 ion-exchange filters, washed with so- were eluted twice with 250 ml of freshly prepared elution buffer (1% SDS dium phosphate buffer, and air dried, and radioactivity was measured by and 0.1 M NaHCO3). The formaldehyde cross-links were reversed in scintillation counting. combined eluates by heating at 65˚C for 4 h. The resulting IP DNA sample was purified, resuspended in 25 ml of filtered water, and 2 ml was used in Luminometric Methylation Assay real-time PCR quantification (Applied Biosystems, Foster City, CA). The primers and probe for human NOS2 or VCAM1 loci reside in the same Analysis of global DNA methylation analysis was performed essentially as proximal promoter regions analyzed by the bisulfite method. The amount described (19). Briefly, this technique takes advantage of an isoschizomer Downloaded from of template present was calculated relative to a standard curve. IP DNA pair of restriction enzymes (MspI/HpaII) that cleave 59-CCGG-39. MspI is was calculated by first subtracting the amount of sequence present in the methylation insensitive, but HpaII is methylation sensitive. Postconfluent no Ab background control or IgG control from the amount present in the IP passage three HUVEC were treated with 10 ng/ml TNF-a for 2, 4, 6, 8, DNA and then dividing by the amount of sequence in the diluted input and 24 h, after which total cellular DNA was extracted using the blood mini material, as described. kit (Qiagen) as per the manufacturer’s protocol. Five hundred nanograms of DNA were digested with EcoRI/MspI and EcoRI/HpaII (New England Biolabs, Ipswitch, MA) in two separate 20-ml reactions. Digests were ChIP bisulfite analysis of transcriptionally active http://www.jimmunol.org/ promoter sequences incubated at 37˚C for 4 h, followed by a 20-min incubation at 80˚C to inactivate restriction enzymes. EcoRI (cleaves 59-GAATTC-39 sequence) Postconfluent passage three HUVEC were stimulated with TNF-a was used for normalization. Pyrosequencing using a PSQ96HS instrument (10 ng/ml) for 4 h, after which chromatin extracts were isolated using (EpigenDx) was used to assess each restriction site. The HpaII/MspI ratio the ChIP Assay Kit (Upstate Biotechnology). The chromatin was sonicated was calculated as (HpaII/EcoRI)/(MspI/EcoRI). to fragments averaging 500 bp in size. ChIP was performed using 15 mgof rabbit polyclonal anti-RNA Pol II Ab (sc-899; Santa Cruz) on ∼9 3 106 Sodium bisulfite genomic sequencing, pyrosequencing TNF-a–stimulated HUVEC. The IP complexes were collected with salmon Given that the nascent strand of replicating DNA is hemimethylated im- sperm DNA/protein A agarose. The supernatant fraction was kept as the mediately following DNA replication, only quiescent postconfluent primary unbound DNA control. Bound and unbound DNA samples were reverse cells were used for DNA isolation. Two micrograms of genomic DNA was cross-linked, phenol/chloroform-extracted, and resuspended in 10 ml and by guest on October 2, 2021 digested with BamHI and then subjected to the sodium bisulfite treatment, 100 ml of nuclease-free water, respectively. Ten microliters of the DNA as described (15). Twenty-five nanograms of the bisulfite-treated DNA was was directly subjected to the sodium bisulfite treatment without prior di- subjected to 35 cycles of PCR amplification in a volume of 50 ml. Two gestion with BamHI. One-fifth of the bisulfite-treated DNA was subjected microliters of the PCR product was used as template for another 35 cycles to 40 cycles of PCR amplification in a volume of 50 ml. PCR primers with of nested PCR amplification in a volume of 50 ml using the nested primers. a short amplicon size were specifically designed to the bisulfite-modified All PCR primers were specifically designed to the sodium bisulfite– sonicated DNA (sense strand). Ten microliters of the PCR product was modified sense strand. The final PCR products were subcloned using the used as template for another 40 cycles of nested PCR amplification in a TA Cloning Kit (Invitrogen) to yield individual strands, followed by se- volume of 50 ml using the nested primers. The final PCR products were quencing. For each cell type, at least 15 randomly chosen subclones were subcloned using the TA Cloning Kit (Invitrogen, Burlington, ON) to yield sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems). individual strands, followed by sequencing. For each DNA sample, at least For pyrosequencing, 2 ml of the first PCR product was subjected to 15 randomly chosen subclones were sequenced using an ABI Prism 377 45 cycles of nested PCR using 10 pmol of pyrosequencing primers, with a DNA sequencer (Applied Biosystems). biotin-labeled antisense primer. These were then sequenced by EpigenDx Confocal immunofluorescence assays using a PSQ instrument (Worcester, MA). HUVEC were seeded onto 0.2% gelatin–coated 22 3 22–mm glass cov- Cell transfection a erslips and treated with 10 ng/ml TNF- postconfluence. For VCAM HUVEC were grown to ∼90% confluence and removed from the plate via analysis, coverslips were fixed in 4% paraformaldehyde and rinsed with 2/2 2/2 trypsinization. They were transfected in DMEM containing 10% FBS as PBS , followed by blocking with PBS containing 1% BSA. Cells recommended by Herna´ndez et al. (20) and plated on 100-mm culture were stained using mouse monoclonal VCAM1 (catalog no. ab24628; dishes and used in experimental protocols 48 h posttransfection. Abcam) Ab and Dylight 594–conjugated goat polyclonal secondary Ab (catalog no. ab96881; Abcam). For RelA/p65 staining, cells were fixed and FACS and luciferase assays permeabilized with 0.2% Triton X-100 followed by blocking with 0.3 M glycine and 1% BSA in PBS2/2. Cells were stained with rabbit polyclonal Cells were cotransfected with either pGL2-basic, pGL2-control, pGL2– to NF-kB RelA/p65 (catalog no. ab16502; Abcam) Ab and Dylight 488– NF-kB, or pGL2-2167/+25 VCAM1 Firefly luciferase and the SV40- conjugated goat polyclonal secondary Ab (catalog no. ab96899; Abcam). Renilla