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• W2897051414 abstract "•KMT2B is critical for neuronal transdifferentiation, whereas KMT2A is dispensable•KMT2B is responsible for the activation of the neuronal maturation gene program•KMT2B represses the myocyte fate unleashed upon defective transdifferentiation•KMT2B dependence reveals candidate dystonia-causative genes Transdifferentiation of fibroblasts into induced neuronal cells (iNs) by the neuron-specific transcription factors Brn2, Myt1l, and Ascl1 is a paradigmatic example of inter-lineage conversion across epigenetically distant cells. Despite tremendous progress regarding the transcriptional hierarchy underlying transdifferentiation, the enablers of the concomitant epigenome resetting remain to be elucidated. Here, we investigated the role of KMT2A and KMT2B, two histone H3 lysine 4 methylases with cardinal roles in development, through individual and combined inactivation. We found that Kmt2b, whose human homolog’s mutations cause dystonia, is selectively required for iN conversion through suppression of the alternative myocyte program and induction of neuronal maturation genes. The identification of KMT2B-vulnerable targets allowed us, in turn, to expose, in a cohort of 225 patients, 45 unique variants in 39 KMT2B targets, which represent promising candidates to dissect the molecular bases of dystonia. Transdifferentiation of fibroblasts into induced neuronal cells (iNs) by the neuron-specific transcription factors Brn2, Myt1l, and Ascl1 is a paradigmatic example of inter-lineage conversion across epigenetically distant cells. Despite tremendous progress regarding the transcriptional hierarchy underlying transdifferentiation, the enablers of the concomitant epigenome resetting remain to be elucidated. Here, we investigated the role of KMT2A and KMT2B, two histone H3 lysine 4 methylases with cardinal roles in development, through individual and combined inactivation. We found that Kmt2b, whose human homolog’s mutations cause dystonia, is selectively required for iN conversion through suppression of the alternative myocyte program and induction of neuronal maturation genes. The identification of KMT2B-vulnerable targets allowed us, in turn, to expose, in a cohort of 225 patients, 45 unique variants in 39 KMT2B targets, which represent promising candidates to dissect the molecular bases of dystonia. The conversion of murine embryonic fibroblasts (MEFs) into induced neuronal cells (iNs) through forced expression of the Brn2, Ascl1, and Myt1l transcription factors (TFs) (hereafter called BAM [Brn2, Ascl1, Mytl1] factors) defined the paradigm of transdifferentiation across germ layers (Vierbuchen et al., 2010Vierbuchen T. Ostermeier A. Pang Z.P. Kokubu Y. Südhof T.C. Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors.Nature. 2010; 463: 1035-1041Crossref PubMed Scopus (2258) Google Scholar). BAM factors synergistically cooperate in this process (∼20% efficiency), which does not require either proliferation or the transient reacquisition of pluripotency (Vierbuchen et al., 2010Vierbuchen T. Ostermeier A. Pang Z.P. Kokubu Y. Südhof T.C. Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors.Nature. 2010; 463: 1035-1041Crossref PubMed Scopus (2258) Google Scholar). ASCL1 instructs transdifferentiation as a pioneer TF, recognizing trivalent chromatin states marked by H3 lysine 4 monomethylation (H3K4me1), lysine 9 trimethylation (H3K9me3), and lysine 27 acetylation (H3K27ac) (Wapinski et al., 2013Wapinski O.L. Vierbuchen T. Qu K. Lee Q.Y. Chanda S. Fuentes D.R. Giresi P.G. Ng Y.H. Marro S. Neff N.F. et al.Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.Cell. 2013; 155: 621-635Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). BRN2 and MYT1L are, instead, critical for iN maturation and the suppression of alternative cell fates; upon Ascl1-only infection, MEFs that fail to become iNs deviate toward myocytes, an outcome that is prevented by co-transduction with Brn2 and Myt1l (Mall et al., 2017Mall M. Kareta M.S. Chanda S. Ahlenius H. Perotti N. Zhou B. Grieder S.D. Ge X. Drake S. Euong Ang C. et al.Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates.Nature. 2017; 544: 245-249Crossref PubMed Scopus (118) Google Scholar, Treutlein et al., 2016Treutlein B. Lee Q.Y. Camp J.G. Mall M. Koh W. Shariati S.A. Sim S. Neff N.F. Skotheim J.M. Wernig M. Quake S.R. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.Nature. 2016; 534: 391-395Crossref PubMed Scopus (269) Google Scholar, Wapinski et al., 2013Wapinski O.L. Vierbuchen T. Qu K. Lee Q.Y. Chanda S. Fuentes D.R. Giresi P.G. Ng Y.H. Marro S. Neff N.F. et al.Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.Cell. 2013; 155: 621-635Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). ASCL1 has been recently shown to effect massive remodeling of chromatin accessibility and nucleosome phasing, with open chromatin loci enriched for neuronal and muscle pathway genes (Wapinski et al., 2017Wapinski O.L. Lee Q.Y. Chen A.C. Li R. Corces M.R. Ang C.E. Treutlein B. Xiang C. Baubet V. Suchy F.P. et al.Rapid Chromatin Switch in the Direct Reprogramming of Fibroblasts to Neurons.Cell Rep. 2017; 20: 3236-3247Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Moreover, MEF-to-iN conversion features a major chromatin reconfiguration in the first 5 days after Ascl1 induction, following which further chromatin transitions represent less than 20% of all changes underlying direct neuronal conversion (Wapinski et al., 2017Wapinski O.L. Lee Q.Y. Chen A.C. Li R. Corces M.R. Ang C.E. Treutlein B. Xiang C. Baubet V. Suchy F.P. et al.Rapid Chromatin Switch in the Direct Reprogramming of Fibroblasts to Neurons.Cell Rep. 2017; 20: 3236-3247Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This suggests that these epigenetic transitions are not as gradual as during reprogramming to pluripotency (Cacchiarelli et al., 2015Cacchiarelli D. Trapnell C. Ziller M.J. Soumillon M. Cesana M. Karnik R. Donaghey J. Smith Z.D. Ratanasirintrawoot S. Zhang X. et al.Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency.Cell. 2015; 162: 412-424Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, Chronis et al., 2017Chronis C. Fiziev P. Papp B. Butz S. Bonora G. Sabri S. Ernst J. Plath K. Cooperative Binding of Transcription Factors Orchestrates Reprogramming.Cell. 2017; 168: 442-459.e20Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), but the role of specific chromatin regulators in these accompanying waves of chromatin resetting remains elusive. Here, we investigated the roles of KMT2A and KMT2B in MEF-to-iN conversion in light of their critical function in lineage decisions. KMT2A and KMT2B belong to Set1-Trithorax type H3K4 methylases, specifically responsible for the deposition of histone H3 lysine 4 trimethylation (H3K4me3) at gene promoters (Denissov et al., 2014Denissov S. Hofemeister H. Marks H. Kranz A. Ciotta G. Singh S. Anastassiadis K. Stunnenberg H.G. Stewart A.F. Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant.Development. 2014; 141: 526-537Crossref PubMed Scopus (174) Google Scholar). Both associate with MENIN, which mediates their localization at specific loci, such as HOX genes (Glaser et al., 2006Glaser S. Schaft J. Lubitz S. Vintersten K. van der Hoeven F. Tufteland K.R. Aasland R. Anastassiadis K. Ang S.L. Stewart A.F. Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development.Development. 2006; 133: 1423-1432Crossref PubMed Scopus (228) Google Scholar, Hughes et al., 2004Hughes C.M. Rozenblatt-Rosen O. Milne T.A. Copeland T.D. Levine S.S. Lee J.C. Hayes D.N. Shanmugam K.S. Bhattacharjee A. Biondi C.A. et al.Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus.Mol. Cell. 2004; 13: 587-597Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, Lee et al., 2006Lee S. Lee D.K. Dou Y. Lee J. Lee B. Kwak E. Kong Y.Y. Lee S.K. Roeder R.G. Lee J.W. Coactivator as a target gene specificity determinant for histone H3 lysine 4 methyltransferases.Proc. Natl. Acad. Sci. USA. 2006; 103: 15392-15397Crossref PubMed Scopus (127) Google Scholar). Despite their high homology, KMT2A and KMT2B are spatially and temporally non-redundant. Thus, knockout (KO) of either Kmt2a (Ernst et al., 2004Ernst P. Fisher J.K. Avery W. Wade S. Foy D. Korsmeyer S.J. Definitive hematopoiesis requires the mixed-lineage leukemia gene.Dev. Cell. 2004; 6: 437-443Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) or Kmt2b (Glaser et al., 2006Glaser S. Schaft J. Lubitz S. Vintersten K. van der Hoeven F. Tufteland K.R. Aasland R. Anastassiadis K. Ang S.L. Stewart A.F. Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development.Development. 2006; 133: 1423-1432Crossref PubMed Scopus (228) Google Scholar) is embryonic lethal at embryonic day 12.5 (E12.5) or E.10.5, respectively. Furthermore, KMT2A and KMT2B have specific roles during mammalian neuronal differentiation. During retinoic acid-based differentiation, the two enzymes regulate different HOX genes (Denissov et al., 2014Denissov S. Hofemeister H. Marks H. Kranz A. Ciotta G. Singh S. Anastassiadis K. Stunnenberg H.G. Stewart A.F. Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant.Development. 2014; 141: 526-537Crossref PubMed Scopus (174) Google Scholar), and Kmt2b−/− embryonic stem cells (ESCs) show a severe delay in in vitro differentiation toward the ectodermal lineage (Lubitz et al., 2007Lubitz S. Glaser S. Schaft J. Stewart A.F. Anastassiadis K. Increased apoptosis and skewed differentiation in mouse embryonic stem cells lacking the histone methyltransferase Mll2.Mol. Biol. Cell. 2007; 18: 2356-2366Crossref PubMed Scopus (84) Google Scholar). Adult neurogenesis and function are also specifically affected. Indeed, Kmt2a−/− subventricular zone neural stem cells (SVZ NSCs) are impaired selectively in neuronal differentiation, whereas both KMT2A and KMT2B contribute to memory formation, albeit through distinct target pathways and with no apparent effects on brain and neuronal morphology (Kerimoglu et al., 2013Kerimoglu C. Agis-Balboa R.C. Kranz A. Stilling R. Bahari-Javan S. Benito-Garagorri E. Halder R. Burkhardt S. Stewart A.F. Fischer A. Histone-methyltransferase MLL2 (KMT2B) is required for memory formation in mice.J. Neurosci. 2013; 33: 3452-3464Crossref PubMed Scopus (98) Google Scholar, Kerimoglu et al., 2017Kerimoglu C. Sakib M.S. Jain G. Benito E. Burkhardt S. Capece V. Kaurani L. Halder R. Agís-Balboa R.C. Stilling R. et al.KMT2A and KMT2B Mediate Memory Function by Affecting Distinct Genomic Regions.Cell Rep. 2017; 20: 538-548Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, Lim et al., 2009Lim D.A. Huang Y.C. Swigut T. Mirick A.L. Garcia-Verdugo J.M. Wysocka J. Ernst P. Alvarez-Buylla A. Chromatin remodelling factor Mll1 is essential for neurogenesis from postnatal neural stem cells.Nature. 2009; 458: 529-533Crossref PubMed Scopus (310) Google Scholar). Finally, mutations in KMT2A and KMT2B cause two distinct brain disorders: Wiedemann-Steiner syndrome, featuring intellectual disability, and a newly recognized molecular subset of early-onset generalized dystonia, respectively (Meyer et al., 2017Meyer E. Carss K.J. Rankin J. Nichols J.M. Grozeva D. Joseph A.P. Mencacci N.E. Papandreou A. Ng J. Barral S. et al.UK10K ConsortiumDeciphering Developmental Disorders StudyNIHR BioResource Rare Diseases ConsortiumMutations in the histone methyltransferase gene KMT2B cause complex early-onset dystonia.Nat. Genet. 