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• W2090185129 abstract "•A single factor, C-MYC, induces self-renewal in SOX2-expressing otic progenitors•C-MYC transcriptionally amplifies SOX2 target genes•SOX2 modulates transcription of cell-cycle genes•Immortalized multipotent otic progenitors can differentiate into otic cell types Sensorineural hearing loss is caused by the loss of sensory hair cells and neurons of the inner ear. Once lost, these cell types are not replaced. Two genes expressed in the developing inner ear are c-Myc and Sox2. We created immortalized multipotent otic progenitor (iMOP) cells, a fate-restricted cell type, by transient expression of C-MYC in SOX2-expressing otic progenitor cells. This activated the endogenous C-MYC and amplified existing SOX2-dependent transcripts to promote self-renewal. RNA-seq and ChIP-seq analyses revealed that C-MYC and SOX2 occupy over 85% of the same promoters. C-MYC and SOX2 target genes include cyclin-dependent kinases that regulate cell-cycle progression. iMOP cells continually divide but retain the ability to differentiate into functional hair cells and neurons. We propose that SOX2 and C-MYC regulate cell-cycle progression of these cells and that downregulation of C-MYC expression after growth factor withdrawal serves as a molecular switch for differentiation. Sensorineural hearing loss is caused by the loss of sensory hair cells and neurons of the inner ear. Once lost, these cell types are not replaced. Two genes expressed in the developing inner ear are c-Myc and Sox2. We created immortalized multipotent otic progenitor (iMOP) cells, a fate-restricted cell type, by transient expression of C-MYC in SOX2-expressing otic progenitor cells. This activated the endogenous C-MYC and amplified existing SOX2-dependent transcripts to promote self-renewal. RNA-seq and ChIP-seq analyses revealed that C-MYC and SOX2 occupy over 85% of the same promoters. C-MYC and SOX2 target genes include cyclin-dependent kinases that regulate cell-cycle progression. iMOP cells continually divide but retain the ability to differentiate into functional hair cells and neurons. We propose that SOX2 and C-MYC regulate cell-cycle progression of these cells and that downregulation of C-MYC expression after growth factor withdrawal serves as a molecular switch for differentiation. The six sensory organs of the inner ear—the cochlea, utricle, saccule, and three semicircular canals—mediate our ability to hear and balance. Within these organs, sensory hair cells mediate the conversion from mechanical to neural signals, releasing neurotransmitter onto neurons of the eighth nerve. Built for exquisite sensitivity, hair cells have a high metabolic demand and delicate mechanosensory hair bundles. A variety of insults, such as loud noises and ototoxic drugs, can cause hair cell death. They can also cause acute loss of afferent nerve terminals and delayed degeneration of the auditory nerve (Kujawa and Liberman, 2009Kujawa S.G. Liberman M.C. Adding insult to injury: cochlear nerve degeneration after “temporary” noise-induced hearing loss.J. Neurosci. 2009; 29: 14077-14085Crossref PubMed Scopus (1628) Google Scholar). Degeneration of hair cells and neurons significantly contributes to hearing loss, as these cells are not replaced. To regenerate auditory hair cells and neurons, we must understand how progenitor cells give rise to these cell types. Sox2 and the Myc family of transcription factors are crucial for the proper development of inner ear hair cells and neurons. Human mutations in Sox2 cause anophthalmia, a severe eye malformation, and bilateral sensorineural hearing loss (Fantes et al., 2003Fantes J. Ragge N.K. Lynch S.A. McGill N.I. Collin J.R. Howard-Peebles P.N. Hayward C. Vivian A.J. Williamson K. van Heyningen V. FitzPatrick D.R. Mutations in SOX2 cause anophthalmia.Nat. Genet. 2003; 33: 461-463Crossref PubMed Scopus (435) Google Scholar, Hagstrom et al., 2005Hagstrom S.A. Pauer G.J. Reid J. Simpson E. Crowe S. Maumenee I.H. Traboulsi E.I. SOX2 mutation causes anophthalmia, hearing loss, and brain anomalies.Am. J. Med. Genet. A. 2005; 138A: 95-98Crossref PubMed Scopus (100) Google Scholar). Mouse mutants that express low levels of SOX2 in the inner ear have fewer cochlear hair cells and neurons (Kiernan et al., 2005Kiernan A.E. Pelling A.L. Leung K.K. Tang A.S. Bell D.M. Tease C. Lovell-Badge R. Steel K.P. Cheah K.S. Sox2 is required for sensory organ development in the mammalian inner ear.Nature. 