UBC Faculty Research and Publications

Epigenetic modifying enzyme expression in asthmatic airway epithelial cells and fibroblasts Stefanowicz, Dorota; Ullah, Jari; Lee, Kevin; Shaheen, Furquan; Olumese, Ekiomoado; Fishbane, Nick; Koo, Hyun-Kyoung; Hallstrand, Teal S; Knight, Darryl A; Hackett, Tillie-Louise Jan 31, 2017

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
52383-12890_2017_Article_371.pdf [ 1.83MB ]
Metadata
JSON: 52383-1.0362095.json
JSON-LD: 52383-1.0362095-ld.json
RDF/XML (Pretty): 52383-1.0362095-rdf.xml
RDF/JSON: 52383-1.0362095-rdf.json
Turtle: 52383-1.0362095-turtle.txt
N-Triples: 52383-1.0362095-rdf-ntriples.txt
Original Record: 52383-1.0362095-source.json
Full Text
52383-1.0362095-fulltext.txt
Citation
52383-1.0362095.ris

Full Text

RESEARCH ARTICLE Open AccessEpigenetic modifying enzyme expression inasthmatic airway epithelial cells andfibroblastsDorota Stefanowicz1 , Jari Ullah1, Kevin Lee1, Furquan Shaheen1, Ekiomoado Olumese2, Nick Fishbane1,Hyun-Kyoung Koo1, Teal S. Hallstrand3, Darryl A. Knight4,5 and Tillie-Louise Hackett1,5*AbstractBackground: Recognition of the airway epithelium as a central mediator in the pathogenesis of asthma hasnecessitated greater understanding of the aberrant cellular mechanisms of the epithelium in asthma. The architectureof chromatin is integral to the regulation of gene expression and is determined by modifications to the surroundinghistones and DNA. The acetylation, methylation, phosphorylation, and ubiquitination of histone tail residues has thepotential to greatly alter the accessibility of DNA to the cells transcriptional machinery. DNA methylation can alsointerrupt binding of transcription factors and recruit chromatin remodelers resulting in general gene silencing.Although previous studies have found numerous irregularities in the expression of genes involved in asthma, thecontribution of epigenetic regulation of these genes is less well known. We propose that the gene expression ofepigenetic modifying enzymes is cell-specific and influenced by asthma status in tissues derived from the airways.Methods: Airway epithelial cells (AECs) isolated by pronase digestion or endobronchial brushings and airwayfibroblasts obtained by outgrowth technique from healthy and asthmatic donors were maintained in monolayerculture. RNA was analyzed for the expression of 82 epigenetic enzymes across 5 families of epigenetic modifyingenzymes. Western blot and immunohistochemistry were also used to examine expression of 3 genes.Results: Between AECs and airway fibroblasts, we identified cell-specific gene expression in each of the familiesof epigenetic modifying enzymes; specifically 24 of the 82 genes analyzed showed differential expression. Wefound that 6 histone modifiers in AECs and one in fibroblasts were differentially expressed in cells from asthmaticcompared to healthy donors however, not all passed correction. In addition, we identified a correspondingincrease in Aurora Kinase A (AURKA) protein expression in epithelial cells from asthmatics compared to thosefrom non-asthmatics.Conclusions: In summary, we have identified cell-specific variation in gene expression in each of the families ofepigenetic modifying enzymes in airway epithelial cells and airway fibroblasts. These data provide insight into thecell-specific variation in epigenetic regulation which may be relevant to cell fate and function, and diseasesusceptibility.Keywords: Asthma, Histone modification, Epigenetics, Airway epithelium, Airway epithelial cells, Airway Fibroblasts,Epigenome, Histone code, Post-translational modification, DNA methylation* Correspondence: Tillie.Hackett@hli.ubc.ca1UBC Centre for Heart Lung Innovation, St. Paul’s Hospital, 1081 BurrardStreet, Vancouver, BC V6Z 1Y6, Canada5Department of Anesthesiology, Pharmacology and Therapeutics, Universityof British Columbia, Vancouver, BC, CanadaFull list of author information is available at the end of the article© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Stefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 DOI 10.1186/s12890-017-0371-0BackgroundAsthma is a chronic inflammatory condition of the air-ways that affects around 300 million people worldwide[1]. The airway epithelium, derived from the endoderm,is the first structural barrier to the inhaled environmentin the airway mucosa. In asthma, the airway epitheliumhas an altered phenotype displaying altered cell cyclekinetics and increased numbers of basal cells [2, 3]. Inaddition, the lamina propria of asthmatic donors con-tains resident fibroblasts, derived developmentally fromthe mesoderm, that have been shown to exhibit an inva-sive and synthetic phenotype [4–8]. How these alter-ations in cellular phenotype occur in the disease isunknown but it is clear from the many genomic studiesthat asthma involves both genetic and environmentalcomponents.The epigenetic landscape is essential in determining cellfate through histone modification and DNA methylationpatterns that regulate the expression of genes integral tocellular development and differentiation [9–11]. Covalentmodifications of the histone N-terminal tails can regulategene expression and include acetylation, methylation,phosphorylation, and ubiquitination [12, 13]. Histoneacetylation and phosphorylation are associated with amore open chromatin structure and gene expression,whereas