luciferase as recommended by Promega. Cells were treated with Coverslips were mounted on glass slides using Vectashield mounting 10 ng/ml TNF-a for 4 h 48 h posttransfection. For VCAM-high and -low media with DAPI (catalog no. H-1500; Vector Laboratories), visualized readings, FACS was performed as previously indicated above. Protein using a Zeiss Axio LSM 700 confocal microscope, and analyzed using the extracts were made and subjected to luminescence measurements with a Zeiss ZEN software. Perkin Elmer ENVISION, as recommended by Promega. Methyl-accepting assay RNA extraction of VCAM1 high/low cells HUVEC genomic DNA was used in methyl-accepting assays, as previously FACS was performed as previously indicated above. Total RNA was described (16, 17). Briefly, 500 ng of DNA was incubated with 3[H]-methyl extracted using the RNeasy Plus Mini Kit from Qiagen. A predetermined SAM (2 mCi) and SssI methylase (3 U) in 13 reaction buffer (120 mM amount of in vitro synthesized, capped, and polyadenylated luciferase NaCl, 10 mM Tris-HCl [pH 7.9], 10 mM EDTA, 1 mM DTT) at 30˚C mRNA was added to each sample immediately before RNA extraction and The Journal of Immunology 3 measured by quantitative real-time PCR (qRT-PCR) to control for ef- visualization were performed using R packages, SCATER (v1.2.0), ficiencies of RNA extraction and first-strand cDNA synthesis. For CELLRANGERRKIT (v1.1.0), RTSNE (v0.11), SC3 (v1.3.14), EDGER mRNA and heterogeneous nuclear RNA (hnRNA) studies, qRT-PCR (v3.16.5), SEURAT (v2.1) (24, 25), and PCAMETHODS (v1.50.0). We was done as previously described. For microRNA analysis, TaqMan obtained steady-state mRNA transcript profiles of 8363 cells (4 h, TNF-a Human microRNA Assays and TaqMan microRNA Reverse Tran- treatment) that passed quality control and filtering, for which an average scription kit (Applied Biosystems) were used for microRNA mea- of 3574 genes per cell were measured with 35,283 mean reads per cell. surements according to the manufacturer’s recommendations. Results Additionally, the transcriptional profile from 8281 untreated cells that were normalized to cyclophilin A mRNA levels. mRNA relative fold passed filtering yielded an average of 3627 genes per cell measured with changes were calculated using either absolute quantification with 37,151 mean reads per cell. plasmid standard curves or the comparative cycle threshold method, which was used for microRNAs. Quantitative TaqMan real-time RT-PCR Preparation of single HUVEC for single-cell RNA sequencing One microgram of total cellular RNA was reverse transcribed with random hexamer primers using the Invitrogen SuperScript II kit Single-donor HUVEC were isolated, as previously described (21). Primary (ThermoFisher Scientific) following the manufacturer’s protocol. cultures of EC were plated on 100-mm tissue culture plates precoated with cDNA was diluted to a final volume of 50 ml. Two microliters of the 0.2% gelatin. Cells were maintained in Endothelial Cell Growth Medium reverse transcription reaction mixture was subsequently used as a 2 (C-22011; PromoCell) containing the following: 0.02 ml/ml FCS, template for RT-PCR quantification. Please contact the corresponding 5 ng/ml recombinant human epidermal growth factor (rhEGF), 10 ng/ml author for the sequences of the primers and the TaqMan probes used for recombinant human basic fibroblast growth factor (rhbFGF), 20 ng/ml the quantitative analysis of the human NOS2 and GAPDH mRNA as insulin-like growth factor (Long R3 IGF), 0.5 ng/ml recombinant human well as the Sybr Green primers for the quantification of human VCAM1 vascular endothelial growth factor 165 (rhVEGF165), 1 mg/ml ascorbic mRNA. The human NOS2 primers and probe set spans 4 (26). acid, 22.5 mg/ml heparin, and 0.2 mg/ml hydrocortisone. One hundred The human GAPDH primers and probe set spans 2 and 3. The

units per milliliter of penicillin and 100 U/ml streptomycin were added VCAM1 primers span intron 8 and thus are capable of detecting the two Downloaded from to the supplemented EC growth media. Cell cultures were maintained at major splice variants (which include or exclude 5) in cytokine- 37˚C in a humidified 5% CO atmosphere, and the media replenished activated HUVEC (27). The RelA/p65 primers span 6–7 and de- every 48 h. One plate of confluent passage three HUVEC were stimu- tect all splice variants. Measurements were performed in triplicate using lated with TNF-a (10 ng/ml; R&D systems) for 4 h, after which cells an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). were detached with 0.05% trypsin-EDTA as described previously. Cells All real-time PCR assays were performed in the presence of serial dilu- were resuspended in 5 ml of media and filtered through a cell strainer to tions of reference plasmids for determination of template copy number. eliminate clumps and debris. An untreated cell suspension was similarly Copies of the NOS2 or VCAM1 transcript per microgram of total RNA prepared. All samples for single-cell RNA sequencing (scRNA-seq) were were assessed as raw copy number and were normalized to GAPDH. http://www.jimmunol.org/ processed the same day. Sequencing libraries were prepared, targeting Primers. Please contact authors for primer sequences. ∼ 6000 cells per sample at the Princess Margaret Genomics Centre fol- Statistical analysis. All data sets represent the mean 6 SEM of at least 9 lowing the Chromium Single Cell 3 Reagent Kits v2 User Guide three independent experiments, unless otherwise stated. Statistical analysis (CG00052). This platform uses droplet cell capture technology and ap- was performed using a two-tailed t test or ANOVA, where appropriate. plies a 39 end counting sequencing approach. The libraries were se- quenced on an Illumina HiSeq 2500 instrument (run parameters: read 1–26 cycles, read 2–98 cycles, index 1–8 cycles) targeting 100,000 reads Results per cell. (Actual result for untreated: 8281 cells sequenced, 37,151 mean Heterogeneous expression of VCAM1 after TNF-a induction reads per cell, 3627 mean genes per cell, 15,719 median unique mo- ∼ Populations of isogenic primary EC grown in culture displayed