2005; 434: 1031-1035Crossref PubMed Scopus (419) Google Scholar, Puligilla et al., 2010Puligilla C. Dabdoub A. Brenowitz S.D. Kelley M.W. Sox2 induces neuronal formation in the developing mammalian cochlea.J. Neurosci. 2010; 30: 714-722Crossref PubMed Scopus (103) Google Scholar). Myc genes, including c-Myc and N-Myc, are expressed in the inner ear (Domínguez-Frutos et al., 2011Domínguez-Frutos E. López-Hernández I. Vendrell V. Neves J. Gallozzi M. Gutsche K. Quintana L. Sharpe J. Knoepfler P.S. Eisenman R.N. et al.N-myc controls proliferation, morphogenesis, and patterning of the inner ear.J. Neurosci. 2011; 31: 7178-7189Crossref PubMed Scopus (43) Google Scholar, Kopecky et al., 2011Kopecky B. Santi P. Johnson S. Schmitz H. Fritzsch B. Conditional deletion of N-Myc disrupts neurosensory and non-sensory development of the ear.Dev. Dyn. 2011; 240: 1373-1390Crossref PubMed Scopus (58) Google Scholar). Deletion of N-Myc in the developing inner ear causes reduced proliferative growth, and abnormal morphology and differentiation of both sensory and nonsensory cells (Domínguez-Frutos et al., 2011Domínguez-Frutos E. López-Hernández I. Vendrell V. Neves J. Gallozzi M. Gutsche K. Quintana L. Sharpe J. Knoepfler P.S. Eisenman R.N. et al.N-myc controls proliferation, morphogenesis, and patterning of the inner ear.J. Neurosci. 2011; 31: 7178-7189Crossref PubMed Scopus (43) Google Scholar, Kopecky et al., 2011Kopecky B. Santi P. Johnson S. Schmitz H. Fritzsch B. Conditional deletion of N-Myc disrupts neurosensory and non-sensory development of the ear.Dev. Dyn. 2011; 240: 1373-1390Crossref PubMed Scopus (58) Google Scholar). Studies aimed at producing new hair cells and otic neurons have used embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). iPSCs are generated by converting somatic cells into pluripotent stem cells that possess properties of both self-renewal and pluripotency (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (19304) Google Scholar). This involves transient expression of c-Myc, Sox2, Klf4, and Oct4 to activate expression of the endogenous factors. The endogenous factors function to promote self-renewal, maintain pluripotency, and prevent differentiation. Among the four transcription factors used to generate iPSCs, C-MYC and SOX2 have been implicated in maintaining self-renewal in ESCs (Cartwright et al., 2005Cartwright P. McLean C. Sheppard A. Rivett D. Jones K. Dalton S. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism.Development. 2005; 132: 885-896Crossref PubMed Scopus (599) Google Scholar). Sox2 is also essential for maintaining multipotency in neural stem cells (Suh et al., 2007Suh H. Consiglio A. Ray J. Sawai T. D’Amour K.A. Gage F.H. In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the adult hippocampus.Cell Stem Cell. 2007; 1: 515-528Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar), and knockout or knockdown of Sox2 in ESCs results in differentiation (Ivanova et al., 2006Ivanova N. Dobrin R. Lu R. Kotenko I. Levorse J. DeCoste C. Schafer X. Lun Y. Lemischka I.R. Dissecting self-renewal in stem cells with RNA interference.Nature. 2006; 442: 533-538Crossref PubMed Scopus (806) Google Scholar). Although c-Myc is dispensable for direct reprogramming of somatic cells into pluripotent cells, inclusion of c-Myc increases the number of reprogrammed cells and accelerates the formation of iPSCs (Wernig et al., 2008Wernig M. Meissner A. Cassady J.P. Jaenisch R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts.Cell Stem Cell. 2008; 2: 10-12Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Recent genome-wide binding studies implicated C-MYC as a global transcription amplifier (Lin et al., 2012Lin C.Y. Lovén J. Rahl P.B. Paranal R.M. Burge C.B. Bradner J.E. Lee T.I. Young R.A. Transcriptional amplification in tumor cells with elevated c-Myc.Cell. 2012; 151: 56-67Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar, Nie et al., 2012Nie Z. Hu G. Wei G. Cui K. Yamane A. Resch W. Wang