histone methylation and ubiquitination can workboth in a gene repressive and expressive manner depend-ing on the target residue [13–17]. The enzymes respon-sible for the addition/removal of these modificationsinclude: histone acetyltransferases (HATs)/deacetylases(HDACs), protein kinases/phosphatases, histone methyl-transferases (HMTs)/demethylases (HDMs), and ubiquitinligases/deubiquitinating enzymes (DUBs) [13, 17]. DNAmethylation is facilitated by DNA methyltransferases(DNMTs) that add a methyl group to cytosine bases,forming 5-methylcytosine (5-mC) [12]. Addition of thismark at a gene promoter is generally associated with tran-scriptional repression and gene silencing [12, 18]. Further-more, the epigenome is adaptable; it has the capability torespond to and be modified by environmental factors [10].The outcome of this interaction depends on the environ-mental stressor and can be a normal physiological re-sponse or deregulation of the epigenome producing anabnormal phenotype [10, 19].Abnormal epigenetic control of gene expression hasbeen identified in both fibroblasts and epithelial cells innumerous pathologies [20–25]. However, very little isknown about the expression and regulation of epigeneticmodifying enzymes in asthma. Indeed, dysregulation ofepigenetic mechanisms in asthma has been identified ina variety of cells but most studies have been performedin tissues from outside of the lung [26]. While dysregula-tion of enzymes involved in histone acetylation wasidentified in the airways of asthmatics, there is stilldisagreement on the exact enzymes responsible [27–30].We have additionally identified unique DNA methyla-tion patterns in airway epithelial cells (AECs) from asth-matic donors [31] yet research on the variability of theenzymes responsible for these changes is lacking.To further elucidate the mechanisms driving the epi-genetic alterations observed in the asthmatic airways, abetter understanding of the gene expression profiles ofepigenetic modifying enzymes in airway tissues is re-quired. We hypothesize that the gene expression ofepigenetic modifying enzymes is cell-specific and influ-enced by asthma status in tissues derived from the air-ways. Specifically, the aim of this study was to identify ifthe expression profiles of epigenetic modifying enzymesis cell- and disease-specific by profiling 82 genes across5 families of epigenetic enzymes in AEC and fibroblastsfrom healthy and asthmatic donors. We identified 24cell-specific and 7 disease-specific differentiallyexpressed genes (6 in AECs and one in fibrolasts). Al-though not all of the disease-specific genes passed cor-rection, we were able to identify a corresponding changein AURKA protein expression in asthmatic compared tohealthy individuals.MethodsSample collectionAECs and airway fibroblasts obtained from de-identifiedhuman lungs from asthmatic and healthy donors notsuitable for transplantation and donated for medical re-search were obtained though the International Institutefor the Advancement of Medicine (Edison, NJ). A lungwas identified as healthy if the donor had no history ofasthma or other pulmonary disease or damage. Conduct-ing airways down to the 5th generation were used forAECs isolation by pronase digestion and airway fibro-blasts were obtained by outgrowth technique as previ-ously described [32, 33]. Endobronchial airway brushingsfrom patients were also used to obtain AECs as previ-ously described [34, 35]. AECs were grown in BronchialEpithelial Growth Medium (BEGM, Lonza, Walkersville,MD) containing 100U/mL penicillin and 100ug/mLstreptomycin, whereas fibroblasts were grown in Dulbec-co’s Modified Eagle’s medium (DMEM) (Invitrogen, Bur-lington, ON, Canada) supplemented with 10% FBS,2 mM L-glutamine, and 1% antibiotic/antimycotic solu-tion. Cultures were maintained at 37 °C in a humidified95% air/5% CO2 atmosphere to passage 2. Donor demo-graphics are provided in Table 1.Gene expressionAECs and airway-derived fibroblasts were grown in 6-well plates to 80% confluence, at which point RNA wascollected using RNeasy Mini Kits (Qiagen). 500 ng ofRNA was used to synthesize cDNA using the RT2 FirstStefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 2 of 11Strand Kit (Qiagen). cDNA was then combined with 2×RT2 SYBR Green Mastermix (Qiagen) and RNase-freewater and distributed onto a manufacturer optimized384-well Human Epigenetic Chromatin ModificationEnzymes Focused Array (PAHS-085E-4, Qiagen) pre-loaded with primers targeting 84 genes encoding epigen-etic enzymes and 5 housekeeping genes as per manufac-turer’s protocol. A complete list of the genes that wereanalyzed is available in Table S1 (Additional file 1).Additionally, to identify gene expression of CREBBP andEP300, cDNA was combined with 2× RT2 SYBR GreenMastermix, RNase-free water, and primers targetingCREBBP (PPH00324F-200, Qiagen), EP300 (PPH00319A-200, Qiagen), hypoxanthine phosphoribosyltransferase 1(HPRT1, PPH01018C, Qiagen), ribosomal protein L13a(RPL13A, PPH01020B-200, Qiagen), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, PPH00150F, Qia-gen) and loaded onto 384-well reaction plates. Data clean-ing and housekeeping gene selection is described inSupplementary Methods (Additional file 2). Target geneexpression was calculated using the delta Ct method:2^(CtHousekeeping Gene – CtTarget Gene)*10000.Sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and