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• W2899056105 abstract "•Gene perturbation screens identify a role for ZMAT2 in human epidermal differentiation•ZMAT2 functionally interacts with chromatin regulators in epidermal differentiation•ZMAT2 interacts with the pre-spliceosome and a specific subset of RNA transcripts•ZMAT2 regulates splicing of differentiation and adhesion-associated transcripts Epidermal homeostasis requires balanced progenitor cell proliferation and loss of differentiated cells from the epidermal surface. During this process, cells undergo major changes in their transcriptional programs to accommodate new cellular functions. We found that transcriptional and post-transcriptional mechanisms underlying these changes jointly control genes involved in cell adhesion, a key process in epidermal maintenance. Using siRNA-based perturbation screens, we identified DNA and/or RNA binding regulators of epidermal differentiation. Computational modeling and experimental validation identified functional interactions between the matrin-type 2 zinc-finger protein ZMAT2 and the epigenetic modifiers ING5, SMARCA5, BRD1, UHRF1, BPTF, and SMARCC2. ZMAT2 is an interactor of the pre-spliceosome that is required to keep cells in an undifferentiated, proliferative state. RNA immunoprecipitation and transcriptome-wide RNA splicing analysis showed that ZMAT2 associates with and regulates transcripts involved in cell adhesion in conjunction with ING5. Thus, joint control by splicing regulation, histone, and DNA modification is important to maintain epidermal cells in an undifferentiated state. Epidermal homeostasis requires balanced progenitor cell proliferation and loss of differentiated cells from the epidermal surface. During this process, cells undergo major changes in their transcriptional programs to accommodate new cellular functions. We found that transcriptional and post-transcriptional mechanisms underlying these changes jointly control genes involved in cell adhesion, a key process in epidermal maintenance. Using siRNA-based perturbation screens, we identified DNA and/or RNA binding regulators of epidermal differentiation. Computational modeling and experimental validation identified functional interactions between the matrin-type 2 zinc-finger protein ZMAT2 and the epigenetic modifiers ING5, SMARCA5, BRD1, UHRF1, BPTF, and SMARCC2. ZMAT2 is an interactor of the pre-spliceosome that is required to keep cells in an undifferentiated, proliferative state. RNA immunoprecipitation and transcriptome-wide RNA splicing analysis showed that ZMAT2 associates with and regulates transcripts involved in cell adhesion in conjunction with ING5. Thus, joint control by splicing regulation, histone, and DNA modification is important to maintain epidermal cells in an undifferentiated state. As our understanding of gene expression regulation improves, so does our awareness of its complex dynamics and timing. The transcription machinery and its co-factors, chromatin state, RNA splicing, and microRNAs (miRNAs) are only some of the myriad of processes contributing to the versatile functions of a cell. Regulation of gene expression is of particular importance for governing the delicate balance between proliferation and differentiation in stratified epithelial tissues such as the skin, breast, or intestine. The high renewal rate in these tissues requires tight regulation of gene expression programs to avoid the generation of aberrantly behaving cells giving rise to diseases. Here, we use primary human keratinocytes as a model to study the regulation of gene expression programs governing epidermal proliferation and differentiation. The epidermal layer of the skin is completely replenished each month in a process driven by epidermal stem cells (keratinocytes), which reside attached on the basal membrane. Upon initiation of differentiation, these cells stop proliferating, release their integrin anchors, and move through the different layers of the skin, traveling upward through the spinous and the granular layers (Moreno-Layseca and Streuli, 2014Moreno-Layseca P. Streuli C.H. Signalling pathways linking integrins with cell cycle progression.Matrix Biol. 2014; 34: 144-153Crossref PubMed Scopus (185) Google Scholar). Eventually, they end up denucleated