R. Green D.R. Tessarollo L. Casellas R. et al.c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells.Cell. 2012; 151: 68-79Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar), providing an elegant explanation of the diverse roles of C-MYC in reprogramming and in various cellular functions. We exploited C-MYC to activate the endogenous c-Myc gene and enhance gene expression in neurosensory cell types. By doing so, we derived a self-renewing immortalized multipotent otic progenitor (iMOP) line from SOX2-expressing neurosensory precursors of the inner ear. We show that the endogenous C-MYC binds to most of the same promoters as SOX2 and amplifies transcripts that promote cell-cycle progression. This enhanced expression contributes to self-renewal but allows iMOP cells to retain their capacity to differentiate into hair cells, supporting cells and neurons. During embryonic development of the murine cochlea, progenitors begin exiting the cell cycle at embryonic day 12.5 (E12.5). Terminal mitosis spreads in a wave-like manner from the apex to the base of the cochlea, completing cell-cycle exit by E14.5 (Lee et al., 2006Lee Y.S. Liu F. Segil N. A morphogenetic wave of p27Kip1 transcription directs cell cycle exit during organ of Corti development.Development. 2006; 133: 2817-2826Crossref PubMed Scopus (162) Google Scholar, Ruben, 1967Ruben R.J. Development of the inner ear of the mouse: a radioautographic study of terminal mitoses.Acta Otolaryngol. 1967; 220: 1-44Google Scholar). Progenitors stop dividing and express the cell-cycle inhibitor Cdkn1b (p27KIP) at E14.5 to initiate differentiation (Chen and Segil, 1999Chen P. Segil N. p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti.Development. 1999; 126: 1581-1590Crossref PubMed Google Scholar). We obtained and dissociated cochleas from E11.5–12.5 embryos into single cells. Dissociated cells were cultured in defined medium supplemented with basic fibroblast growth factor (bFGF). Cells were plated on untreated tissue culture dishes to produce both adherent cells and colony-forming cells (Figure S1A available online). Colony-forming otic cells (known as otospheres) were enriched by gently superfusing the cultures and collecting the suspension of otospheres (Figure S1B). In 50 otospheres examined, ∼60% of cells expressed detectable levels of SOX2, and in culture they incorporated the nucleotide analog 5-ethynyl-2′-deoxyuridine (EdU), indicating they were dividing (Figure S1C). Cells from postnatal cochleas can form three types of otospheres: solid, transitional, and hollow (Diensthuber et al., 2009Diensthuber M. Oshima K. Heller S. Stem/progenitor cells derived from the cochlear sensory epithelium give rise to spheres with distinct morphologies and features.J. Assoc. Res. Otolaryngol. 2009; 10: 173-190Crossref PubMed Scopus (56) Google Scholar). Otospheres from postnatal vestibular and auditory organs contain dividing cells that can become neurons and sensory cells (Oshima et al., 2007Oshima K. Grimm C.M. Corrales C.E. Senn P. Martinez Monedero R. Géléoc G.S. Edge A. Holt J.R. Heller S. Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.J. Assoc. Res. Otolaryngol. 2007; 8: 18-31Crossref PubMed Scopus (258) Google Scholar). To identify spheres derived from embryonic cochlea, we dissociated cochleas and cultured cells for 3–5 days until otospheres were observed. Primary otospheres were fixed, embedded in plastic, serially sectioned, and observed by transmission electron microscopy (TEM). In 100 otospheres examined, we found cells in all sections, suggesting that embryonic primary otospheres are similar to solid spheres from postnatal inner ear organs (Figure S1D). We next sought a way to promote long-term self-renewal, and asked whether a single gene, c-Myc, could amplify the underlying gene-expression profile to promote self-renewal. We used a retrovirus to introduce exogenous C-MYC into SOX2-expressing neurosensory precursors and assessed activation of endogenous C-MYC in these cells. Primers were designed to detect and distinguish total c-Myc, endogenous c-Myc, and transgenic c-Myc transcripts. As controls, we used ESCs cultured under normal conditions and in otic progenitor media used for culturing SOX2-expressing