immunoblotProtein was collected from AECs and airway fibroblastsin culture and electrophoresed on a 12.5% SDS-polyacrylamide gel. Membranes were first incubatedovernight with primary antibody (Table 2), then withgoat anti-mouse IR-800 (1:2500, Vector Laboratories) orgoat anti-rabbit Alexa 680 (1:2500, Invitrogen) secondaryantibody, and finally imaged on the LI-COR Odysseysystem. Odyssey software 1.1 was used to perform densi-tometry (LI-COR Biotechnology, Lincoln, NE, USA).Data for AURKA and SMYD3 were normalized to β-tubulin and hsp-90 respectively. A two-tailed unpaired t-test was performed, a p-value of less than 0.05 was con-sidered significant.Immunohistochemical stainingAirway sections were formalin fixed and paraffin embed-ded prior to immunohistochemical staining. Sectionswere deparaffinized, rehydrated, processed for antigenretrieval and incubated overnight at 4 °C with CREBBPantibody (Table 2). Sections were subsequently incu-bated with a biotinylated goat anti-rabbit secondary anti-body (1:100, Vector Laboratories, Burlingame, CA, USA)prior to visualization with Streptavidin-HRP (Dako) and3,3-diaminobenzidine (Dako). Slides were counterstainedwith Harris Hematoxylin solution (Sigma, St. Louis,MO, USA) and dehydrated before coverslipping withCytoseal 60 medium (Richard-Allan Scientific, Kalama-zoo, MI, USA).Using the Nikon Eclipse 700 (Nikon Instruments, Mel-ville, NY, USA) with a 60× objective and SPOT Ad-vanced software (Diagnostic Instruments, SterlingHeights, MI, USA), five images were obtained from eachsection. These images were analyzed for positively andnegatively stained nuclei using ImagePro Plus software(Media Cybernetics, Rockville, MD, USA).Principal component analysis (PCA)Principal components analysis was performed to assesssources of variation in our gene expression dataset. Prin-cipal components were obtained as a new set of orthog-onal variables by extracting eigenvectors from singularvalue decomposition of the expression matrix, andranked by the size of their respective eigenvalue, repre-senting the component of overall variation.Table 1 Donor demographics including disease status, age, cell type, and sexCell type Disease status Number Cell source (W/B) Sex (M/F) Average Age° (range)AEC Asthmatic 11 7/4 5/6 18.8 (8–29)Healthy 13 8/5 6/7 22.6 (11–42)Fb Asthmatic 6 6/0 6/0 20.8 (10–36)Healthy 6 6/0 6/0 18.2 (5–43)Airway epithelial cells (AECs) and airway fibroblasts (Fb) were collected from healthy and asthmatic donors. Cell source is identified by whole lung (W) or brushing(B). There were no differences for age between all groups by one-way ANOVA; p = 0.69Table 2 Antibodies used in experimentsEpitope Host Company Catalogue number Primary antibody dilutionSMYD3 Rabbit Abcam ab155018 1/1000AURKA Mouse Cell Signaling 12100 1/500Hsp90 Mouse BD Biosciences 610418 1/1000β-tubulin Mouse Millipore 05–661 1/2000CREBBP Rabbit Santa Cruz Biotechnology sc-369 1/50Stefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 3 of 11Co-expression analysisCo-expression analysis was performed by calculatingSpearman correlations between genes using pairwise-complete observations. This correlation matrix was thenused to generate heatmaps showing co-expression be-tween genes. Using the correlation matrix, we calculatedthe effective number of independent variables (ENIV) ineach data set using spectral decomposition [36]. For ana-lyses using both AECs and airway fibroblasts, only AECs,and only airway fibroblasts, the adjustment value wasfound to be 21.38, 21.52, and 15.25 respectively.Statistical analysisAll data were log2 transformed prior to statistical ana-lysis. Sex was correlated with the expression of most tar-get genes, so was included as a covariate whereapplicable. Linear regression including sex and diseasestatus as covariates was used to test the association be-tween gene expression and cell-type. Since the fibroblastsamples were isolated only from males, the interpret-ation of this model can only be generalized to the malepopulation. Linear regression was also used to test theassociation of disease in AECs adjusting for gender as acovariate. For fibroblasts, we did not adjust for any co-variates and a t-test was employed to identify signifi-cantly different gene expression. All statistical analysisand figures were generated using the R software version3.0.2 [37] and the ggplots2 package [38].ResultsAirway cell-specific expression of epigenetic modifyingenzymesWe profiled the expression of 82 genes across 5 familiesof epigenetic modifying enzymes in AECs and airway fi-broblasts from healthy and asthmatic donors (Fig. 1).We observed that 53.81% of the variation across bothcell types could be accounted for by the first principalcomponent, PC2 accounted for 15.53% of the variation,and PC1 and PC2 combined explained 69.34% of thetotal variation in the data. Due to limitations in access toclinical characteristics of our cohort, we could not iden-tify the variable resulting in the greatest source of vari-ation PC1. However, given expression differences existedbetween cell types, we next proceeded to determine cell-specific gene expression profiles.Fig. 1 Principal component analysis (PCA) of epigenetic modifier enzymes in airway epithelial cells (AECs) and airway fibroblasts. Gene expressionlevels of 82 genes were used to construct the PCA