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Volume 14, Number 5,December 2021ISSN 1995-6673Pages 1035 – 1043https://doi.org/10.54319/jjbs/140524JJBSJordan Journal of Biological SciencesTranscriptional Impact of E-cadherin Loss on Embryonic StemCellsHani Alotaibi1,2,*1Izmir Biomedicine and Genome Center, Izmir, Turkey; 2 Izmir International Biomedicine and Genome Institute, Dokuz Eylül University,Izmir, TurkeyReceived: April 22, 2021; Revised: August 15, 2021; Accepted: August 24, 2021AbstractIn embryonic stem cells, E-cadherin plays a crucial role in sustaining the pluripotent state; this is achieved by thesimultaneous presence of active core pluripotency transcriptional network, proper cell-cell adhesion, and an undifferentiatedstate chromatin signature. In contrast, N-cadherin is linked to a more differentiated cell state but can support pluripotency ifexpressed as a knock-in allele from the E-cadherin locus. This study describes the N-cadherin ki/ki embryonic stem cells,which lack E-cadherin expression, to identify transcriptional changes distinct from the known changes observed in Ecadherin knockout embryonic stem cells. As a result, a remarkable similarity in the expression profiles of N-cadherin ki/kiand wild-type embryonic stem cells was observed. Further analysis of the slight differences revealed significant alterations inseveral biological processes such as chromatin organization and epithelial-mesenchymal transition. The findings presentedhere shed light on a new aspect of E-cadherin biology in embryonic stem cells and lay the foundations for comprehensiveunderstanding of E-cadherin's functional relevance beyond the prominent role in maintaining pluripotency.Keywords: E-cadherin, Epithelial to Mesenchymal Transition, EMT, Embryonic Stem Cells, Gene replacement.1. IntroductionEmbryonic stem (ES) cells can self-renew, maintain anundifferentiated state of pluripotency, and generate cellsthat can differentiate into the various cell types of the body(Niwa, 2007). The pluripotent state's maintenance requiresthe function of a core transcriptional network comprised ofthree main transcription factors, Nanog, Sox2, and Oct4(Ying et al., 2008). The functions of these transcriptionfactors keep the cells in an undifferentiated statedistinguished by growth in compact colonies, an activepluripotency transcriptional network, and the presence ofbivalent histone modifications on the promoters of severalessential genes (Mikkelsen et al., 2007). The transcriptionfactors Sox2 and Oct4, Klf4, and c-Myc induce thepluripotent state in mouse somatic cells by establishingES-like cells called induced pluripotent stem cells (iPSCs)(Takahashi and Yamanaka, 2006). Nanog's activation is anessential step in establishing the pluripotent state (Silva etal., 2009). The reprogramming to the pluripotent staterequires completing a well-orchestrated series of events,including changes in the chromatin landscape and DNAmethylation states (Boyer et al., 2006, Maherali et al.,2007). These events coincide with the initiation of themesenchymal to epithelial transition (MET), during whichthe somatic cells lose their fibroblast-specificmesenchymal morphology and gradually establishpolarization and gain the ES cell-like appearance (Li et al.,2010). This MET event is a crucial step and depends on E*cadherin's (E-cad) activation, a calcium-dependentadhesion molecule. E-cad is essential for maintainingstemness and the pluripotent state since its loss can causealterations in homophilic adhesion, causing a scattered cellgrowth of ES cells in culture (Larue et al., 1996, Soncin etal., 2009). Moreover, exogenous E-cad expression cansubstitute Oct4 in the mixture of reprogramming factors,which results in effective iPSC reprogramming (Redmer etal., 2011).ES cell homeostasis fundamentally requires the E-cadmediated adhesion, an indispensable event for cell-cellcommunication and signaling (Chen et al., 2010). On theother hand, in mesenchymal cells, such as fibroblasts, cellcell adhesion is achieved by N-cadherin (N-cad). Theprotein sequences of both cadherins show strikingconservation; thus, they have a similar role in mediatinghomophilic adhesion and even interact with similarintracellular proteins such as beta-catenin. However, theyare primarily present in a mutually exclusive fashion.These molecules also promote contrasting phenotypes; Ecad expression will