lecular index [UMI] counts per cell, and 20% sequencing saturation. by guest on October 2, 2021 Actual result for TNF-a treated: 8363 cells sequenced, 35,283 mean a time-dependent increase in VCAM1 transcription during stimu- reads per cell, 3574 mean genes per cell, 15,600 median UMI counts lation with TNF-a. Time-course studies showed peak VCAM1 ∼ per cell, and 20% sequencing saturation). mRNA inducible in bulk RNA extracted from populations of scRNA-seq computational analyses cells following 4 h of TNF-a stimulation, which was associated with peak RNA Pol II loading on the proximal promoter region Data from the treated and untreated samples were analyzed individually and also aggregated. Raw Illumina sequencing data from Chromium Single Cell (Fig. 1A, 1B). In contrast, we detected no changes in NOS2 libraries were preprocessed using the CellRanger (v2.1.0) pipeline from mRNA expression, which is notoriously hard to induce in human 103 Genomics. First, CellRanger mkfastq was used to demultiplex raw EC and suggests specificity in gene expression (Fig. 1C, 1D) base call files based on sample indices and converted the cell barcodes and (28). Interestingly, we consistently observed marked variability in read data to FASTQ files. Each transcript has a library barcode (sample index) that allows identification of pooled samples on one sequencing lane. VCAM1 cell surface induction between individual cells within the Cell barcodes (16 bp) are used to identify the cell the read came from, and population during TNF-a stimulation (10 ng/ml; 4 h) by confocal a 10-bp UMI uniquely identifies each RNA molecule. CellRanger count imaging (Fig. 1E, 1F). Such variability in VCAM1 at the protein takes FASTQ files from CellRanger mkfastq and performs alignment, fil- level was explained by heterogeneity in transcription as high tering, and UMI counting. FASTQ sequences were mapped to the GRCh38 VCAM1 protein–expressing cells also contained increased VCAM1 reference genome using STAR aligner (STAR v2.5.2b) (22). After aligning sequence data to the genome, the quality of the mapping was assessed mRNA and unspliced hnRNA as detected by FISH (Fig. 1G–I). The using RNA-SeQC (v1.1.7) and SAMTOOLS (v1.3.1). The primary anal- homogeneity of the EC preparations was confirmed morpho- ysis output of CellRanger is gene-barcode matrices. Two types of gene- logically and by expression of CD31 (PECAM-1), as previously barcode matrices were generated. The first (unfiltered) contained every described (14). barcode from the fixed list of known barcode sequences, including back- ground (droplets with no cells) and noncellular barcodes (from ambient To explore the determinants of VCAM1 variability, we employed RNA). The second (filtered) contained only the detected cellular barcodes. fluorescent cell sorting techniques to isolate the extreme upper and To remove cells representing background noise, the cumulative fraction of lower 5% of VCAM1 high and low cells (Fig. 1J). We confirmed uniquely mapped reads was plotted against the cell barcodes ordered by elevated levels of VCAM1 mRNA and hnRNA in the high VCAM1– descending number of reads, and the inflection point of the curve was used expressing fraction compared with the low VCAM1 sorted cells as a cutoff. Gene-barcode matrices were normalized according to the method by Lun et al. (23) using the R package SCRAN (v1.2.2). Low- (Fig. 1K, 1L), consistent with FISH studies and distinct when quality cells with log library sizes greater than four median absolute de- compared with the whole population. Importantly, no significant viations below the median log library size, log gene expression greater differences were noted in the ratio of VCAM1 hnRNA/mRNA than four median absolute deviations below the median, or high mito- between these two subpopulations, indicating an analogous pro- chondrial gene expression were removed from the data sets. A total of 8281 cells in the untreated sample and 8363 cells in the TNF-a–treated duction of mature mRNA from hnRNA precursor in both fractions sample were removed from further analysis. PCA, clustering, differen- (Fig. 1M), demonstrating heterogeneity in the transcriptional in- tial gene expression, and t-distributed stochastic neighbor embedding duction of the VCAM1 gene. 4 EPIGENETIC HETEROGENEITY IN ENDOTHELIAL VCAM1 EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. TNF-a induces heterogeneous VCAM1 transcription. (A)RNAPolIIChIPanalysisoftheVCAM1 proximal promoter after TNF-a (10 ng/ml) treatment. (B) qRT-PCR of VCAM1 mRNA normalized to 1,000,000 copies of GAPDH mRNA, which did not change after TNF-a. (C) qRT-PCR of human NOS2, VCAM1 mRNA. Values were normalized to GAPDH mRNA levels. (D)ChIPassaywithanAbdirectedagainstthelarge subunit of Pol II. IP DNA was analyzed using qPCR with NOS2 and VCAM1 primers. (E) Vehicle-treated HUVEC were stained with propidium iodide (PI; left) and VCAM1 (center). Merge shown on right. (F) HUVEC were treated with TNF-a (10 ng/ml) for 4 h and were stained with PI (left) and VCAM1 (center). Merge shown on right. (G) Fluorescence in situ hybridizations using a VCAM1 mRNA probe and (Figure legend continues) The Journal of Immunology 5