plot. Healthy samples are identified with a triangle, asthmatic samples with a circle. Filledsymbols indicate male samples whereas open symbols indicate female samples. AECs are shown in black and airway fibroblasts (Fb) are shownin blueStefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 4 of 11Co-expression analysis within healthy donors fromAECs and fibroblasts showed that the epigenetic modi-fier enzyme genes which we examined were heavily co-expressed, with the majority showing positive co-expression (Fig. 2).Examination of differentially expressed genes betweenAECs and airway fibroblasts revealed 39 genes, of which24 passed ENIV correction (Fig. 3 and Additional file 3:Table S3). Of the 24 genes, all showed increased expres-sion in AECs as compared to airway fibroblasts. The dif-ferentially expressed genes were part of the DNAmethylation (2 genes), histone methylation (6 genes), his-tone phosphorylation (3 genes), histone ubiquitination (2genes), and histone acetylation (11 genes) families.Disease specific alterations in gene expression ofepigenetic modification enzymes in airway epithelial cellsTo identify if asthma status influences epigenetic modi-fying enzymes, we compared the gene expression of the82 genes in AECs derived from healthy and asthmaticdonors (Additional file 4: Table S4). Although onlyCREBBP passed ENIV correction, linear regression iden-tified differential expression of 6 genes: down regulationof the acetyltransferases CREBBP and EP300 and upregulation of the kinase AURKA, the ligases DZIP3 andthe methyltransferases EHMT2 and SUV39H1 (Fig. 4).To confirm whether the observed changes in CREBBPand AURKA mRNA expression in AECs correspond toprotein expression we performed immunohistochemistryand immunoblot. Although protein expression ofCREBBP was not different between asthmatic (24.76 ±3.47) and healthy donors (26.76 ± 5.46, p = 0.77, Fig. 5aand b), AURKA was significantly elevated in AECs fromasthmatic (0.025 ± 0.003) as compared to healthy donors(0.017 ± 0.002, p = 0.04, Fig. 5c and d).Disease specific alterations in expression of epigeneticmodification enzymes in airway fibroblastsNext, we investigated if any differences existed in the 82genes in airway fibroblasts isolated from healthy andFig. 2 Co-expression heatmap of epigenetic modifying genes. Gene expression from both AECs and fibroblast cells from healthy individuals wasused to analyze degree of co-expression of 82 genes involved in epigenetic mechanisms. Genes are listed on the x- and y-axis, blue indicatespositive co-expression and pink indicates negative co-expression of genesStefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 5 of 11asthmatic donors. We found increased mRNA expres-sion of the histone methyltransferase SMYD3 in airwayfibroblasts from asthmatics. However, this statistical sig-nificance did not pass ENIV correction (p = 0.02 and0.37 after correction, Fig. 6a). When assessing proteinexpression of SMYD3, we found no significant differencein SMYD3 protein expression in airway fibroblasts iso-lated from healthy (0.85 ± 0.07) and asthmatic donors(0.94 ± 0.04, p = 0.23, Fig. 6b and c).DiscussionThis is the first study to evaluate the gene expressionlevels of histone and DNA modifier enzymes in AECsand airway fibroblasts derived from human lung tissue.We found significantly higher expression for 24 of theseenzymes in AECs compared to airway fibroblasts fromhealthy individuals. Further, we demonstrate thatAURKA is differentially regulated in AECs from asth-matic compared to healthy donors. In addition, we iden-tified a corresponding increase in AURKA proteinexpression in AECs from asthmatic compared to healthydonors, further supporting our findings. Even thoughAECs and fibroblasts reside in close proximity withinthe airway mucosa, the function of each cell is very dif-ferent. These data support the notion that epigeneticmodulation of gene expression may be important for celltype specificity, and may potentially influence suscepti-bility to diseases such as asthma.Multiple studies have documented differential DNAmethylation in relation to tissue and cell specificity, andhow this is altered in diseases [31, 39–41]. Yet very fewstudies have focused on the global expression of en-zymes responsible for DNA methylation expression. Inour study, DNMT3a and MBD2 were both elevated inAECs compared to airway fibroblasts. DNMT3a is notonly integral for mammalian development but also re-sponsible for de novo DNA methylation [42]. It is pos-sible that the elevated DNMT3a seen in AECs mayreflect the cell’s geographical position. The airway epi-thelium is constantly in contact with external environ-mental factors thus must be responsive and adaptable toincoming stimuli. Elevated DNMT3a allows the cell tomethylate genes de novo in response to these environ-mental stimuli. The increased expression of MBD2 maybe a response to the increase in DNMT3a as MBD2 is atranscriptional repressor which binds methylated DNA[43]. To further support this theory, the complex whichMBD2 forms to repress gene expression is not stronglybound to the DNA [43] suggesting a transient visit aswould be expected from a responsive reaction.The outcome of an epigenetic change can be variabledepending on the particular modification that occurs.Methylation of lysine and arginine residues on histoneFig. 