and heavily interconnected in the cornified layer, where they are shed from the surface. This process is marked by, among others, downregulation of integrins and upregulation of differentiation genes such as envoplakin (ENV), periplakin (PPL) (Ruhrberg et al., 1997Ruhrberg C. Hajibagheri M.A. Parry D.A. Watt F.M. Periplakin, a novel component of cornified envelopes and desmosomes that belongs to the plakin family and forms complexes with envoplakin.J Cell Biol. 1997; 139: 1835-1849Crossref PubMed Scopus (177) Google Scholar), involucrin (INV), and transglutaminase 1 (TGM1) (Eckert et al., 2005Eckert R.L. Sturniolo M.T. Broome A.M. Ruse M. Rorke E.A. Transglutaminase function in epidermis.J. Invest. Dermatol. 2005; 124: 481-492Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Throughout this transition they continuously change and fine-tune their expression programs using transcriptional and post-transcriptional processes in a differentiation state-dependent manner. These transitions in gene expression programs are controlled by regulatory mechanisms provided by epigenetic factors, transcription factors, and post-transcriptional processes such as splicing. Epigenetic factors regulate chromatin accessibility through remodeling (BPTF, SMARCA5) or by adding or removing histone or DNA modifications (ING5, UHRF1) (Mulder et al., 2012Mulder K.W. Wang X. Escriu C. Ito Y. Schwarz R.F. Gillis J. Sirokmány G. Donati G. Uribe-Lewis S. Pavlidis P. et al.Diverse epigenetic strategies interact to control epidermal differentiation.Nat. Cell Biol. 2012; 14: 753-763Crossref PubMed Scopus (115) Google Scholar). Open chromatin structures allow for transcription factors to bind their motifs and enable activation of gene expression programs. There are several transcription factors that have a known role in keratinocyte biology (AP1, ETS family [Eckert et al., 1997Eckert R.L. Crish J.F. Banks E.B. Welter J.F. The epidermis: genes on – genes off.J.Invest. Dermatol. 1997; 109: 501-509Abstract Full Text PDF PubMed Scopus (175) Google Scholar, Nagarajan et al., 2010Nagarajan P. Chin S.S. Wang D. Liu S. Sinha S. Garrett-Sinha L.A. Ets1 blocks terminal differentiation of keratinocytes and induces expression of matrix metalloproteases and innate immune mediators.J. Cell Sci. 2010; 123: 3566-3575Crossref PubMed Scopus (26) Google Scholar]) to regulate the expression of differentiation markers (e.g., IRF6 [Biggs et al., 2012Biggs L.C. Rhea L. Schutte B.C. Dunnwald M. Interferon regulatory factor 6 is necessary, but not sufficient, for keratinocyte differentiation.J. Invest. Dermatol. 2012; 132: 50-58Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, Botti et al., 2011Botti E. Spallone G. Moretti F. Marinari B. Pinetti V. Galanti S. De Meo P.D. De Nicola F. Ganci F. Castrignanò T. et al.Developmental factor IRF6 exhibits tumor suppressor activity in squamous cell carcinomas.Proc. Natl. Acad. Sci. USA. 2011; 108: 13710-13715Crossref PubMed Scopus (110) Google Scholar] and MAFB [Miyai et al., 2016Miyai M. Hamada M. Moriguchi T. Hiruma J. Kamitani-Kawamoto A. Watanabe H. Hara-Chikuma M. Takahashi K. Takahashi S. Kataoka K. Transcription factor MafB coordinates epidermal keratinocyte differentiation.J. Invest. Dermatol. 2016; 136: 1848-1857Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]). The emerging RNA transcript is then found by RNA-binding proteins that edit and protect it on its way to being translated into a protein. One of the most important RNA editing processes is RNA splicing, in which introns are excised from the immature transcript, generating a mature RNA transcript. More extensive editing via alternative splicing may be important in the skin, as there are several genes that display isoform specific expression in different keratinocyte cell states. For instance, dermokine, a gene that is highly expressed in the granular layer of the epidermis, is heavily spliced, generating multiple isoforms with different functions (Naso et al., 2007Naso M.F. Liang B. Huang C.C. Song X.Y. Shahied-Arruda L. Belkowski S.M. D’Andrea M.R. Polkovitch D.A. Lawrence D.R. Griswold D.E. et al.Dermokine: an extensively differentially spliced gene expressed in epithelial cells.J. Invest. Dermatol. 