The contribution of stochastic signaling to heterogeneous VCAM1 (2116 to +7; Supplemental Fig. 2A). A modest amount of expression. Heterogeneous VCAM1 induction by TNF-a could H3K9me2 and its agonist H3K4me2 was detected at the theoretically relate to stochastic effects in TNF-a–mediated signal VCAM1 promoter but consistent with the previous findings, transduction or posttranscriptional variation. Others have previ- H3K9me3 was not (Supplemental Fig. 2B–D) (28, 33). The ously detected intercellular variability in TNF-a–induced NF-kB amount of H3K9me3 at the VCAM1 promoter may be func- nuclear translocation using an information theory mathematical tionally insufficient to recruit heterochromatin protein 1 (HP1) approach (10). Other work showed preferential RelA/p65 nuclear and MeCP2 binding at the VCAM1 promoter (Supplemental translocation and heterogeneous expression of VCAM1 protein in Fig. 2E, 2F). Overall, these observations suggest that a lack of mouse aortic arch in vivo, possibly mediated by hemody- repressive histone modifications at the VCAM1 promoter may namic influences on NF-kB priming (5). To explore the possible enable trans-factors, such as NF-kB, to selectively activate sources of variability in VCAM1 expression, we examined various VCAM1 in EC. pre- and posttranscriptional events. We did not detect substantial We wished to examine whether changes occur in epigenetic differences in the quantity of various NF-kB signaling compo- marks at the VCAM1 promoter in response to TNF-a and the nents (Supplemental Fig. 1A–H). Specifically, the VCAM1-high kinetics of such changes. Histone (H3/H4) acetylation and H3K4 and -low subpopulations showed no significant difference in methylation are known to be involved in transcriptional acti- RelA/p65 mRNA and protein levels after TNF-a treatment vation in addition to histone eviction (34–36). We found that (Supplemental Fig. 1A–D). We observed IkBa and IkBb induc- TNF-a induced a strong and rapid increase in histones H3/H4 tion and relative enrichment in the VCAM1 high-expressing cells, acetylationandH3K4me2at30minand1h(Supplemental which would dampen VCAM1 transcription (Supplemental Fig. Fig. 2G). These data were normalized to the remaining histones 1E–H). Confocal imaging of TNF-a–stimulated cells showed lit- present because histone N-terminal tail acetylation is known to Downloaded from tle variability in basal and TNF-a–stimulated cellular location of precede histone eviction (36). We also found that TNF-a induces RelA/p65, describing a novel endothelial phenotype (Fig. 2A, 2B). nucleosome eviction at the VCAM1 promoter, as histone H3 density To examine the functionality of TNF-a signal transduction, EC decreased (Supplemental Fig. 2H), with parallel decreases in the were transfected with a promoter luciferase reporter plasmid con- levels of H2A, H2B, and H4 histones (Supplemental Fig. 2H). We taining the human VCAM1 promoter to determine differences in reasoned that histone modifications do not drastically contribute NF-kB signaling between cells. Transfected cells were stimulated to heterogeneity of VCAM1 expression, as near-complete his- http://www.jimmunol.org/ with TNF-a, sorted based on endogeneous VCAM1 expression into tone eviction at the VCAM1 promoter occurred (Supplemental high and low populations, and assayed for luciferase activity as a Fig. 2H), whereas VCAM1 mRNA and protein showed a large surrogate for episomal promoter activity. We did not detect any distribution in expression levels. differences in luciferase activity between populations, which was also seen using a synthetic promoter reporter containing Heterogeneous DNA methylation of the VCAM1 promoter tandem repeats of the NF-kB transcription response elements In the active chromatin state, variable gene expression between (Fig. 2C, 2D). cells could result from alterations in DNA methylation patterns. We Last, we assessed the possibility that posttranscriptional

previously found DNA methylation to be critical in regulating the by guest on October 2, 2021 mechanisms regulate VCAM1 mRNA and protein heterogeneity cytokine-inducible NOS2 gene; however, little is known about its directly or indirectly through microRNAs. We examined miR-126 role in VCAM1 transcriptional regulation (28). Using bisulfite and miR-146a, both shown to play a role in EC VCAM1 ex- sequencing, we explored the methylation patterns of the VCAM1 pression and let-7a, based on in silico predictions (29, 30). Para- promoter. We encountered a broad distribution and heterogeneous doxically, higher levels of miR-126, miR-146, and let-7a were methylation patterns of individual CpGs on single alleles within detected in the VCAM1 high-expressing cells compared with populations similar to VCAM1 mRNA and protein expression the low-expressing subpopulation (Fig. 2E–G). These micro- (Fig. 3A–C, open circles and closed circles, respectively). We did were not all inducible in the total population with TNF-a not find common sequence diversity in the VCAM1 promoter. treatment (Supplemental Fig. 1I–K) (29–31). Together, these Alleles ranged from sparsely methylated to densely methylated, results argue against current paradigms describing a major role in which individual CpG sites showed variation in methylation for stochasticity in receptor–ligand interactions, intracellular with no discernible pattern (Fig. 3D–F). Allelic heterogeneity in signal transduction, or posttranscriptional mechanisms in in- VCAM1 methylation can be illustrated using a histogram of the ducing heterogeneity in VCAM1 expression and favor further number of methylated CpGs located between 2521 and +50 in exploration. the VCAM1 allele (Fig. 3G–I). Importantly, compared with the Characterization of the VCAM1 promoter histone code. We have VCAM1 promoter, the NOS2 promoter showed only a single pat- previously shown that plays a role in endothelial tern of hypermethylation (Fig. 3J). There were no differences in gene expression in human EC, in the regulation of NOS3, VCAM1 methylation patterns between EC analyzed directly from vascular endothelial–cadherin, and vWF, among others (11, the umbilical cord and prolonged cell culture (Fig. 3K, 3L). Hence, 15, 32). We explored the epigenetic regulation of VCAM1 the VCAM1 methylation variability observed in vitro is reflective induction by characterizing nucleosomal regulation and his- of the endothelium in vivo. We also asked whether the proximal tone modifications that encompass the VCAM1 core promoter VCAM1 promoter was methylated in other endothelial and non-EC