3 Differentially expressed epigenetic modifying genes in airway epithelial cells (AECs) compared to airway fibroblasts. Linear modeling wasused to identify genes that were differentially expressed in AECs compared to airway fibroblasts. Genes are shown on the y-axis, p-values areshown on the x-axis. Solid line indicates significance threshold meeting ENIV criteria, dotted line indicates p = 0.05Stefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 6 of 11Fig. 5 Expression of CREB-binding protein (CREBBP) and aurora kinase A (AURKA) in airway epithelial cells (AEC) from healthy and asthmaticdonors. a CREBBP staining of formalin fixed, paraffin embedded airway sections from healthy and asthmatic donors. Scale bar is equal to 50 μm.b Data are presented as percent of CREBBP positive cell nuclei ± SD (n = 9 Healthy, n = 8 Asthmatic). c and d AURKA protein expression normalized toβ-tubulin (± SD) in AECs from healthy (n = 7) and asthmatic donors (n = 6). A two tailed unpaired t-test was performed, * indicates p < 0.05Fig. 4 Differentially expressed epigenetic modifying genes in asthmatic compared to healthy airway epithelial cells (AECs). Healthy donors areshown in white whereas asthmatic donors are shown in grey. Linear regression was performed and found all 6 of these genes were significant,however only CREBBP met ENIV criteria. * indicates p < 0.05 after correctionStefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 7 of 11tails is facilitated by enzymes which are specific to bothresidue and site yet the outcome can activate or represstranscription [13]. In contrast, histone acetylation, com-monly associated with gene expression, is regulated byenzymes that have been described as promiscuous intheir substrate specificity [14]. We identified differentialexpression of enzymes involved in both histone methyla-tion and acetylation in AECs compared to airway fibro-blasts. Of the 6 enzymes involved in histone methylation,half target the activating mark H3K4me; SETD3 methyl-ates while KDM5B and KDM1A demethylate H3K4. Thismay indicate that AECs preferentially utilize H3K4 methy-lation over others to control gene expression. A similarobservation was seen with histone acetylation as 5 HATsand 6 HDACs were identified. Three of the HDACs thatwere elevated in AECs comprise 75% of the class I HDACfamily of enzymes important in controlling proliferation,differentiation, and tissue development programs [44].Higher expression of the majority of the class I HDACfamily of enzymes in epithelial cells may be a reflection oftheir considerable specialization as they have the capacityto differentiate and develop into a variety of epithelial celltypes, which requires manipulation of the processes men-tioned above.We found elevated expression of 3 histone kinases and2 DUBs when we compared AECs to airway fibroblasts.Although histone phosphorylation is commonly associ-ated with gene activation, histone ubiquitination can re-sult in both permissive and repressive states dependingon the residue. However, all of the resulting histonemodifications from the 5 above enzymes are associatedwith gene expression. This suggests there may be an im-balance in the regulation of these activating marks inAECs, potentially indicating lower levels of cellular tran-scriptional activity in airway fibroblasts compared toAECs.Through its interaction with β-catenin, CREBBP hasrecently been identified as a pivotal component of themachinery maintaining an undifferentiated and prolifera-tive state [45]. Inhibition of this interaction facilitates β-catenin and EP300 pairing which is thought to controlcell differentiation [45, 46]. Our findings of decreasedgene expression of CREBBP in AECs from asthmaticsmay indicate a divergence away from a proliferative statetowards an initiated, but incomplete differentiation path-way. This imbalance of proliferation/differentiationmechanisms may contribute to the phenotypically im-mature epithelium seen in asthmatic airways.In the context of disease, aurora kinases have beenlinked to spermatogenic arrest, chromosomal instability,and tumorigenicity in pathologies such as infertility,chronic inflammation, and a wide range of cancers [47–49]. AURKA is capable of phosphorylating H3S10, a siteimplicated in both gene activation and cell division [15,50]. In a murine model of wound repair, rapid and sus-tained phosphorylation of H3S10 was associated withwound healing in intestinal epithelial cells [51]. Further,although the mechanism is not fully clear,Fig. 6 SET and MYND domain containing 3 (SMYD3) expression in asthmatic compared to healthy airway fibroblasts. SMYD3 expression wasanalyzed at the RNA (a) and protein (b) level in airway fibroblasts. c SMYD3 expression is normalized to HSP90 (± SD). For gene expression data, at-test found SMYD3 to be significant however it did not pass ENIV correction. A two tailed unpaired t-test was performed on protein data (n = 6)Stefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 8 of 11phosphorylation of H3S10 is a critical component ofchromatin compaction during mitosis [52]. Given thatAECs from asthmatics are mitotically dyssynchronous[53], show defects in cell cycle regulation [54], and ex-hibit abnormal proliferation and delayed wound repair[55–57], our finding of increased AURKA expressionmay indicate aberrant regulation of these processes inasthma.We identified elevated