2007; 127: 1622-1631Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Another example is desmoplakin (DSP), which has two major isoforms that seem to have distinct functions in controlling desmosomal adhesion in the skin (Cabral et al., 2012Cabral R.M. Tattersall D. Patel V. McPhail G.D. Hatzimasoura E. Abrams D.J. South A.P. Kelsell D.P. The DSPII splice variant is crucial for desmosome-mediated adhesion in HaCaT keratinocytes.J. Cell Sci. 2012; 125: 2853-2861Crossref PubMed Scopus (23) Google Scholar). These examples illustrate the importance of proper regulation of both transcriptional and post-transcriptional processes. However, our current understanding of how these processes coordinately govern epidermal biology is limited. Here, we investigated the role of 145 putative DNA and/or RNA binding factors in human epidermal differentiation using small interfering RNA (siRNA)-based perturbation screens. We identified the matrin type-2 zinc-finger protein ZMAT2 as being important to maintain cells in an undifferentiated state and as a splicing regulator of adhesion-related transcripts. Moreover, computational predictions and experimental validation uncovered a previously unappreciated connection between epigenetic and post-transcriptional control of keratinocyte differentiation. To investigate the role of putative nucleic acid-binding factors in the regulation of epidermal stem cell renewal and differentiation, we performed siRNA-based knockdown screens (Figure 1A). For this, we selected 145 genes based on their expression levels in keratinocytes (reads per kilobase million [RPKM] >5), differential expression during differentiation (Figures S1A and S1B) (Kouwenhoven et al., 2015Kouwenhoven E.N. Oti M. Niehues H. van Heeringen S.J. Schalkwijk J. Stunnenberg H.G. van Bokhoven H. Zhou H. Transcription factor p63 bookmarks and regulates dynamic enhancers during epidermal differentiation.EMBO Rep. 2015; 16: 863-878Crossref PubMed Scopus (95) Google Scholar), and DNA or RNA binding potential. To test the contribution of these genes to the process of differentiation, we silenced each gene using a pool of 3 independent siRNAs in triplicate and subsequently induced differentiation. Transfected cells were cultured in different conditions for 48 hr to induce differentiation with the EGF inhibitor AG1478, BMP 2/7, a combination of these compounds, 10% fetal bovine serum, and a vehicle control, as previously described (Mulder et al., 2012Mulder K.W. Wang X. Escriu C. Ito Y. Schwarz R.F. Gillis J. Sirokmány G. Donati G. Uribe-Lewis S. Pavlidis P. et al.Diverse epigenetic strategies interact to control epidermal differentiation.Nat. Cell Biol. 2012; 14: 753-763Crossref PubMed Scopus (115) Google Scholar). These treatments led to the robust induction of terminal differentiation marker TGM1 (Mulder et al., 2012Mulder K.W. Wang X. Escriu C. Ito Y. Schwarz R.F. Gillis J. Sirokmány G. Donati G. Uribe-Lewis S. Pavlidis P. et al.Diverse epigenetic strategies interact to control epidermal differentiation.Nat. Cell Biol. 2012; 14: 753-763Crossref PubMed Scopus (115) Google Scholar). Endogenous TGM1 protein was quantified using a fluorescent In-Cell Western assay with an antibody (BC.1), whose specificity was confirmed using 2 independent siRNAs (Figure S1C). To account for differences in cell number among the knockdown populations in the screen, TGM1 measurements were normalized on DNA content (DRAQ5 signal) for each well. After Z score transformation, the results were compiled into a single dataset covering all 145 genes, 5 conditions, and replicates (Data S1). High correlation between replicates highlighted the reproducibility of our findings and the quality of our dataset (Figure S1D). Using a random selection of 11 genes, we estimated the median knockdown efficiency to be 88% (range: 28%–93%) and the false-negative rate at <10% (Figure S1E). In addition, we performed deconvolution experiments in which the individual siRNAs were tested in parallel with the pool of 3 siRNAs that was used in the screen. This indicated that 71%–87% of the siRNA pools contained at least 2 siRNAs that recapitulated screen results (Figure S1F), which argues against widespread off-target effects. Our dataset constitutes a high-quality resource of nucleic acid binding factors to further characterize for their role in epidermal self-renewal and