result in a polarized epithelial form; incontrast, N-cad expression correlates with a depolarizedmotile state (Wheelock et al., 2008). Several studiesaddressed the functional redundancy of the two cadherins,their impact on critical developmental stages, and theirimpact on the pluripotent state in ES cells. One reportinvestigated the impact of replacing E-cad by N-cad duringtrophectoderm formation and concluded that E-cadfunctions could not be restored by N-cad (Kan et al.,2007). Besides, the expression of N-cad from the E-cadCorresponding author e-mail: hani.alotaibi@ibg.edu.trAbbreviations: Embryonic stem (ES), induced pluripotent stem cells (iPSCs), E-cadherin (E-cad), N-cadherin (N-cad), knockout (ko),knock-in (ki), Gene Expression Omnibus (GEO).**

1036 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 5locus was sufficient to maintain the pluripotent state andwas also suitable for reprogramming fibroblasts into iPSCs(Bedzhov et al., 2013). The maintenance of adhesion wasenough for the preservation of pluripotency. Maintainingthe pluripotent state by N-cad in ES cells provided aunique opportunity to study the impact of E-cad loss on EScells that retain the pluripotent state. In recent years, agreat deal of research has been dedicated to studying EScells and iPSCs, notably for their potential use inpersonalized medicine (Yamanaka, 2020). So, it is ofinterest to have a better understanding of stem cell biologyin general, and a better understanding of the outcomes ofpotential gene expression signature changes would be ofutmost importance for the design and planning of suchtherapies. The current study reveals the changes in EScells' gene expression profiles expressing N-cad instead ofE-cad. This is addressed by performing in silico analysesand then comparing the expression profiles of E-cadknockout (ko) cells and N-cad knock-in (ki) cells to thewild-type ES cells.2. Materials and Methods2.1. ES cell cultureES cells used in this work were described previously;in summary, N-cad ki/ki, E-cad ko/ko, and wt ES cellswere prepared from E2.5 embryos as described in(Bedzhov et al., 2013). ES cells were grown on feedercells (mitotically inactivated mouse embryonic fibroblasts)in ES cell medium prepared with DMEM (Biochrom) andsupplemented with 15% FCS (PAN), 10 U/ml Penicillinand Streptomycin, 0.1 mM non-essential amino acids, twomM L-glutamine (Gibco), 0.15 mM β-mercaptoethanol(Sigma), and 500 U/ml Lif (Millipore). ES cells weregrown at 37 C in the presence of 10% CO 2 . The mediumwas changed every two days, and the cells were subcultured or processed at sub-confluence.2.2. RNA isolation and gene expression analysisCells were seeded in 6-well plates (105 cells per well),incubated to sub-confluence, and collected for RNAisolation. For gene expression analysis, RNA extractionand preparation of cDNA were performed using themethods and reagents described previously (Bedzhov etal., 2013). Primers used in this study and theircorresponding UPL probes are presented in Table 1.Relative expression was calculated using the 2-ΔΔCt method(Livak and Schmittgen, 2001) relative to the wt controland normalized to the housekeeping gene Gapdh. Datarepresent at least three biological replicates. One-wayANOVA, followed by Tukey's multiple comparison tests,was performed for statistical analysis of groupcomparisons.Table 1: Primers and corresponding UPL probes used in qPCRexperiments.GeneNamePrimer SequenceUPL probe IDCdh1F: atcctcgccctgctgattR: accaccgttctcctccgtaUPL No: 18Cdh2F: tccctgagatacagcgtcactR: ataatgaagatgcccgttggUPL No: 17EomesF: accggcaccaaactgagaR: aagctcaagaaaggaaacatgcUPL No: 9Klf4F: cgggaagggagaagacactR: gagttcctcacgccaacgUPL No: 62Neurod1F: cgcagaaggcaaggtgtcR: tttggtcatgtttccacttccUPL No: 1VimF: ccaaccttttcttccctgaaR: ccaaccttttcttccctgaaUPL No: 109Snai1F: gtctgcacgacctgtggaaR: caggagaatggcttctcaccUPL No: 71Zeb1F: gccagcagtcatgatgaaaaR: tatcacaatacgggcaggtgUPL No: 48GapdhF: agcttgtcatcaacgggaagR: tttgatgttagtggggtctcgUPL No: 92.3. Microarray analysisMicroarray analysis for the ki/ki and wt2 cells wasdescribed before (Bedzhov et al., 2013). In short, RNAfrom two independent samples per genotype washybridized to the GeneChip Mouse Genome 430 v2.0array (Affymetrix, Inc.). The raw data can be downloadedfrom the NCBI GEO database (Barrett et al., 2013) (GEOaccession number GSE42008). Raw data for the E-cadknockout and the corresponding