mRNA expression of the his-tone methyltransferase SMYD3 in airway fibroblastsfrom asthmatics. SMYD3 is integral to cell cycle regu-lation through interactions with RNA polymerase IIand methylation of H3K4 [58]. In addition to geneactivation through H3K4 methylation, SMYD3 is cap-able of gene repression through H2K20 methylation[59], suggesting a complex role for this enzyme. How-ever, although differences in gene expression wereseen, we were unable to replicate these findings atthe protein level possibly indicating a further level oftranscriptional control.While we found many cell-specific and some diseasespecific changes in the enzymes involved in epigeneticmodification, there are limitations to our study. We useda cell culture model that does not necessarily representthe complexity of cell – cell interactions known to be in-tegral to airway mucosal homeostasis. However, a cellculture model allowed us to identify differences in theepigenetic modification families in relatively undifferen-tiated epithelial cells and fibroblasts under controlledconditions. Although we examined gene and protein ex-pression of the epigenetic modifiers associated withasthma, we did not assess the activity of these enzymes,which has been shown to differ in disease. In addition,due to the sample size, we were unable to examine sexdifferences within our samples. Lastly, we did not lookat the targeted epigenentic changes as a result of the dif-ferential expression of the epigenetic modifying enzymesand further studies would need to be performed to solid-ify the functional effects of the cell and disease specificchanges we described in our cohort.ConclusionsIn summary, we identified cell-specific variation ingene expression in each of the families of epigeneticmodifying enzymes in AECs and airway fibroblasts.These data provide insight into the cell-specific vari-ation in epigenetic regulation which may impact thefunctions of different cell types. We identified diseasespecific dysregulation of the histone kinase AURKAin AECs, which may play a role in processes import-ant in the pathogenesis of asthma such as prolifera-tion and inflammation. These findings provide furtherevidence of the importance of the epigenome in celldevelopment and function.Additional filesAdditional file 1: Table S1. Epigenetic modification genes includingfamily, full name, and alias. (DOCX 19 kb)Additional file 2: Supplementary Methods. (DOCX 24 kb)Additional file 3: Table S3. Comparison of epigenetic modifier geneexpression between epithelial cells and fibroblasts from healthy donors.(DOCX 17 kb)Additional file 4: Table S4. Comparison of epigenetic modifier geneexpression between airway epithelial cells from asthmatic and healthydonors. (DOCX 15 kb)AbbreviationsAEC: Airway epithelial cell; AURKA: Aurora kinase A; CREBBP: CREB bindingprotein; DNMT: DNA methyltransferase; DUB: Deubiquitinating enzyme;DZIP3: DAZ interacting zinc finger protein 3; EHMT2: Euchromatic histone-lysine N-methyltransferase 2; ENIV: Effective number of independentvariables; EP300: E1A binding protein p300; Fb: Airway fibroblast;GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; H2K20: Histone 2Lysine 20; H3K4me: Histone 3 lysine 4 monomethylation; H3S10: Histone 3serine 10; HAT: Histone acetyltransferase; HDAC: Histone deacetylase;HDM: Histone demethylase; HMT: Histone methyltransferase;HPRT1: Hypoxanthine phosphoribosyltransferase 1; KDM: Lysine (K)-specificdemethylase; MBD2: Methyl CpG binding domain protein 2; PCA: Principalcomponent analysis; RPL13A: Ribosomal protein L13a; SMYD3: SET andMYND domain containing 3; SUV39H1: Suppressor of variegation 3–9homolog 1AcknowledgementsNot applicable.FundingThis work was funded by a grant from the Canadian Institutes of HealthResearch (MOP-82745). The funding body had no role in study design, datacollection and analysis, interpretation of data, writing of the manuscript, or inthe decision to submit the manuscript for publication.Availability of data and materialsThe datasets during and/or analysed during the current study available fromthe corresponding author on reasonable request.Authors’ contributionsDS, JU, KL, FS, EO, and HKK performed the experiments. DS and NF analyzedthe data. DS wrote the manuscript. DS, TLH, and DAK designed the study.TSH provided samples from brushings. TLH, TSH, and DAK edited themanuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable.Ethics approval and consent to participatePrimary cells were obtained from de-identified donor lungs donated forresearch and not suitable for transplantation though the InternationalInstitute for the Advancement of Medicine (Edison, NJ, USA). The study wasapproved (#H0-50110) by the Providence Research Ethics committee, TheUniversity of British Columbia.Author details1UBC Centre for Heart Lung Innovation, St. Paul’s Hospital, 1081 BurrardStreet, Vancouver, BC V6Z 1Y6, Canada. 2Department of Biological Sciences,Southern Methodist University, Dallas, TX, USA. 3Department of Medicine,Division of Pulmonary and Critical Care, University of Washington, Seattle,USA. 4School of Biomedical Sciences and Pharmacy, Faculty of Health andMedicine, University of Newcastle, Callaghan, NSW, Australia. 