(H) hnRNA probe in 4-h TNF-a–treated HUVEC. (I) VCAM1 mRNA FISH (top, left) and immunostaining (top, right) with DAPI (bottom, left). Merge shown on bottom, right. (J) Representative gating strategy for isolating the VCAM1 high- and low-expressing subpopulations (5% top and bottom) after 4-h TNF-a treatment using fluorescence-activated cell sorting. (K) qRT-PCR determination of VCAM1 mRNA, (L) hnRNA, and (M) the ratio of hnRNA to mRNA in VCAM1 high- and low-expressing subpopulations. HUVEC were either treated with TNF-a (10 ng/ml) for 0.5, 1, 2, 4, 12, and 24 h (A and B) or 4 h (D–M). (A and B) Data represent the mean 6 SD of triplicate measurements from one of four independent experiments with similar results. (C, D, and K–M) Data represent the mean 6 SEM of three or four independent experiments. (E–I) Representative images of one of at least four independent experiments. *p , 0.05. 6 EPIGENETIC HETEROGENEITY IN ENDOTHELIAL VCAM1 EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Known regulators of VCAM1 mRNA do not contribute to VCAM1 expression heterogeneity. (A) HUVEC were treated with vehicle or (B) TNF-a (10 ng/ml) for 30 min and were stained with propidium iodide (PI; left) and RelA/p65 (center). Merge shown on right. RelA/p65 is not hetero- geneous. (C–G) VCAM1 high- and low- (5% top and bottom, respectively) expressing cells were isolated using FACS and subject to analysis. (C) Relative amount of VCAM1 22167/+95 episomal promoter luciferase activity assay in VCAM1 high- and low-expressing cells. (D) Relative amount of tandem NF-kB episomal promoter luciferase activity assay in VCAM1 high- and low-expressing cells. Luciferase activities were normalized for transfection efficiency by cotransduction through SV40-Renilla. (E) Relative amount of hsa-miR-126, (F) hsa-miR-146, and (G) hsa-let-7a in VCAM1 high- and low-expressing subpopulations. Data represent the mean 6 SEM of three independent experiments. *p , 0.05. types. As shown in Fig. 3, graded levels of methylation were evi- hypomethylation when compared with the founding master dent in a variety of primary human cell types. As expected, we population (Fig. 4A). Such differential methylation states re- observed no DNA methylation heterogeneity in cell types in main in VCAM1-high and -low cells after 1 wk of culture which VCAM1 promoter methylation was either almost com- (Fig. 4B). Moreover, we failed to detect any significant changes pletely unmethylated (e.g., microvascular EC) or completely in global bulk genomic methylation levels following TNF-a methylated (e.g., primary human hepatocytes). using methyl-accepting assays, cytosine extension assay, and To further assess the consequences of promoter methyl- Luminometric Methylation Assay (LUMA) (19) (Fig. 4C–E). ation on VCAM1 expression, we compared the methylation We also found no consistent methylation changes at any of the status in sorted VCAM1 high- and low-expressing subpopula- six CpG dinucleotides after TNF-a treatment (Fig. 4F), indi- tions after treatment with TNF-a. We focused on the four cating that VCAM1 promoter DNA methylation is not affected CpG dinucleotides within the VCAM1 proximal core promoter by short-term TNF-a treatment. Prior work in our laboratory using bisulfite pyrosequencing. Interestingly, the VCAM1 pro- has profiled DNA replication timing patterns at EC-enriched moter was hypermethylated in VCAM1 low-expressing cells, gene promoters with differentially methylated regions and whereas the high-expressing cells showed VCAM1 promoter found that they replicate early in S phase (32). We reasoned The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Heterogeneity in VCAM1 proximal promoter DNA methylation in HUVEC. (A–L) DNA methylation of three independent HUVEC lines. (A–C) Lollipop diagrams showing the methylation patterns of individually sequenced alleles. Each row represents a single allele, and each circle represents a specific CpG site (open circle, unmethylated CpG; filled circle, methylated CpG). Arrow indicates the transcription start site. (D–F) Analysis of CpG site- specific methylation of the sequenced alleles. (G–I) Histogram of the DNA methylation frequency at each of the CpG sites in the VCAM1 promoter shown in (D)–(F), respectively. (J) HUVEC methylation frequency histogram of NOS2. DNA methylation of VCAM1 promoter in (K) passage zero and (L) passage three cells. that heterogeneity in the VCAM1 promoter DNA methylation replication timing across S phase, indicating its replication couldbecausedbyheterogeneousreplicationoftheVCAM1 pattern across EC is also heterogeneous. locus during cell division. To investigate the DNA replication timing, we used a BrdU pulse labeling and quantitative PCR DNA methylation of the VCAM1 promoter and RNA Pol II and (qPCR)–based approach. Both control genes NOS3 (Fig. 4G) RelA/p65 binding and CD31 (Fig. 4H) exhibited early S phase replication (32). To determine whether VCAM1 promoter methylation was func- We found NOS2 (Fig. 4I), which shows dense promoter DNA tionally relevant with respect to VCAM1 expression, we com- methylation to be replicated late S phase (28). Interestingly, bined bisulfite genomic analysis with ChIP DNA (ChIP-BA). VCAM1 (Fig. 4J) was unique as it showed broad promoter Briefly, chromatin fragments were IP, followed by bisulfite 8 EPIGENETIC HETEROGENEITY IN ENDOTHELIAL VCAM1 EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. VCAM1 high-expressing cells are specifically hypomethylated at the VCAM1 promoter. HUVEC were stimulated with TNF-a (10 ng/ml) for 4 h and sorted into VCAM1-high and -low fractions and compared with the parental (master) population. (A) Pyrosequencing histogram of DNA methylation at the VCAM1 promoter from VCAM1-high and -low cells and the master. (B) Pyrosequencing histogram of DNA methylation at the VCAM1 promoter from VCAM1-high and -lowcellsafter1wkofgrowth.