wild-type cells (wt1) arepublicly available and were obtained from theArrayExpress, accession number E-MEXP-2836 (Soncinet al., 2011). Data analysis was performed in R v4.0.3. Thedata set's quality was examined with the simpleaffypackage v2.66.0 (Wilson and Miller, 2005), andexpression values and normalization were performed bythe GCRMA function, with quantile normalization andexcluding features with expression values of 4 in allsamples. Differential expression analysis was performedwith the limma package v 3.46.0 (Ritchie et al., 2015),applying the eBayes function. Heatmaps were plottedusing the Heatmap.2 function of the gplots package ots/index.html). Using Log2-transformed and normalized expressionvalues, heatmap images were generated, applyingPearson's correlation to calculate hierarchical clustering.Significant genes were calculated by measuring thestandard deviation for each row, then subsetting theshortest interval covering half of the values in the data setusing the shorth function of the genefilter package bioc/html/genefilter.html), and then calculating the adjusted p-valueaccording to FDR. PCA plots were generated using thestats package's prcomp command and plotted using theautoplot command of the ggplot2 package v3.3.2(Wickham, 2009). GO enrichment analysis was performedusing the GOexpress package v1.24.0 (Rue-Albrecht et al.,2016) with the following parameters: seed 4543,permutations 1000, and random forest method wasselected.

2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 53. ResultsIn order to understand the impact of E-cad deletion inES cells, the previously generated mouse models and EScells where E-cad was replaced for N-cad as a knock-inallele were used (Bedzhov et al., 2013). In the presence ofN-cad (either as ki/wt or ki/ki), ES cells retained thepluripotency state as opposed to the E-cad ko/ko cells,which lose the pluripotency. The functional loss of E-cadexpression in ES cells is not limited to loss ofpluripotency; this is why the N-cad knock-in (ki/ki) EScells present a unique model for studying the consequencesof E-cad depletion beyond pluripotency.3.1. High correlation between wt and N-cad ki/ki EScellsWe have previously shown that the N-cad ki/ki cellsresembled wt cells morphologically; they grow as compactcolonies on feeder cells and express key pluripotencyspecific genes (Bedzhov et al., 2013). This is acontinuation of the previous analysis to shed light on theglobal changes in ki/ki and ko/ko cells. The first analysis1037revealed that ki/ki cells were closely related to wt cellsrather than ko/ko cells (Figure 1A). This remarkablesimilarity is, of course, reflected by the previous findingsregarding pluripotency genes and is also reflected by themorphological features mentioned earlier. Moreover,correlation analysis revealed a remarkable 97% correlation(Figure 1B). As expected, there was no statisticallysignificant correlation between the ko/ko cells compared toeither ki/ki or wt cells. These results confirmed once morethe functional redundancy of E-cad and N-cad in ES cells.The global resemblance of gene expression profiles canalso be recognized by looking at a heatmap comparing thethree cellular models (Figure 1C). The three-way analysisof samples highlighted the similarities and differencesbetween the three cell lines. However, careful examinationof the heatmap also unveils the slight differences betweenki/ki and wt ES cells (Figure 1C). This analysis wasfollowed by qPCR experiments to validate the expressionprofiling results. Accordingly, the status of Cdh1 and Cdh2(The official gene symbols for E-cad and N-cad,respectively) expression was confirmed and reflected thegenotypic changes in both cell lines (Figure 1D)Figure 1. The N-cad ki/ki ES cells are highly similar to the wt ES cells. A) Principal component analysis showing the high similaritybetween ki/ki and wt cells. PC2 corresponds to less than 3% difference, while PC1 corresponds to more than 95% difference. B) Correlationmatrix indicating the percentages of similarity between the samples. C) Heatmap drawing of the expression values of all samples used in thisstudy, D-F) qPCR analysis of genes selected to verify the expression data obtained from microarray analysis. Experiments were performedat least 3 times in triplicates. Error bars correspond to the standard error of the mean. Significant values were labeled with the asterisk.