5Department ofAnesthesiology, Pharmacology and Therapeutics, University of BritishColumbia, Vancouver, BC, Canada.Stefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 9 of 11Received: 8 September 2016 Accepted: 18 January 2017References1. Masoli M, Fabian D, Holt S, Beasley R. Global initiative for asthma (GINA)program. The global burden of asthma: executive summary of the GINAdissemination committee report. Allergy. 2004;59(5):469–78.2. Barbato A, Turato G, Baraldo S, Bazzan E, Calabrese F, Panizzolo C, et al.Epithelial damage and angiogenesis in the airways of children with asthma.Am J Respir Crit Care Med. 2006;174(9):975–81.3. Hackett TL, Singhera GK, Shaheen F, Hayden P, Jackson GR, Hegele RG, et al.Intrinsic phenotypic differences of asthmatic epithelium and itsinflammatory responses to respiratory syncytial virus and air pollution. Am JRespir Cell Mol Biol. 2011;45(5):1090–100.4. Choe MM, Sporn PH, Swartz MA. Extracellular matrix remodeling bydynamic strain in a three-dimensional tissue-engineered human airway wallmodel. Am J Respir Cell Mol Biol. 2006;35(3):306–13.5. Brewster CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Roche WR.Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J RespirCell Mol Biol. 1990;3(5):507–11.6. Gizycki MJ, Adelroth E, Rogers AV, O’Byrne PM, Jeffery PK. Myofibroblastinvolvement in the allergen-induced late response in mild atopic asthma.Am J Respir Cell Mol Biol. 1997;16(6):664–73.7. Lewis CC, Chu HW, Westcott JY, Tucker A, Langmack EL, Sutherland ER, et al.Airway fibroblasts exhibit a synthetic phenotype in severe asthma. J AllergyClin Immunol. 2005;115(3):534–40.8. Ingram JL, Huggins MJ, Church TD, Li Y, Francisco DC, Degan S, et al. Airwayfibroblasts in asthma manifest an invasive phenotype. Am J Respir Crit CareMed. 2011;183(12):1625–32.9. Goldberg AD, Allis CD, Bernstein E. Epigenetics: a landscape takes shape.Cell. 2007;128(4):635–8.10. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how thegenome integrates intrinsic and environmental signals. Nat Genet. 2003;33(Suppl):245–54.11. Waddington CH. The strategy of the genes; a discussion of some aspects oftheoretical biology. London: Allen & Unwin; 1957.12. Tammen SA, Friso S, Choi SW. Epigenetics: the link between nature andnurture. Mol Aspects Med. 2013;34(4):753–64.13. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128(4):693–705.14. Bhaumik SR, Smith E, Shilatifard A. Covalent modifications of histonesduring development and disease pathogenesis. Nat Struct Mol Biol. 2007;14(11):1008–16.15. Rossetto D, Avvakumov N, Cote J. Histone phosphorylation: a chromatinmodification involved in diverse nuclear events. Epigenetics. 2012;7(10):1098–108.16. Grant PA. A tale of histone modifications. Genome Biol. 2001;2(4):REVIEWS0003.17. Cao J, Yan Q. Histone ubiquitination and deubiquitination in transcription,DNA damage response, and cancer. Front Oncol. 2012;2:26.18. Tate PH, Bird AP. Effects of DNA methylation on DNA-binding proteins andgene expression. Curr Opin Genet Dev. 1993;3(2):226–31.19. Vaissiere T, Sawan C, Herceg Z. Epigenetic interplay between histonemodifications and DNA methylation in gene silencing. Mutat Res. 2008;659(1–2):40–8.20. Karouzakis E, Gay RE, Gay S, Neidhart M. Epigenetic control in rheumatoidarthritis synovial fibroblasts. Nat Rev Rheumatol. 2009;5(5):266–72.21. Horiuchi M, Morinobu A, Chin T, Sakai Y, Kurosaka M, Kumagai S. Expressionand function of histone deacetylases in rheumatoid arthritis synovialfibroblasts. J Rheumatol. 2009;36(8):1580–9.22. Coward WR, Watts K, Feghali-Bostwick CA, Knox A, Pang L. Defectivehistone acetylation is responsible for the diminished expression ofcyclooxygenase 2 in idiopathic pulmonary fibrosis. Mol Cell Biol. 2009;29(15):4325–39.23. Enkhbaatar Z, Terashima M, Oktyabri D, Tange S, Ishimura A, Yano S, et al.KDM5B histone demethylase controls epithelial-mesenchymal transition ofcancer cells by regulating the expression of the microRNA-200 family. CellCycle. 2013;12(13):2100–12.24. Ramadoss S, Chen X, Wang CY. Histone demethylase KDM6B promotesepithelial-mesenchymal transition. J Biol Chem. 2012;287(53):44508–17.25. Bartling TR, Drumm ML. Loss of CFTR results in reduction of histonedeacetylase 2 in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol.2009;297(1):L35–43.26. Yang IV, Schwartz DA. Epigenetic mechanisms and the development ofasthma. J Allergy Clin Immunol. 2012;130(6):1243–55.27. Ito K, Caramori G, Lim S, Oates T, Chung KF, Barnes PJ, et al. Expression andactivity of histone deacetylases in human asthmatic airways. Am J RespirCrit Care Med. 2002;166(3):392–6.28. Ito K, Ito M, Elliott WM, Cosio B, Caramori G, Kon OM, et al. Decreasedhistone deacetylase activity in chronic obstructive pulmonary disease. NEngl J Med. 2005;352(19):1967–76.29. Butler CA, McQuaid S, Taggart CC, Weldon S, Carter R, Skibinski G, et al.Glucocorticoid receptor beta and histone deacetylase 1 and 2 expression inthe airways of severe asthma. Thorax. 2012;67(5):392–8.30. Bergeron C, Fukakusa M, Olivenstein R, Lemiere C, Shannon J, Ernst P, et al.Increased glucocorticoid receptor-beta expression, but not decreasedhistone deacetylase 2, in severe asthma. J Allergy Clin Immunol. 