(C) Methyl-accepting assay in which the amount of 3[H]-methyl incorporation (DPM) per 80 ng of genomic DNA was used to measure changes in methyl content after TNF-a. (Figure legend continues) The Journal of Immunology 9 genomic sequencing of the VCAM1 core promoter to establish Our data did not identify a global methylation phenomenon, as the methylation patterns of the bound DNA (Fig. 5A). Using there was no correlation between global DNA methylation levels RNA Pol II, we observed enrichment in binding to unmethy- and VCAM1 methylation in clones (Supplemental Fig. 4I, 4J), lated or lightly methylated VCAM1 core promoter (Fig. 5B, regardless of the DNA methylation method used (Supplemental 5C). In contrast, VCAM1 promoter in the Pol II–unbound Fig. 4K, 4L). In total, we worked with five independent distinct fraction was significantly hypermethylated, typically with EC sources and generated a total of 36 single cell–derived clones. three or more methylated CpGs (Fig. 5D, 5E). We also wanted In support of the importance of methylation state rather than any to determine if this was due to differential trans factor binding. specific pattern, we were not able to identify any specific CpG site Using ChIP studies, RelA/p65 was enriched at the VCAM1 within the core promoter that could impede VCAM1 transcription proximal promoter following TNF-a stimulation (Fig. 5F). when methylated. Taken together, these results suggest that Similar to RNA Pol II, pyrosequencing of RelA/p65-occupied methylation state is a mitotically inherited trait within the VCAM1 DNA showed marked hypomethylation of the VCAM1 pro- promoter that confers responsiveness to cytokine stimulation from moter (Fig. 5G), suggesting that DNA methylation of the a parent cell to its progeny. VCAM1 promoter is critical in facilitating both RelA/p65 and We next assessed the relationship between clonal DNA methyl- Pol II binding after TNF-a stimulation. We also found that ation and VCAM1 expression. Shown in Fig. 7, bisulfite genomic differences in VCAM1 promoter methylation persisted during sequencing again reveals heterogeneity in methylation between the prolonged cell culture between VCAM1-high and -low sub- parental population and single cell–derived endothelial clones populations following VCAM1 sorting. RelA/p65 ChIP analysis (Fig. 7A–F). For example, Clone i, a hypomethylated line, consisted of long-term cultures again showed that RelA/p65 selectively of sparsely methylated VCAM1 promoter, with a 6.9% overall engaged on hypomethylated VCAM1 promoter regions in methylation and fewer than two methylated CpGs (Fig. 7C, 7D). In Downloaded from plated high cells compared with low cells (Fig. 5H–J), sug- contrast, Clone ii showed heavy methylation, with a 48.3% overall gesting RelA/p65 binding is not compatible with VCAM1- methylation and primarily contained three to five methylated methylated DNA, although there is no CpG in RelA/p65 CpGs (Fig. 7E, 7F). These results were confirmed with quantita- binding sequences of the VCAM1 promoter. We also detected a tive bisulfite pyrosequencing, which correlated with strand anal- rare single-nucleotide variant in one of our HUVEC prepara- ysis of single alleles (Supplemental Fig. 4M). Additionally, we tions (NC_000001.11:g.1007119649del, GRCh38.p12 chr1 found no differences in methylation of the NOS3 promoter http://www.jimmunol.org/ build 153). This single-nucleotide deletion was present in a (Supplemental Fig. 4N), arguing against global genomic effects heterogeneous state in a single preparation. This gave us the from clonality. We measured the expression levels of VCAM1, opportunity to define whether there was a difference in DNA NOS2,andNOS3 in these low and high VCAM1 promoter– methylation of the two distinct haplotypes of the proximal methylated clones before or after TNF-a treatment. We observed promoter. In 18 distinct DNA strands, no association was an inverse relationship between VCAM1 promoter methylation observed between DNA methylation and, what we assume, is and mRNA expression (Fig. 7G). For example, VCAM1 mRNA an informative DNA marker. expression in hypomethylated clone i was 13-fold higher than that of hypomethylated clone ii. In contrast, there was no significant Mitotic inheritance of DNA methylation at the by guest on October 2, 2021 difference in NOS2 and NOS3 mRNA expression between the VCAM1 promoter clones (Fig. 7G). Of note, global methylation status of the clones We then wanted to further examine whether heterogeneous indi- was not indicative of the methylation status of the VCAM1 promoter vidual VCAM1 promoter methylation patterns were conserved and (Fig. 7H). Similar RNA findings were observed in 15 single-cell heritable following mitosis. We used limiting dilution of cell clonal populations derived from three genetically independent EC sorting to establish single-cell EC clones. Single cells were ex- preparations (Fig. 7I, 7J). In comparison with NOS3, VCAM1 7 panded and propagated (Fig. 6A) to ∼2 3 10 cells, representing mRNA induction varied markedly across clones. at least 25–30 mitotic cellular divisions. Clonality was validated a using HUMARA-MSP assay in female cell preparations (37, 38). Heterogeneity of other TNF- –regulated endothelial genes Surprisingly, we found that each clonal population contained We considered whether heterogeneous gene expression is unique various VCAM1 methylation patterns that differed between clones to VCAM1 or is a generalized phenomenon to other cytokine- and differed from the parental master plate used to generate regulated genes. We compared global changes in gene expres- single-cell isolates. Shown is a representative experiment with sionbetweentheVCAM1high-andlow-expressingcellsafter seven single cell–derived EC clonal populations (Supplemental 4hofTNF-a and compared these results with bulk RNA levels Fig. 4A–H). Using the method of Ushijima et al. (37), we esti- of the master population both 4 and 24 h after TNF-a treatment mated the fidelity in maintaining VCAM1 methylation pattern to (Gene Expression Omnibus accession: GSE141374). All com- be 99.26–99.84% per CpG per cell generation, comparable with parisons were done using matched pair analysis. After 4 h of their methylation fidelities. Because each clone is unique, espe- TNF-a treatment, 141 and 12 protein coding transcripts showed cially compared with the parental line, we infer that this reflects a .2-fold increase or decrease (corrected p value # 0.05), re- the individuality of the cells that initiated the clone and mainte- spectively. Surprisingly, the VCAM1-high and -low subpopulations nance of a specific heterogeneous epigenetic identity (Fig. 6B–I). showed distinct differentially expressed genes (2-fold difference,