1038 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 5We have previously studied the expression levels ofpluripotency-related genes (Bedzhov et al., 2013). Here,other developmentally relevant genes, such as Eomes, Klf4,and Neurod1 (Figure 1E), as well as genes relevant to thecadherin switching, were selected (Figure 1F). Overall, theexpression levels detected using qPCR confirmed thesignals obtained from microarray experiments' expressionprofiles.3.2. Common genes differentially expressed in ki/ki andko/ko ES cellsStudying the differentially expressed genes in ko/kocells compared to wt ES cells will reveal the impact of Ecad deletion on gene expression, which is reflected bychanges in ES physiology. These changes include theknown impact on pluripotency. Similarly, examiningdifferentially expressed genes between ki/ki and wt cellswill show the impact of the forced N-cad expression on EScells. Differential expression analyses of ko/ko-wt andki/ki-wt ES cells were performed using the R-packagelimma with a cutoff of log2 0.6. The number ofdifferentially expressed genes in ko/ko ES cells was 3527(Figure 2A), and in ki/ki ES cells, it was 1976 (Figure 2B).To better understand E-cad's impact on ES cells, thedifferentially expressed genes of ko/ko-wt and ki/ki-wt EScells were compared first. The common genes werecategorized into different groups, as illustrated in Figure 2.Figure 2. Common differentially expressed genes. A-B) Heatmaps show 1335 differentially expressed genes in ki/ki and ko/ko ES cells.C-D) Numeric representation of commonly expressed genes.In total, 1335 genes differentially expressed in bothki/ki-wt and ko/ko-wt comparisons are common. Thenumber of commonly upregulated genes was 245, while206 genes were found downregulated. The Venn diagramin Figure 2C shows the common and unique areas with thenumber of genes depicted; the complete data is alsosummarized as a matrix in Figure 2C. Of note here is thedifference in differentially expressed genes in bothcomparisons. The number of differentially expressed genesin ko/ko cells was about two times higher than in ki/kicells, attesting to the similarities between ki/ki and wt cells(Figure 2C). As described in materials and methods,

2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 5common genes differentially expressed were subjected tosequential filtering to find significant genes. The numberof remaining genes that remained after applying theadjusted p-value of 0.01 filter was 280 (SupplementaryTable S1). These genes were also plotted in the heatmap insupplementary Figure S1 and revealed the remarkableresemblance of ki/ki ES cells to their wt counterparts. Theheatmap view also revealed the subtle differences between1039ki/ki ES cells and wt cells. To better understand thesedifferences, subsetting the common differentiallyexpressed genes for the ki/ki and wt samples from thedataset was performed and followed by applying thefiltering as done in the previous analysis. As a result, 179differentially expressed genes in the ki/ki ES cells with anadjusted p-value of 0.05 were found (Figure 3A andSupplementary Table S2).Figure 3. Commonly upregulated and downregulated genes in the ki/ki ES cells. A) Heatmap of the common differentially expressedgenes in ki/ki cells. One hundred seventy-nine genes with an adjusted p-value of 0.05 or less were plotted here. Upregulated ordownregulated genes in both genotypes were subjected to GO enrichment analysis. The heatmap in B represents 95 genes that were found asenriched.3.3. GO enrichment analysis of common genesdifferentially expressed in ki/ki and ko/ko ES cellsTo determine the relevance of the differentiallyexpressed genes in ki/ki cells, commonly expressed upand downregulated genes were included in the GOenrichment analysis. A total of 451 genes (245 upregulatedand 206 downregulated) were subjected to GO enrichmentanalysis using the R-package GOexpress. This analysisrevealed significant enrichment in 95 genes belonging to19 different biological processes. The expression pattern ofthese genes is presented in the heatmap in Figure 3B. TheGO enrichment analysis revealed significant changes ingenes within the response to the drug biological process(20 genes: 14%).Moreover, chromatin organization (18 genes: 12%)related genes and genes related to angiogenesis (14 genes: 10%) were also significantly represented. A summary ofthe GO enrichment analysis results is depicted in the piechart in figure 4A. Eighteen genes belonging to thechromatin organization biological process were mainlydownregulated in response to Cdh1 depletion in the ki/kicells (Figure 4B), suggesting a positive correlationbetween these biological processes and E-cad function. Onthe other hand, genes related to EMT were found to beupregulated in the ki/ki cells, suggesting a tendency toshift to a mesenchymal state in the absence of E-cad(Figure 4C).