differentiation. From these data we identified putative nucleic acid binding factors that significantly affected TGM1 levels (Benjamini-Hochberg [BH] false discovery rate [FDR] p < 0.001) compared to the average across all siRNAs in the screen in any of the conditions (Data S1; Figures 1B–1F, blue datapoints). The normalized effect size per gene is plotted on the x axis, whereas the statistical significance is depicted on the y axis. These representations of the data highlight the factors that modulate differentiation in the top left and top right quadrants, respectively. Notably, the effects and significance of individual factors were condition dependent, indicating that not all identified factors play equivalent roles in the different conditions tested (Data S1; Figure S2A). This is represented by the partial correlation between the effect sizes across the conditions (Figure S2A, scatterplots in bottom half) and the significant differences when comparing different conditions (Figure S2A, volcano plots in top half). In total, our screens revealed 57 genes that display a significant effect in at least 1 of the conditions (Data S1), indicating that our experiments identified factors that have a potential regulatory role in keratinocyte differentiation. To explore these individual hits, we selected the top 5 most significant genes (Benjamini-Hochberg FDR p < 0.001) for each condition (Figure 1G). These represent 21 distinct nucleic acid binding factors, one-third of which (7) have previously been implicated in epidermal renewal and/or differentiation, confirming the validity of our approach (Nagarajan et al., 2010Nagarajan P. Chin S.S. Wang D. Liu S. Sinha S. Garrett-Sinha L.A. Ets1 blocks terminal differentiation of keratinocytes and induces expression of matrix metalloproteases and innate immune mediators.J. Cell Sci. 2010; 123: 3566-3575Crossref PubMed Scopus (26) Google Scholar, Botti et al., 2011Botti E. Spallone G. Moretti F. Marinari B. Pinetti V. Galanti S. De Meo P.D. De Nicola F. Ganci F. Castrignanò T. et al.Developmental factor IRF6 exhibits tumor suppressor activity in squamous cell carcinomas.Proc. Natl. Acad. Sci. USA. 2011; 108: 13710-13715Crossref PubMed Scopus (110) Google Scholar, Kolev et al., 2008Kolev V. Mandinova A. Guinea-Viniegra J. Hu B. Lefort K. Lambertini C. Neel V. Dummer R. Wagner E.F. Dotto G.P. EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.Nat. Cell Biol. 2008; 10: 902-911Crossref PubMed Scopus (161) Google Scholar, Tsuji et al., 2018Tsuji G. Ito T. Chiba T. Mitoma C. Nakahara T. Uchi H. Furue M. The role of the OVOL1-OVOL2 axis in normal and diseased human skin.J. Dermatol. Sci. 2018; 90: 227-231Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, van den Bogaard et al., 2015van den Bogaard E.H. Podolsky M.A. Smits J.P. Cui X. John C. Gowda K. Desai D. Amin S.G. Schalkwijk J. Perdew G.H. Glick A.B. Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation.J. Invest. Dermatol. 2015; 135: 1320-1328Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, Mehic et al., 2005Mehic D. Bakiri L. Ghannadan M. Wagner E.F. Tschachler E. Fos and jun proteins are specifically expressed during differentiation of human keratinocytes.J. Invest. Dermatol. 2005; 124: 212-220Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, Marthaler et al., 2017Marthaler A.M. Podgorska M. Feld P. Fingerle A. Knerr-Rupp K. Grässer F. Smola H. Roemer K. Ebert E. Kim Y.J. et al.Identification of C/EBPα as a novel target of the HPV8 E6 protein regulating miR-203 in human keratinocytes.PLoS Pathog. 2017; 13: e1006406Crossref Scopus (28) Google Scholar, Tribioli et al., 2002Tribioli C. Robledo R.F. Lufkin T. The murine fork head gene Foxn2 is expressed in craniofacial, limb, CNS and somitic tissues during embryogenesis.Mech. Dev. 2002; 118: 161-163Crossref Scopus (23) Google Scholar, Bourke et al., 2017Bourke L.M. Del Monte-Nieto G. Outhwaite J.E. Bharti V. Pollock P.M. Simmons D.G. Adam A. Hur S.S. Maghzal G.J. Whitelaw E. et al.Loss of Rearranged L-Myc Fusion (RLF) results in defects in heart development in the mouse.Differentiation. 