(D) Cytosine extension assay showing a ratio of HpaII/MspI digestion of genomic DNA after TNF-a expressed as a percentage of total CCGG sites. (E) LUMA results showing the effects of TNF-a on global DNA methylation in HUVEC over a variety of different time points. (F) Representative pyrosequencing analysis showing site-specific methylation at the VCAM1 promoter in HUVEC treated with TNF-a for the time points shown. The relative abundance (percentage IP) of the proximal promoter of (G) NOS3, (H) CD31, (I) NOS2, and (J) VCAM1 in each cell cycle fraction was quantified by qPCR in HUVEC, as we have previously reported [n = 4 for (G), n = 3 for (H)–(J), (32)]. Data represent mean 6 SEM. The statistical significance of each result is calculated compared with the control untreated sample using ANOVA. ANOVA for cell cycle assays were compared with G2. Results represent three independent experiments unless otherwise stated. *p , 0.05. 10 EPIGENETIC HETEROGENEITY IN ENDOTHELIAL VCAM1 EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. RNA Pol II, a surrogate for transcription, preferentially binds to hypomethylated VCAM1 alleles. (A) Schematic diagram of ChIP with an RNA Pol II Ab followed by bisulfite sequencing (RNA Pol II ChIP-BA) at the VCAM1 promoter, where RNA Pol II ChIP from HUVEC was isolated after 4-h TNF-a treatment into bound and unbound fractions, followed by sodium bisulfite sequencing. (B and C) RNA Pol II–bound fraction. (B) Histogram showing site-specific CpG methylation in the VCAM1 promoter in the Pol II–bound fractions. (C) Frequency histogram showing site-specific CpG methylation in the VCAM1 promoter in the Pol II–bound fractions. Lollipop diagrams for VCAM1 alleles (inset). (D and E) RNA Pol II–unbound fraction. (D) Histogram showing site-specific CpG methylation in the VCAM1 promoter in the Pol II–unbound fractions. (E) Frequency histogram showing site- specific CpG methylation in the VCAM1 promoter in the Pol II–unbound fractions. Lollipop diagrams for VCAM1 alleles (inset). (F–J) RelA/p65 ChIP-BA of the VCAM1 promoter of whole dish after 2-h TNF-a treatment. (F) Relative enrichment of RelA/p65 at the VCAM1 promoter. (G) Pyrosequencing of ChIP input and RelA/p65 IP DNA. (H–J) ChIP-BA of the VCAM1 promoter of VCAM1-high and -low cells after 2-h TNF-a treatment. (H) RelA/p65 ChIP DNA enrichment after 2 h of TNF-a treatment in VCAM1-low and -high cells 7 d after physical sorting and culture. (I) Lollipop diagrams and (J) site- specific CpG methylation of VCAM1 promoter of RelA/p65 pulled down DNA of RelA/p65 ChIP DNA 7 d after physical sorting and culture. Results represent three independent experiments unless otherwise stated. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. Differences in DNA methylation patterns between genetically identical HUVEC clones and within the original master population. Clones were generated from single cells and analyzed using sodium bisulfite genomic sequencing. (A) Schematic showing generation and expansion of HUVEC clones. Site-specific DNA methylation analysis of parental line [(B), top] and randomly chosen clones [(C–I), top]. Frequency histograms of master population [(B), bottom] and randomly chosen clones [(C–I), bottom]. p # 0.05), with 247 and 121 genes overexpressed and underex- such as SOD2, were coregulated with VCAM1 (Fig. 8C). This pressed in the VCAM1 high- versus VCAM1 low-expressing heterogeneous expression was not a general phenomenon, as other cells, respectively (Fig. 8A, 8B). A total of 31 genes were found genes that showed TNF-a regulation (such as SERPINE-1 [PAI-1] to be preferentially upregulated by TNF-a and present in the and CSF2 [GM-CSF]) showed only trivial heterogeneity between VCAM1 high-expressing subpopulation (Fig. 8A, 8C). These rep- VCAM1-high and -low subpopulations (Fig. 8C). Even more resent 22% of genes that were induced by TNF-a in EC. Intrigu- importantly, some TNF-a–induced genes at the whole population ingly, we found that other TNF-a–induced adhesion molecules, level, such as urokinase-type plasminogen activator (PLAU1), such as SELE and ICAM1, as well as other TNF-a–induced genes, showed an inverse relationship between whole dish levels in 12 EPIGENETIC HETEROGENEITY IN ENDOTHELIAL VCAM1 EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021 FIGURE 7. DNA methylation patterns inversely correlate with VCAM1 inducibility. Multiple clones were generated from single cells and analyzed using sodium bisulfite genomic sequencing, and mRNA inducibility was analyzed using qRT-PCR (n = 36). Site-specific percentage of methylation (A) and frequency histogram (B) at the VCAM1 promoter in master population and two representative HUVEC clones, i (C and D) and ii (E and F). (G) qRT-PCR data showing VCAM1, eNOS, and iNOS mRNA levels in master and clones i and ii after TNF-a treatment (4 h, 10 ng/ml). (H) Global DNA methylation levels in the master population and HUVEC clones i and ii using long interspersed nuclear element-1 repetitive DNA methylation with LUMA. (I and J) Boxplot of NOS3 and VCAM1 mRNA expression in clones after TNF-a treatment (4 h, 10 ng/ml) normalized to vehicle-treated cells and GAPDH. This distribution of changes in VCAM1 expression is marked compared with NOS3.(G) mRNA levels are normalized to GAPDH, and parental levels were set at 1. (I and J) Data represent mean 6 SD of all clones studied (n = 9).