1040 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 5Figure 4. GO enrichment analysis identifies significant biological processes affected by E-cad depletion. A) Nineteen biologicalprocess GO terms were enriched in ki/ki ES cells, and their percentage is represented in the pie chart. B) Chromatin organization genes weremostly downregulated in ki/ki ES cells. C) EMT-related genes were upregulated ki/ki ES 1 cells.Other gene groups are also presented in SupplementalFigure 2. Of note, genes belonging to three developmentalprocesses were mapped in supplementary Figure S2; theossification gene group was mainly upregulated in theabsence of E-cad. In contrast, genes belonging to thepituitary gland development were mainly downregulated inthe ki/ki cells. The neural tube development genes weremixed (Figure S2A). Genes in the angiogenesis (FigureS2B), apoptosis (Figure S2C), and response to drugs(Figure S2D) were mainly upregulated as a result of E-cadabsence. A detailed description of all biological processesrepresented by the 95 enriched genes is displayed in Table2.

2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 51041Table 2: Detailed descriptions of the identified GO enrichment analysis showing significant biological process terms and correspondinggenes.DataTotalp.valGO IDGO NameGene IDcountcountGO:0001503ossificationSparc, Bmp4, Gpm6b, Ifitm1, Ptn, Csf160.009GO:0001525angiogenesisCcn2, Ptgs2, Hs6st1, Jun, Syk, Itgav, Pecam1,459Bmp4, Casp8, Cemip2, Parva, Gab1, Pknox1, Nrp2177140.009GO:0001837epithelial to mesenchymaltransitionMir7040, Tgfbr3, Hmga2, Rflnb8440.001GO:0006325chromatin organizationSatb1, Epc1, Dnmt3a, Dppa3, Prkaa2, Cecr2,Asxl1, Kdm5a, Rcor1, Chd4, Brd1, Nsd1, Tbl1xr1,Ctcf, L3mbtl3, Eya1, Smarca1, Nr3c1610180.001GO:0008203cholesterol metabolic processApoa2, App, Cyp7b1, Abcg1, Prkaa2, Vldlr, Cln617070GO:0009314response to radiationPtgs2, Jun, App, Polh, Col3a1, Plk36560.001GO:0009749response to glucoseCasp6, Ccn2, Apoa2, Trh, Fos, Nnat, Neurod1,Selenot, Thbs114390.002GO:0010942positive regulation of cell deathPrnp, Ccn2, Ptgs2, Cdkn1a, Bmp4, Dnmt3a10660.002GO:0021915neural tube developmentHes1, Sema3c, Cecr2, Sfrp19440.006GO:0021983pituitary gland developmentHes1, Bmp4, Cdh1, Gli1, Hmga26750GO:0034097response to cytokineCasp6, Sparc, Ptgs2, Jun, Fos, Col3a1, Timp2,Cited1, Cd3814190.006GO:0034446substrate adhesion-dependentcell spreadingItgav, Parva, Megf9, Antxr1, Itga8, Sfrp110760.002GO:0035019somatic stem cell populationmaintenanceHes1, Tfap2c, Hmga2, Sfrp17840.007GO:0042493response to drugPtgs2, Jun, Gstt1, Abcb1b, Cdkn1a, Map1b,Dnmt3a, Fos, Slc1a3, Neurod1, Usp47, Timp2,Adipor2, Srr, Ptn, Cdh1, Cd38, Cyba, Sfrp1, Thbs1619200.008GO:0043525positive regulation of neuronapoptotic processCasp6, PrnpvJun, Pmaip1, App, Pak315360.007GO:0045600positive regulation of fat celldifferentiationFrzb, Ccdc3, Sh3pxd2b, Zfp36, Sfrp19050.007GO:0051384response to glucocorticoidAdam9, Sparc, Ptgs2, Cdkn1a, Alpl, Kras, Dusp1,Cdo112680.01GO:0071276cellular response to cadmium ion Jun, Mt1, Fos, Mt2, Mt46850.004GO:1901998toxin transport7830.009Abcg1, Crtc2, Antxr14. DiscussionThis manuscript addresses the impact of E-cad deletionin ES cells while retaining the pluripotency state, takingadvantage of our previously published cellular model inwhich the expression of N-cad as a knock-in allele in theE-cad locus was performed. The expression profiles of EScells with deleted E-cad (the ko/ko cells) or N-cad as aknock-in allele in the E-cad locus (the ki/ki cells) werecompared to the wild type ES cells using an in silicoapproach. The analysis revealed a slight shift to themesenchymal gene signature by upregulating EMT-relatedgenes. On the other hand, a decrease in the expression ofchromatin organization-related genes was observed in theabsence of E-cad. The gene replacement model analyzedhere proved to be a powerful tool to study aspects of E-cadbiology. The first studies of replacing E-cad with N-cadrevealed that N-cad could not replace E-cad functionduring the formation of the trophectoderm (Kan et