2017; 94: 8-20Crossref Scopus (7) Google Scholar, Amendt et al., 2002Amendt C. Mann A. Schirmacher P. Blessing M. Resistance of keratinocytes to TGFbeta-mediated growth restriction and apoptosis induction accelerates re-epithelialization in skin wounds.J. Cell Sci. 2002; 115: 2189-2198PubMed Google Scholar, Albino et al., 2012Albino D. Longoni N. Curti L. Mello-Grand M. Pinton S. Civenni G. Thalmann G. D’Ambrosio G. Sarti M. Sessa F. et al.ESE3/EHF controls epithelial cell differentiation and its loss leads to prostate tumors with mesenchymal and stem-like features.Cancer Res. 2012; 72: 2889-2900Crossref PubMed Scopus (96) Google Scholar). We sought to experimentally verify that the TGM1 measurements in our screen represented bona fide differentiation and not solely regulation of TGM1 levels. To this end, we selected IRF6 and ETS1 as exemplars and used RT-qPCR as an alternative readout of the expression of differentiation and basal cell markers after siRNA-mediated silencing. IRF6 has been shown to be important for the expression of genes critical to epidermal differentiation (Botti et al., 2011Botti E. Spallone G. Moretti F. Marinari B. Pinetti V. Galanti S. De Meo P.D. De Nicola F. Ganci F. Castrignanò T. et al.Developmental factor IRF6 exhibits tumor suppressor activity in squamous cell carcinomas.Proc. Natl. Acad. Sci. USA. 2011; 108: 13710-13715Crossref PubMed Scopus (110) Google Scholar). In addition, ETS1 is involved in keeping the cells in an undifferentiated state by repressing genes involved in the formation of the cornified envelope (Nagarajan et al., 2010Nagarajan P. Chin S.S. Wang D. Liu S. Sinha S. Garrett-Sinha L.A. Ets1 blocks terminal differentiation of keratinocytes and induces expression of matrix metalloproteases and innate immune mediators.J. Cell Sci. 2010; 123: 3566-3575Crossref PubMed Scopus (26) Google Scholar). RT-qPCR analysis confirmed the effects of the knock down of these genes on TGM1 protein levels in our experiments (Figure S1G). Moreover, we found that silencing IRF6 resulted in lower expression of the differentiation markers PPL, ENV, and IVL, whereas ETS1 silencing resulted in induction of these differentiation markers and a reduction in the basal cell markers ITGA6 and ITGB1. This is in line with the literature in which IRF6 is thought to regulate differentiation and ETS1 is important in the self-renewing state. These results also confirm that the effects of silencing IRF6 or ETS1 in our screen reflect the cellular differentiation state and not merely deregulation of TGM1 expression. A powerful feature of the approach we took is that our siRNA-based screen setup allows us to combine the current results on 145 DNA and/or RNA binding factors with published data characterizing ∼330 epigenetic regulators (Mulder et al., 2012Mulder K.W. Wang X. Escriu C. Ito Y. Schwarz R.F. Gillis J. Sirokmány G. Donati G. Uribe-Lewis S. Pavlidis P. et al.Diverse epigenetic strategies interact to control epidermal differentiation.Nat. Cell Biol. 2012; 14: 753-763Crossref PubMed Scopus (115) Google Scholar). Moreover, it enables us to use a previously developed Bayesian statistical framework that reveals genes that functionally interact (Wang et al., 2012Wang X. Castro M.A. Mulder K.W. Markowetz F. Posterior association networks and functional modules inferred from rich phenotypes of gene perturbations.PLoS Comput. Biol. 2012; 8: e1002566Crossref PubMed Scopus (15) Google Scholar). This means identifying sets of genes that share functionality through the regulation of similar cell biological processes—in this case epidermal differentiation. Applying this statistical approach to a combined epigenetic and DNA and/or RNA binding factor dataset should therefore allow identification of functional interactions among these groups of genes, leading to insights into their joint regulation of epidermal biology. The data distribution and variation of the two datasets are highly comparable, allowing us to combine them for further analysis (Figures 1B–1F, boxplots, and S2B). This resulted in a rich dataset comprising 473 genes describing their effects on the expression of TGM1 in 5 conditions. Application of the Bayesian network algorithm to this joint dataset revealed strong predicted functional interactions between ZMAT2, a matrin-type 2 like zinc-finger with potential DNA or RNA binding capacity, and components of a previously identified network of epigenetic regulators involved in epidermal renewal (Mulder et al., 2012Mulder