otospheres to detect all four transcription factors that induce pluripotency. We found that expression of the four transcription factors was not altered in ESCs. In progenitor cells, Oct4, a crucial factor for pluripotent ESCs and iPSCs (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (19304) Google Scholar), was not detected, whereas Sox2 was detected in all samples. This suggests that the iMOP cells, unlike iPSCs, are not pluripotent but are fate restricted. Viral c-Myc was transiently upregulated along with Klf4 2 days after infection but decreased after the cells were cultured for 2 weeks. Endogenous c-Myc and total c-Myc were present in iMOP cells and did not show a large upregulation in transcript levels even after integration of the c-Myc retrovirus (Figure 1A). To determine the contribution of endogenous and viral c-Myc to total c-Myc levels, we performed quantitative RT-PCR (qPCR) and normalized the transcript levels to total c-Myc levels. At 2 days postinfection, endogenous and viral c-Myc represented 37.6% and 62.4% of total c-Myc, respectively. At 2 weeks postinfection, viral c-Myc was 1.4% of total, indicating that the c-Myc retrovirus had been silenced (Figure 1B), similar to what was previously observed in factor-based reprogramming of iPSCs (Hotta and Ellis, 2008Hotta A. Ellis J. Retroviral vector silencing during iPS cell induction: an epigenetic beacon that signals distinct pluripotent states.J. Cell. Biochem. 2008; 105: 940-948Crossref PubMed Scopus (123) Google Scholar). To compare c-Myc transcript levels with endogenous levels in the inner ear, we performed qPCR and normalized the transcript levels to E12.5 cochleas. ESCs and c-Myc-infected-progenitor cells showed increased c-Myc transcript compared with uninfected progenitor cells (Figure S1E). To select for dividing cells that still maintained their otic identity, we cultured cells in defined medium with bFGF. bFGF promotes the proliferation of inner ear epithelia cultures (Zheng et al., 1997Zheng J.L. Helbig C. Gao W.Q. Induction of cell proliferation by fibroblast and insulin-like growth factors in pure rat inner ear epithelial cell cultures.J. Neurosci. 1997; 17: 216-226Crossref PubMed Google Scholar) and also induces otic cell identity (Groves and Bronner-Fraser, 2000Groves A.K. Bronner-Fraser M. Competence, specification and commitment in otic placode induction.Development. 2000; 127: 3489-3499Crossref PubMed Google Scholar). Mesenchymal and pluripotent stem cells that were previously used to generate hair cells were also treated with bFGF to propagate cultures and induce otic cell fate (Hu and Corwin, 2007Hu Z. Corwin J.T. Inner ear hair cells produced in vitro by a mesenchymal-to-epithelial transition.Proc. Natl. Acad. Sci. USA. 2007; 104: 16675-16680Crossref PubMed Scopus (45) Google Scholar, Oshima et al., 2010Oshima K. Shin K. Diensthuber M. Peng A.W. Ricci A.J. Heller S. Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells.Cell. 2010; 141: 704-716Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, Koehler et al., 2013Koehler K.R. Mikosz A.M. Molosh A.I. Patel D. Hashino E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture.Nature. 2013; 500: 217-221Crossref PubMed Scopus (283) Google Scholar). We continuously expanded the cells in bFGF medium for up to 36 months to maintain otospheres. Otosphere clones derived from single cells were compared with ESCs (Figures S2A and S2B). To ensure that these were proliferative proneurosensory cells that retained a restricted identity, we assessed the expression of endogenous alkaline phosphatase, a marker for pluripotent cells (Stadtfeld and Hochedlinger, 2010Stadtfeld M. Hochedlinger K. Induced pluripotency: history, mechanisms, and applications.Genes Dev. 2010; 24: 2239-2263Crossref PubMed Scopus (604) Google Scholar). Unlike pluripotent ESCs, progenitor cells derived from otospheres did not express endogenous alkaline phosphatase and were unlikely to be pluripotent (Figures S2C and S2D). To determine the expression level of proneurosensory markers relative to the inner ear, we performed qPCR for Sox2, Pax2, and Isl1 using progenitors and c-Myc-infected progenitor cells. Transcript levels were normalized to E12.5 embryonic cochlea. Both