al.,2007); this was illustrated by the specific crosstalkbetween E-cad and Igf1r signaling providing a survivalsignal that cannot be reproduced when N-cad is expressedinstead of E-cad (Bedzhov et al., 2012). Several reportsdescribed the essential role E-cad plays in defining theidentity of ES cells and the maintenance of stemness(Chen et al., 2010, Soncin et al., 2011).Furthermore, E-cad was also described as an essentialfacilitator of the reprogramming of iPSCs; it could evenreplace members of the Yamanaka cocktail used in iPSCderivation (Li et al., 2010, Redmer et al., 2011). The highhomology between E-cad and N-cad results in a significantredundancy in ES cells' pluripotency. As opposed totrophectoderm development, ki/ki ES cells injected inblastocysts can develop up to embryonic day E8.5,showing that N-cad could replace E-cad in these chimericembryos (Bedzhov et al., 2013). Notably, the N-cad ki/kiES cells maintained pluripotency, and the resemblance ofthe gene expression profile to the wild-type ES cells wasdistinctive. Despite the global comparability of geneexpression profiles of N-cad ki/ki ES cells and the wildtype ES cells, subtle differences were observed. Thesedifferences are evident when looking at subsettedheatmaps encompassing genes within the biologicalprocesses enriched using the GO enrichment analysis.The results presented here suggest that the loss of Ecad in ES cells results in the foundation of a partial EMTlike state; while cells retain adhesive characteristics, due toN-cad expression, they upregulate specific EMTassociated genes such as Zeb1 and Tgfbr3 anddownregulate essential epithelial genes such as Grhl3.

1042 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 5Loss of Grhl3 was previously shown to result in a robustmesenchymal phenotype during MET initiation (Alotaibiet al., 2015). The effects of E-cad depletion have beenstudied in the non-tumorigenic MCF10A cells. The cellspresented significant changes associated with tissueremodeling and cell-substrate attachment proteins such asITGA1. On the other hand, they did not show a similarincrease in EMT-related genes (Chen et al., 2014). Theobserved increase in Itga8 and Itgav gene expression is inagreement with their findings. E-cad deletion also affectedseveral developmental genes, particularly the ossificationgenes, and some of the neural tube development-relatedgenes were mainly upregulated. This was associated withchanges in differentiation genes and suggested a linkbetween the changes in the EMT-associated genes and thedevelopment-related or differentiation-related genes.Cellular reprogramming is known to include EMT andMET (Li et al., 2010), and the differentiation state of cellsis also expressed in terms of epithelial-like ormesenchymal-like. A recent report showed that asequential EMT/MET is required to differentiate hESCs tothe hepatic lineage. This process involves an autocrineTGFβ signaling loop resulting in an upregulation of EMTinducers and downregulation of E-cad expression (Li etal., 2017). Concerning reprogramming, the observeddownregulation in the chromatin organization genesignature was profound, except for Ctcf and Dnmt3a,which regulate chromatin architecture and DNAmethylation. Epigenetic reprogramming is closelyassociated with fate determination, differentiation, andcellular reprogramming. Previous studies indicated acritical role for E-cad in reprogramming MEFs into iPSCs(Li et al., 2010, Redmer et al., 2011).Furthermore, genetic ablation of E-cad prevented iPSCreprogramming (Redmer et al., 2011, Bedzhov et al.,2013). This reprogramming is associated with globalepigenetic reorganization, resetting the chromatininfrastructure from a differentiated somatic state to anundifferentiated pluripotent state (Hochedlinger andJaenisch, 2015). E-cad's importance to ES cells in terms ofpluripotency is well known, and its essential role in thereprogramming to the pluripotent state is central to theestablishment of MET that precedes the pluripotencynetwork activation. In the absence of E-cad, the tendencyto shift to