K.W. Wang X. Escriu C. Ito Y. Schwarz R.F. Gillis J. Sirokmány G. Donati G. Uribe-Lewis S. Pavlidis P. et al.Diverse epigenetic strategies interact to control epidermal differentiation.Nat. Cell Biol. 2012; 14: 753-763Crossref PubMed Scopus (115) Google Scholar) (Figures 2A and S3, full network). This subnetwork contained multiple members of different protein complexes representing diverse epigenetic mechanisms such as MORF complex members ING5 and BRD1, NURF complex members BPTF and SMARCA5, and SMARCC2 and UHRF1 (Mulder et al., 2012Mulder K.W. Wang X. Escriu C. Ito Y. Schwarz R.F. Gillis J. Sirokmány G. Donati G. Uribe-Lewis S. Pavlidis P. et al.Diverse epigenetic strategies interact to control epidermal differentiation.Nat. Cell Biol. 2012; 14: 753-763Crossref PubMed Scopus (115) Google Scholar). This implies that ZMAT2 plays a role in epidermal differentiation in conjunction with these epigenetic modifiers. These predicted functional interactions prompted us to functionally characterize ZMAT2 further. RT-qPCR analysis showed that silencing ZMAT2 in keratinocytes resulted in increased expression of differentiation markers (PPL, EVPL, INV, TGM1) and concordant downregulation of basal-cell markers ITGB1 and ITGA6 (Figure 2B). In addition, depletion of ZMAT2 resulted in a strong reduction in the number and size of clones in the colony formation assay (Figure 2C), reflecting a loss of cell renewal and proliferation capacity. These effects were not associated with increased cell death or the induction of apoptosis following ZMAT2 depletion (Figures S4A and S4B). Furthermore, cell-cycle analysis showed that there was no immediate defect in proliferation (72 hr post-siRNA transfection), indicating that the decrease in colony number and size in the colony formation assay (CFA) is caused by an effect on long-term proliferation (Figure S4C). This is also recapitulated in 3D organotypic cultures (Figure 2D), showing that knock down of ZMAT2 causes a marked decrease in the number of proliferative cells (Ki67+ cells quantified by immunostaining) and a disorganized epidermis compared to non-targeting control siRNA (siControl) (Figure 2E, 2–4 independent cultures with 2 independent siRNAs). These results indicated that ZMAT2 plays a role in maintaining epidermal cells in an undifferentiated, proliferative state and confirms our primary screen results. The Bayesian mixture model predicted that ZMAT2 functionally interacts with the epigenetic regulators ING5, SMARCA5, BPTF, UHRF1, and BRD1. We aimed to confirm these predicted interactions using a double knockdown strategy. In this way, true functional interactions can be identified (Mani et al., 2008Mani R. St Onge R.P. Hartman 4th, J.L. Giaever G. Roth F.P. Defining genetic interaction.Proc. Natl. Acad. Sci. USA. 2008; 105: 3461-3466Crossref PubMed Scopus (312) Google Scholar, Costanzo et al., 2010Costanzo M. Baryshnikova A. Bellay J. Kim Y. Spear E.D. Sevier C.S. Ding H. Koh J.L. Toufighi K. Mostafavi S. et al.The genetic landscape of a cell.Science. 2010; 327: 425-431Crossref PubMed Scopus (1622) Google Scholar, Horn et al., 2011Horn T. Sandmann T. Fischer B. Axelsson E. Huber W. Boutros M. Mapping of signaling networks through synthetic genetic interaction analysis by RNAi.Nat. Methods. 2011; 8: 341-346Crossref PubMed Scopus (148) Google Scholar, Roguev et al., 2013Roguev A. Talbot D. Negri G.L. Shales M. Cagney G. Bandyopadhyay S. Panning B. Krogan N.J. Quantitative genetic-interaction mapping in mammalian cells.Nat. Methods. 2013; 10: 432-437Crossref PubMed Scopus (88) Google Scholar) by comparing the quantitative effects of combinatorial knockdowns on global gene expression with effects that can be expected based on the individual knockdown (Horn et al., 2011Horn T. Sandmann T. Fischer B. Axelsson E. Huber W. Boutros M. Mapping of signaling networks through synthetic genetic interaction analysis by RNAi.Nat. Methods. 2011; 8: 341-346Crossref PubMed Scopus (148) Google Scholar). In cases in which 2 genes are functionally independent (i.e., do not functionally interact), the observed phenotype in the double knockdown is equivalent to the product of the individual knockdown phenotypes, or, when working with log-transformed values, their summed value (Mani et al., 2008Mani R. St Onge R.P. Hartman 4th, J.L. Giaever G. Roth F.P. Defining genetic interaction.Proc. Natl. Acad. Sci. USA. 2008; 105: 3461-3466Crossref PubMed Scopus (312) Google Scholar, Costanzo et al., 2010Costanzo M. Baryshnikova A. Bellay J. Kim Y. Spear E.D. Sevier C.S. Ding H. Koh J.L. Toufighi K. Mostafavi S. et al.The genetic landscape of a cell.Science. 