2006;117(3):703–5.31. Stefanowicz D, Hackett TL, Garmaroudi FS, Gunther OP, Neumann S,Sutanto EN, et al. DNA methylation profiles of airway epithelial cells andPBMCs from healthy, atopic and asthmatic children. PLoS One. 2012;7(9):e44213.32. Karp PH, Moninger TO, Weber SP, Nesselhauf TS, Launspach JL, Zabner J, etal. An in vitro model of differentiated human airway epithelia. Methods forestablishing primary cultures. Methods Mol Biol. 2002;188:115–37.33. Pechkovsky DV, Hackett TL, An SS, Shaheen F, Murray LA, Knight DA.Human lung parenchyma but not proximal bronchi produces fibroblastswith enhanced TGF-beta signaling and alpha-SMA expression. Am J RespirCell Mol Biol. 2010;43(6):641–51.34. Trudeau J, Hu H, Chibana K, Chu HW, Westcott JY, Wenzel SE. Selectivedownregulation of prostaglandin E2-related pathways by the Th2 cytokineIL-13. J Allergy Clin Immunol. 2006;117(6):1446–54.35. Hallstrand TS, Lai Y, Ni Z, Oslund RC, Henderson Jr WR, Gelb MH, et al.Relationship between levels of secreted phospholipase A(2) groups IIA andX in the airways and asthma severity. Clin Exp Allergy. 2011;41(6):801–10.36. Li J, Ji L. Adjusting multiple testing in multilocus analyses using theeigenvalues of a correlation matrix. Heredity. 2005;95(3):221–7.37. R Development Core Team. R: A Language and Environment for StatisticalComputing. In: R Foundation for Statistical Computing. Vienna, Austria;2011. Available from: http://www.R-project.org/.38. Wickham H. ggplot2: Elegant Graphics for Data Analysis: Springer-VerlagNew York; 2009. Available from: http://ggplot2.org.39. Wang Y, Shang Y. Epigenetic control of epithelial-to-mesenchymal transitionand cancer metastasis. Exp Cell Res. 2013;319(2):160–9.40. Devries A, Vercelli D. Epigenetics of human asthma and allergy: promises tokeep. Asian Pac J Allergy Immunol. 2013;31(3):183–9.41. Low D, Mizoguchi A, Mizoguchi E. DNA methylation in inflammatory boweldisease and beyond. World J Gastroenterol. 2013;19(32):5238–49.42. Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a andDnmt3b are essential for de novo methylation and mammaliandevelopment. Cell. 1999;99(3):247–57.43. Ng HH, Zhang Y, Hendrich B, Johnson CA, Turner BM, Erdjument-BromageH, et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histonedeacetylase complex. Nat Genet. 1999;23(1):58–61.44. Reichert N, Choukrallah MA, Matthias P. Multiple roles of class I HDACs inproliferation, differentiation, and development. Cell Mol Life Sci. 2012;69(13):2173–87.45. Teo J-L, Kahn M. The Wnt signaling pathway in cellular proliferation anddifferentiation: a tale of two coactivators. Adv Drug Deliv Rev. 2010;62(12):1149–55.46. Moheimani F, Roth HM, Cross J, Reid AT, Shaheen F, Warner SM, et al.Disruption of beta-catenin/CBP signaling inhibits human airwayepithelial-mesenchymal transition and repair. Int J Biochem Cell Biol.2015;68:59–69.47. Kimmins S, Crosio C, Kotaja N, Hirayama J, Monaco L, Hoog C, et al.Differential functions of the Aurora-B and Aurora-C kinases in mammalianspermatogenesis. Mol Endocrinol. 2007;21(3):726–39.48. Wu SR, Li CF, Hung LY, Huang AM, Tseng JT, Tsou JH, et al. CCAAT/enhancer-binding protein delta mediates tumor necrosis factor alpha-induced Aurora kinase C transcription and promotes genomic instability. JBiol Chem. 2011;286(33):28662–70.Stefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 10 of 1149. Lens SM, Voest EE, Medema RH. Shared and separate functions of polo-likekinases and aurora kinases in cancer. Nat Rev Cancer. 2010;10(12):825–41.50. Crosio C, Fimia GM, Loury R, Kimura M, Okano Y, Zhou H, et al. Mitoticphosphorylation of histone H3: spatio-temporal regulation by mammalianAurora kinases. Mol Cell Biol. 2002;22(3):874–85.51. Karrasch T, Steinbrecher KA, Allard B, Baldwin AS, Jobin C. Wound-inducedp38MAPK-dependent histone H3 phosphorylation correlates with increasedCOX-2 expression in enterocytes. J Cell Physiol. 2006;207(3):809–15.52. Hans F, Dimitrov S. Histone H3 phosphorylation and cell division.Oncogene. 2001;20(24):3021–7.53. Freishtat RJ, Watson AM, Benton AS, Iqbal SF, Pillai DK, Rose MC, et al.Asthmatic airway epithelium is intrinsically inflammatory and mitoticallydyssynchronous. Am J Respir Cell Mol Biol. 2011;44(6):863–9.54. Puddicombe SM, Torres-Lozano C, Richter A, Bucchieri F, Lordan JL,Howarth PH, et al. Increased expression of p21(waf) cyclin-dependent kinaseinhibitor in asthmatic bronchial epithelium. Am J Respir Cell Mol Biol. 2003;28(1):61–8.55. Kicic A, Sutanto EN, Stevens PT, Knight DA, Stick SM. Intrinsic biochemicaland functional differences in bronchial epithelial cells of children withasthma. Am J Respir Crit Care Med. 2006;174(10):1110–8.56. Kicic A, Hallstrand TS, Sutanto EN, Stevens PT, Kobor MS, Taplin C, et al.Decreased fibronectin production significantly contributes to dysregulatedrepair of asthmatic epithelium. Am J Respir Crit Care Med. 2010;181(9):889–98.57. Stevens PT, Kicic A, Sutanto EN, Knight DA, Stick SM. Dysregulated repair inasthmatic paediatric airway epithelial cells: the role of plasminogen activatorinhibitor-1. Clin Exp Allergy. 2008;38(12):1901–10.58. Hamamoto R, Furukawa Y, Morita M, Iimura Y, Silva FP, Li M, et al. SMYD3encodes a histone methyltransferase involved in the proliferation of cancercells. Nat Cell Biol. 2004;6(8):731–40.59. Foreman KW, Brown M, Park F, Emtage S, Harriss J, Das C, et al. Structuraland functional profiling of the human histone methyltransferase SMYD3.PLoS One. 2011;6(7):e22290.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Stefanowicz et al. BMC Pulmonary Medicine  (2017) 17:24 Page 11 of 11

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.52383.1-0362095/manifest

Comment

Related Items