cell types retained expression of all three proneurosensory markers (Figure S2E). We thus named the c-Myc-infected progenitor cells immortalized multipotent otic progenitor (iMOP) cells. To determine the proliferative capacity of iMOP cells after transient C-MYC expression, we clonally derived and expanded three primary otospheres and three iMOP cell lines. Cells (104) were passaged and the cumulative cell counts were tabulated weekly. Primary cells from otospheres initially expanded exponentially (Figure S3), but stopped dividing after 5 weeks in culture, whereas iMOP cells continued to divide at a much faster rate during 10 weeks in culture (Figure 1C), doubling in ∼18 hr at a rate similar to that observed for ESCs. Many immortalized cell types, such as ESCs and iPSCs, acquire chromosomal abnormalities in continuous culture. The karyotype of ESCs is well known to be metastable, with 50%–60% of cells showing a normal karyotype and a high degree of aneuploidy (Rebuzzini et al., 2008Rebuzzini P. Neri T. Zuccotti M. Redi C.A. Garagna S. Chromosome number variation in three mouse embryonic stem cell lines during culture.Cytotechnology. 2008; 58: 17-23Crossref PubMed Scopus (16) Google Scholar). For cells in culture, aneuploidy presumably would be disadvantageous and the cells might not continue growing. We examined one of the clonal iMOP cell lines for chromosomal stability. It displayed a normal karyotype with 19 pairs of autosomes and an XY chromosome (Figure 1D) and had a distribution of chromosome numbers similar to that found for ESCs (n = 40) (Figure S4). Thus, iMOP cells showed a genomic stability similar to that of pluripotent ESCs. To determine whether the cells still retained transcripts for otic neurosensory markers, we conducted qPCR. The proneurosensory transcripts Sox2, Pax2, and Isl1 were enriched relative to pluripotent ESCs (by 31.5%, 79.4%, and 96.0%, respectively), whereas the negative control Isl2, a marker for differentiating sensory and nonsensory cells of the inner ear (Huang et al., 2008Huang M. Sage C. Li H. Xiang M. Heller S. Chen Z.Y. Diverse expression patterns of LIM-homeodomain transcription factors (LIM-HDs) in mammalian inner ear development.Dev. Dyn. 2008; 237: 3305-3312Crossref PubMed Scopus (32) Google Scholar), showed no significant changes in transcript levels (<1%; Figure 1E). Infection of otic progenitors with a c-Myc retrovirus apparently activates the endogenous c-Myc before silencing itself, and allows for prolonged proliferation of cells that retain otic neurosensory transcripts. Because the endogenous c-Myc transcript accounts for most (98.6%) of the total c-Myc transcript (Figure 1B), we asked whether endogenous C-MYC and SOX2 are responsible for self-renewal in iMOP cells. At early stages of otic development, SOX2 could maintain or establish neurosensory cell fate and promote proliferation, while C-MYC transcriptionally amplified SOX2 target genes. To identify their genome-wide targets in iMOP cells, we used chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies against C-MYC or SOX2. In parallel, we defined promoter regions around known transcriptional start sites (TSSs) using reads obtained from RNA polymerase II (POLII) ChIP-seq in iMOP cells. Binding sites of SOX2 and endogenous C-MYC at promoter regions were determined by enrichment of sequences within the ±5 kb region of the TSS. Genes with both RNA POLII and the transcription factors bound in the promoter region were considered target genes. Mapping the overlapping RNA POLII-, C-MYC-, and SOX2-binding sites on the c-Myc and Sox2 genes, we observed three RNA POLII peaks for the c-Myc gene (Figure 2A, arrowheads). These correspond to the promoter regions of three known c-Myc splice variants. In the promoters of the c-Myc gene, both C-MYC and SOX2 were bound. For the Sox2 gene, RNA POLII occupied a broad peak in the promoter. C-MYC and SOX2 both occupied the same promoter region on the Sox2 gene (Figure 2A). These results suggest that C-MYC and SOX2 occupy each other’s promoter regions and may auto- and cross-regulate RNA-POLII-dependent transcription at each other’s promoter. To identify all the genes regulated by C-MYC and SOX2 in iMOP cells, we defined SOX2 and C-MYC target