2010; 327: 425-431Crossref PubMed Scopus (1622) Google Scholar, Horn et al., 2011Horn T. Sandmann T. Fischer B. Axelsson E. Huber W. Boutros M. Mapping of signaling networks through synthetic genetic interaction analysis by RNAi.Nat. Methods. 2011; 8: 341-346Crossref PubMed Scopus (148) Google Scholar, Roguev et al., 2013Roguev A. Talbot D. Negri G.L. Shales M. Cagney G. Bandyopadhyay S. Panning B. Krogan N.J. Quantitative genetic-interaction mapping in mammalian cells.Nat. Methods. 2013; 10: 432-437Crossref PubMed Scopus (88) Google Scholar). Using this “product rule” as the null hypothesis (labeled “exp” for expected in Figure 3A), genetic interactions are statistically defined as aggravating (when the observed phenotype is greater than expected) or alleviating (when the observed phenotype of the combined knockdown is less pronounced than expected), respectively (Figure 3A). In general, alleviating interactions occur between genes involved in the same process and/or pathway, whereas aggravating interactions tend to be associated with functionally redundant processes (Costanzo et al., 2010Costanzo M. Baryshnikova A. Bellay J. Kim Y. Spear E.D. Sevier C.S. Ding H. Koh J.L. Toufighi K. Mostafavi S. et al.The genetic landscape of a cell.Science. 2010; 327: 425-431Crossref PubMed Scopus (1622) Google Scholar, Roguev et al., 2013Roguev A. Talbot D. Negri G.L. Shales M. Cagney G. Bandyopadhyay S. Panning B. Krogan N.J. Quantitative genetic-interaction mapping in mammalian cells.Nat. Methods. 2013; 10: 432-437Crossref PubMed Scopus (88) Google Scholar). In previous work, we showed that the epigenetic regulators within the predicted subnetwork (Figure 2A) display true functional interactions (Mulder et al., 2012Mulder K.W. Wang X. Escriu C. Ito Y. Schwarz R.F. Gillis J. Sirokmány G. Donati G. Uribe-Lewis S. Pavlidis P. et al.Diverse epigenetic strategies interact to control epidermal differentiation.Nat. Cell Biol. 2012; 14: 753-763Crossref PubMed Scopus (115) Google Scholar). Therefore, we decided to experimentally test the predicted interactions between ZMAT2 and ING5, SMARCA5, BPTF, UHRF1, or BRD1 using a combined knockdown approach. As a control, we included EZH2, which is only peripherally associated with the epigenetic factors in this network (Mulder et al., 2012Mulder K.W. Wang X. Escriu C. Ito Y. Schwarz R.F. Gillis J. Sirokmány G. Donati G. Uribe-Lewis S. Pavlidis P. et al.Diverse epigenetic strategies interact to control epidermal differentiation.Nat. Cell Biol. 2012; 14: 753-763Crossref PubMed Scopus (115) Google Scholar). To obtain a detailed quantitative phenotype representing the cell state, we performed RNA-sequencing expression profiling after silencing each of these epigenetic factors individually, as well as in combination with ZMAT2. Two independent siRNAs targeting each gene were used in all of the possible permutations in triplicate. We analyzed a total of 156 knockdown samples using a modified CEL-seq2 method that enables high-throughput 3′ end tag-counting RNA-sequencing (see STAR Methods for details). It is important to note that the relatively shallow RNA-sequencing we performed does not allow us to directly interpret the molecular mechanisms underlying these interactions, but it is powerful for the discovery of functional interactions based on the obtained RNA expression profiles. We examined the data using DESeq 2.0 and identified transcripts that were significantly differentially expressed (p < 0.05) compared to control siRNA-transfected cells. After quality control and filtering (see STAR Methods), we labeled an interaction alleviating" @default.
• W2899056105 created "2018-11-09" @default.
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• W2899056105 date "2018-10-01" @default.
• W2899056105 modified "2023-10-12" @default.
• W2899056105 title "Splicing and Chromatin Factors Jointly Regulate Epidermal Differentiation" @default.
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