VCAM1-low and VCAM1-high cells (Fig. 8C). Especially inter- regulation. scRNA-seq also confirmed the upregulation of esting was the finding that expression of the whole population other genes after 4 h of TNF-a treatment, such as SOD2 and seemingly failed to demonstrate a TNF-a–induced population SERPINE1 (Fig. 8H, 8I). This confirmed initial findings of dif- effect, but heterogeneity still existed in the VCAM1-high versus ferentially regulated 5% VCAM1 high- compared with VCAM1 VCAM1-low fractions. For instance, fibronectin (FN1, Fig. 8C) low-expressing populations and allowed the conclusion that and STAT1 (Fig. 8C) were differentially expressed in VCAM1- coexpression was observed in single cells. These data revealed high versus VCAM1-low populations at 4 h, but 4 h of TNF-a coexpression of SELE, ICAM1, SOD2, CSF2, and FN1 in indi- failed to show a population-based average effect. vidual cells (p value # 0.001) (Fig. 8J, 8K). Taken together, these These results were confirmed using scRNA-seq of EC. In the findings suggest that selective examples of expression heteroge- untreated single-cell population, as expected, we observed a neity exist with TNF-a treatment. limited number of the total cells (8281) with VCAM1 expression (21, 0.25%) compared with the number of cells with VCAM1 Discussion expression after 4 h of TNF-a treatment (5905, 70.6%) The regulation of VCAM1 expression has been of interest since the (Fig. 8D, 8E). As expected, SELE and ICAM1 also showed in- identification of its role in cardiovascular disease (39). Our work creased cell numbers within the population after TNF-a treatment, demonstrates that heterogeneity in VCAM1 expression is evident from 0.06% (5) and 1.4% (112) to 60.3% (5045) and 88.4% within a homogeneous mature cell population. We provide evi- (7393), respectively (Fig. 8F, 8G). Moreover, examination of dence that chromatin remodeling through histone density and changes in mRNA levels revealed VCAM1 mRNA was upregu- posttranslational modifications contributes to VCAM1 gene regu- lated 160-fold (p value = 1.07 3 102112). SELE was upregulated lation. We found that TNF-a elicited acute H3 and H4 hyper- 154-fold (p value = 2.66 3 102110), and ICAM1 was upregulated acetylation as well as H3K4 methylation, followed by eviction of 93-fold (p value = 1.91 3 102101), confirming expected TNF-a the histone octamer. This induction to an active chromatin state The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 8. Expression heterogeneity is not unique to VCAM1 in VCAM1 high- and low-expressing cells. Venn diagrams of genes that are significantly upregulated (A) and downregulated (B) .2-fold at 4 h compared with vehicle and increased in VCAM1-high cells compared with VCAM1-low cells. (C) qRT-PCR was used to examine expression in HUVEC treated with vehicle or TNF-a (10 ng/ml) for 4 h. HUVEC were also treated with TNF-a for 4 h and sorted into VCAM1-high and -low fractions. Genes with similar expression profiles compared with VCAM1 (SELE, ICAM1, SOD2) are shown in addition to genes that show opposite expression profiles (SERPINE1, CSF2, PLAU, FN1, STAT1). Bold denotes p , 0.05. Italics denote p value of 0.05–0.06. (D) t-distributed stochastic neighbor embedding of single-cell sequencing data of EC merged in silico at single-cell level with libraries prepared from TNF-a– (4 h, 10 ng/ml) and vehicle-treated cells. Expression profiles of (E) VCAM1, (F) SELE, (G) ICAM1, (H) SOD2, (I) SERPINE1, (J) CSF2, and (K) FN1 visualized using CLOUPE browser. 14 EPIGENETIC HETEROGENEITY IN ENDOTHELIAL VCAM1 EXPRESSION was subsequently followed by the recruitment of RNA Pol II, methylation at the VCAM1 promoter and suggest this phenomenon increased VCAM1 transcription, and protein expression. A general is heritable for VCAM1 and potentially other genes. Although consensus is emerging that histone eviction tends to accompany separation of the VCAM1 high- and low-expressing subpopula- RNA Pol II recruitment and transcription initiation (35). It has tions was TNF-a dependent, single-cell sequencing highlights that been shown that NFkB1 (p50), the most prominent interacting heterogeneous TNF-a responsiveness of EC exists for a gene but partner of RelA/p65, does not cause histone eviction, and NF-kB not all TNF-a–regulated genes. binding alone is nucleosome independent (40). Therefore, other In summary, our findings provide evidence that VCAM1 is, in mechanisms are also likely to be influencing VCAM1 promoter part, epigenetically regulated through both histone modifications, nucleosomes. Overall, VCAM1 induction requires histone post- but more importantly, DNA methylation. Furthermore, we provide translational modifications as VCAM1 activation is blocked by a evidence that VCAM1 may be the first metastable epiallele histone deacetylase inhibitor. Although key to VCAM1 induction, identified in human EC in which DNA methylation influences changes in histone code or density did not explain heterogeneity gene expression following cytokine activation. This work pro- of VCAM1 induction. vides newer insight into understanding endothelial heterogeneity Our results show that VCAM1 expression heterogeneity can be, in vascular beds. in part, accounted for by heterogeneous DNA methylation at the VCAM1 promoter that is basally present in EC. Mechanisti- Acknowledgments cally, we found that RNA Pol II preferentially bound to an We thank members of the Marsden laboratory for critical review of this unmethylated or very lightly methylated VCAM1 promoter, work and members of the Flow Cytometry cores at the Faculty of Medicine giving rise to EC with increased VCAM1 expression. This was at the University of Toronto and Li Ka Shing Knowledge Institute at confirmed with FACS showing that VCAM1 high-expressing St. Michael’s hospital. Downloaded from cells had hypomethylated VCAM1 alleles. Interestingly, we also found that the main NF-kB subunit in EC, namely RelA/ Disclosures p65, preferentially bound to hypomethylated DNA at the The authors have no financial conflicts of interest. VCAM1 promoter. This leads us to believe that increased DNA methylation at the VCAM1 promoter either directly or indi- References http://www.jimmunol.org/ rectly inhibits the chromatin engagement of RelA/p65 and re- 1. Luscinskas, F. W., M. I. 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