a more differentiated state as illustrated bychanges in chromatin organization genes and EMTassociated genes shed light onto new aspects of E-cadfunctions in ES cells. It will be of great interest toexperimentally corroborate these novel findings, whichwill significantly benefit the field of stem cell biology andeventually personalized medicine. Stem cell therapy andpersonalized medicine are considered the future ofmedicine (Strauer and Kornowski, 2003). The scope ofpersonalized medicine goes beyond common diseases suchas cancer and is considered for developmental disorders orrare diseases as well (Garcia-Castro and Singec, 2017).Hematopoietic stem cells are used to treat leukemia (Tianet al., 2016), and recent examples can also be seen instudies related to Parkinson's disease (Parmar et al., 2020).With this in mind, it is imperative to acknowledge theimportance of understanding changes in molecularpathways and gene signatures when planning noveltherapeutics.AcknowledgmentsWe would like to thank Dr. Gökhan Karakülah andProf. Uygar Tazebay for carefully reading the manuscript.This study was financed by İzmir Biomedicine andGenome Center institutional funds. The author disclosesno conflict of interest that may have influenced either theconduct or the presentation of the research.Supplementary DataSupplementary Data is available online at wd66b4gb4d.2).ReferencesAlotaibi H, Basilicata MF, Shehwana H, Kosowan T, Schreck I,Braeutigam C, Konu O, Brabletz T and Stemmler MP. 2015.Enhancer cooperativity as a novel mechanism underlying thetranscriptional regulation of E-cadherin during mesenchymal toepithelial transition. Biochimica Et Biophysica Acta-GeneRegulatory Mechanisms, 1849(6): 731-742.Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF,Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM,Holko M, Yefanov A, Lee H, Zhang N, Robertson CL, Serova N,Davis S and Soboleva A. 2013. NCBI GEO: archive for functionalgenomics data sets--update. Nucleic Acids Res, 41(Databaseissue): D991-995.Bedzhov I, Alotaibi H, Basilicata MF, Ahlborn K, Liszewska E,Brabletz T and Stemmler MP. 2013. Adhesion, but not a specificcadherin code, is indispensable for ES cell and inducedpluripotency. Stem Cell Res, 11(3): 1250-1263.Bedzhov I, Liszewska E, Kanzler B and Stemmler MP. 2012.Igf1r signaling is indispensable for preimplantation developmentand is activated via a novel function of E-cadherin. PLoS Genet,8(3): e1002609.Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, LeeTI, Levine SS, Wernig M, Tajonar A, Ray MK, Bell GW, OtteAP, Vidal M, Gifford DK, Young RA and Jaenisch R. 2006.Polycomb complexes repress developmental regulators in murineembryonic stem cells. Nature, 441(7091): 349-353.Chen A, Beetham H, Black MA, Priya R, Telford BJ, Guest J,Wiggins GA, Godwin TD, Yap AS and Guilford PJ. 2014. Ecadherin loss alters cytoskeletal organization and adhesion in nonmalignant breast cells but is insufficient to induce an epithelialmesenchymal transition. BMC Cancer, 14: 552.Chen T, Yuan D, Wei B, Jiang J, Kang J, Ling K, Gu Y, Li J,Xiao L and Pei G. 2010. E-cadherin-mediated cell-cell contact iscritical for induced pluripotent stem cell generation. Stem Cells,28(8): 1315-1325.Garcia-Castro J and Singec I. 2017. Prospects of Pluripotent andAdult Stem Cells for Rare Diseases. Adv Exp Med Biol, 1031:371-386.Hochedlinger K and Jaenisch R.

cells' gene expression profiles expressing N-cad instead of E-cad. This is addressed by performing in silico analyses and then comparing the expression profiles of E-cad knockout (ko) cells and N-cad knock-in (ki) cells to the wild-type ES cells. 2. Materials and Methods . 2.1. ES cell culture . ES cells used in this work were described previously;

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SFUSD Math Core Curriculum is licensed under the Creative Commons Attribution 4.0 International License Subject: Math Grade: 2nd Grade Name of Unit: Introduction Length of Unit: 3 days Overview of Unit: The first week of school is focused on setting up the classroom culture for the year and