genes based on binding of RNA POLII and the two transcription factors in the promoter region. A total of 4,994 target genes were identified as direct targets of SOX2 while 5,422 genes were direct targets of C-MYC (Table S1). By comparing the overlap of promoter binding regions, we found that 4,231 genes or ∼85% of SOX2 target gene promoters were also occupied by C-MYC (Figure 2B). We predicted that by amplifying the existing SOX2 transcriptional program, C-MYC helps retain cellular processes attributed to SOX2 in iMOP cells. To determine how much iMOPs and primary otospheres differ, we performed a hierarchical clustering analysis on all detectable transcripts from RNA sequencing (RNA-seq) samples obtained from ESCs, on two independently derived iMOP cell lines, and on three independently derived otospheres (Figure 2C). Based on gene expression, otospheres and iMOP cells cluster together rather than with ESCs. RNA-seq samples from otospheres and iMOP cells showed a high Spearman’s rank correlation coefficient of ρ > 0.8 (where 1.0 suggests perfect correlation). At the level of the transcriptome, iMOP cells appear similar to cells from primary otospheres and only a subset of transcripts are differentially expressed. To determine whether C-MYC occupies the promoter and enhancer regions near the TSS, we assessed binding sites of RNA POLII and C-MYC by mapping the quantile normalized RNA POLII ChIP-seq density ±5 kb around the TSS. The majority of C-MYC binding was within 1 kb of the TSS and had a similar distribution to RNA POLII (Figure 2D). This suggests that C-MYC binds in the promoter proximal sites near the TSS, genome wide. To see whether C-MYC transcriptionally amplifies the SOX2 target genes in iMOP cells relative to primary otospheres, we selected normalized reads from the 4,231 genes that were both C-MYC and SOX2 targets. We ranked and compared individual target genes from two iMOP cell lines and three primary otospheres. To display the relative changes for each gene, we normalized reads from C-MYC and SOX2 target genes by subtracting the median read from each gene and dividing by the median absolute deviation. These reads were compiled on a heatmap, which revealed that transcripts from C-MYC and SOX2 target genes displayed a graded degree of amplification in iMOP cells (shown in red) relative to primary otospheres (Figure 2E). To understand the distribution and extent of the transcript increase in iMOPs compared with otospheres, we plotted the cumulative distribution of reads per kilobase per million (RPKM) from individual genes. A nonlinear, sigmoidal distribution of transcripts was observed in primary otospheres. A similar distribution was also observed in iMOP cells, except that the global distribution of transcripts in iMOP cells was shifted to the right (p < 1.5 × 10−9 in Welch’s two-tailed t test; Figure 2F). The global increase in transcripts of C-MYC and SOX2 targets in iMOP cells relative to primary otospheres is consistent with the universal and nonlinear amplification by C-MYC of this subset of actively transcribed genes. To determine the consequences of amplification of this subset of genes, we wished to identify all the genetic factors attributed to self-renewal, including genes both directly and indirectly affected by C-MYC. We performed RNA-seq on proliferating iMOP cells and primary otospheres cultured with bFGF. To identify these transcripts, we compared ESC, iMOP, and otosphere samples. Gene expression in iMOP cells was much more similar to that in cells derived from otospheres than to ESCs (Figure 3A). We performed a pairwise comparison between iMOP cells and otosphere samples and identified transcripts that were significantly different between iMOP and otosphere samples (p < 0.05). Reads from individual genes were plotted on a heatmap to show the relative changes between iMOP cells and otospheres (Figure 3B). The upper and lower portions of the heatmap showed highly upregulated and downregulated genes. We observed an ∼34-fold increase in c-Myc (p < 10−154) and an ∼5,700-fold increase in Sox2 (p < 10−180) in iMOP cells relative to otospheres. Selected upregulated genes in iMOP cells (labeled) were direct targets of both C-MYC and SOX2 as determined by ChIP-seq. To determine all of the genes that promote self-renewal in iMOP cells, we identified all of the differentially expressed genes and categorized them based on Gene Ontology. Significant functional groups included genes for DNA replication, cell cycle, mitosis, and cell proliferation (Figure 3C). One of these genes was Wdr5, a WD-repeat-containing protein that is essential for histone H3K4 methylation and mediates self-renewal in ESCs (Ang et al., 2011Ang Y.S. Tsai S.Y. Lee D.F. Monk J. Su J. Ratnakumar K. Ding J. Ge Y. Darr H. Chang B. et al.Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network.Cell. 2011; 145: 183-197Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Wdr5 showed an ∼5.9-fold increase (p < 10−5) in iMOP cells compared with otospheres. Many cyclin-dependent kinases were also identified. Cdk1 (2.3-fold increase; p < 10−21) is part of a highly conserved cyclin-dependent protein-kinase complex that is essential for G1/S and G2/M phase transitions of the eukaryotic cell cycle. Cdk2 (3.7-fold increase; p < 10−34) is another cyclin-dependent kinase that allows the G1/S transition. Cdk4 (2.1-fold; p < 10−43) promotes progression through the G1 phase of the cell cycle. Other genes, such as Cdc7 (10-fold; p < 10−65), Mcm2 (4.1-fold; p < 10−58), Cdt1 (4.4-fold; p < 10−50), and Skp2 (1.7-fold; p < 10−8), regulate the initiation of DNA replication. Many of the downregulated genes function to inhibit cell-cycle progression. We observed a decrease in the cyclin-dependent kinase inhibitor Cdkn1a (p21CIP) (−41-fold; p = 0). Similarly, both Lats1 (−2.2-fold; p < 10−31) and Lats2 (−2.4-fold; p < 10−29), tumor suppressors that negatively regulate cell-cycle progression, showed a decrease in transcript levels. Wee2 (−11.4-fold; p < 3 × 10−4), a gene that encodes a kinase that phosphorylates and inhibits CDK1, was also decreased in iMOP cells. The altered levels of both positive and negative regulators of cell-cycle progression could contribute to the increased proliferative capacity of iMOP cells relative to primary otospheres. This constellation of signature cell-cycle genes may contribute to the ability of iMOP cells to continually divide and retain their otic cell identity. We next determined how iMOP cells undergo self-renewal by following the growth of single cells and assessing the retention of neurosensory markers. We dissociated iMOP cells into single-cell suspensions and immobilized individual cells on Matrigel to maintain their positions throughout the growth period. Single-cell cultures were allowed to proliferate and form colonies over 7 days (Figure 4A). Immunostaining showed that 92% (956/1,036) of the cells retained SOX2 labeling (n = 20) (Figure 4B). iMOP cells also maintained expression of other lineage-restricted markers. PAX2 is a neuroectoderm marker that is expressed early in otic vesicle development (at E10.5) and specifies cell types in the cochlea (Burton et al., 2004Burton Q. Cole L.K. Mulheisen M. Chang W. Wu D.K. The role of Pax2 in mouse inner ear development.Dev. Biol. 2004; 272: 161-175Crossref PubMed Scopus (131) Google Scholar, Li et al., 2004aLi H. Liu H. Corrales C.E. Mutai H. Heller S. Correlation of Pax-2 expression with cell proliferation in the developing chicken inner ear.J. Neurobiol. 2004; 60: 61-70Crossref PubMed Scopus (40) Google Scholar). ISL1 is expressed in the proneurosensory domain at E11.5, as the cochlea starts to develop and mature to form hair cells and auditory neurons (Radde-Gallwitz et al., 2004Radde-Gallwitz K. Pan L. Gan L. Lin X. Segil N. Chen P. Expression of Islet1 marks the sensory and neuronal lineages in the mammalian inner ear.J. Comp. Neurol. 2004; 477: 412-421Crossref PubMed Scopus (99) Google Scholar, Li et al., 2004bLi H. Liu" @default.
• W2090185129 created "2016-06-24" @default.
• W2090185129 creator A5010157316 @default.
• W2090185129 creator A5081441104 @default.
• W2090185129 creator A5081539865 @default.
• W2090185129 date "2015-01-01" @default.
• W2090185129 modified "2023-10-16" @default.
• W2090185129 title "C-MYC Transcriptionally Amplifies SOX2 Target Genes to Regulate Self-Renewal in Multipotent Otic Progenitor Cells" @default.
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