2017; 49: 223-237Crossref PubMed Scopus (139) Google Scholar, Strom et al., 2014Strom S.P. Lozano R. Lee H. Dorrani N. Mann J. O’Lague P.F. Mans N. Deignan J.L. Vilain E. Nelson S.F. et al.De Novo variants in the KMT2A (MLL) gene causing atypical Wiedemann-Steiner syndrome in two unrelated individuals identified by clinical exome sequencing.BMC Med. Genet. 2014; 15: 49Crossref PubMed Scopus (40) Google Scholar, Zech et al., 2016Zech M. Boesch S. Maier E.M. Borggraefe I. Vill K. Laccone F. Pilshofer V. Ceballos-Baumann A. Alhaddad B. Berutti R. et al.Haploinsufficiency of KMT2B, Encoding the Lysine-Specific Histone Methyltransferase 2B, Results in Early-Onset Generalized Dystonia.Am. J. Hum. Genet. 2016; 99: 1377-1387Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To study the role of KMT2A and KMT2B during transdifferentiation, we employed conditional mouse strains carrying exon 2 of Kmt2a and/or Kmt2b flanked by LoxP sites, the knockin of the YFP-coding gene into one Rosa26 allele downstream of a LoxP-flanked transcription termination cassette (STOP cassette), and the gene coding for the tamoxifen-inducible version of Cre recombinase knocked into the second Rosa26 allele (Glaser et al., 2006Glaser S. Schaft J. Lubitz S. Vintersten K. van der Hoeven F. Tufteland K.R. Aasland R. Anastassiadis K. Ang S.L. Stewart A.F. Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development.Development. 2006; 133: 1423-1432Crossref PubMed Scopus (228) Google Scholar, Kranz et al., 2010Kranz A. Fu J. Duerschke K. Weidlich S. Naumann R. Stewart A.F. Anastassiadis K. An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase.Genesis. 2010; 48: 512-520Crossref PubMed Scopus (99) Google Scholar, Testa et al., 2004Testa G. Schaft J. van der Hoeven F. Glaser S. Anastassiadis K. Zhang Y. Hermann T. Stremmel W. Stewart A.F. A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles.Genesis. 2004; 38: 151-158Crossref PubMed Scopus (161) Google Scholar). Upon 4-hydroxytamoxifen (4-OHT) administration, Cre is expressed, generating a frameshift mutation in Kmt2a and/or Kmt2b (Figure 1A). MEFs were derived from Kmt2a (and/or Kmt2b)fl/fl Cre+ YFP+ embryos and from Kmt2a+/+Kmt2b+/+ Cre+ YFP+ or Kmt2afl/+ Cre+ YFP+ for Kmt2a conditional KO (cKO) as controls (Figure 1A). After 5 days of 4-OHT treatment followed by either 2 or 7 days in normal medium (Figure 1B), depletion of Kmt2a and/or Kmt2b was assessed by qRT-PCR (Figure 1C) and western blot (Figure 1D). Transdifferentiation was then implemented according to the original protocol (Vierbuchen et al., 2010Vierbuchen T. Ostermeier A. Pang Z.P. Kokubu Y. Südhof T.C. Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors.Nature. 2010; 463: 1035-1041Crossref PubMed Scopus (2258) Google Scholar; Figure 1B). The sustained expression of YFP (Figure S1A) along with permanent loss of Kmt2b exon 2 (Figure S1B) through the end of transdifferentiation excluded selection of Kmt2bfl/fl iNs that might have escaped recombination. To decipher the role of the two KMT2 methylases, we integrated two complementary approaches to capture the acquisition of iN identity along transdifferentiation: morphological analysis through an automated and unbiased imaging method (ScanR) and cytofluorimetric analysis (fluorescence-activated cell sorting [FACS]) for the expression of polysialic acid-neural cell adhesion molecule (PSA-NCAM), a marker of iN induction (Figure 1B). Transcriptomic and epigenomic profiling was then performed at 5 and 13 days of transdifferentiation (Figure 1B). Day 5 was chosen as reference starting point because, at this time, MEFs are still equally competent to become iNs, having all undergone convergent transcriptional changes (Treutlein et al., 2016Treutlein B. Lee Q.Y. Camp J.G. Mall M. Koh W. Shariati S.A. Sim S. Neff N.F. Skotheim J.M. Wernig M. Quake S.R. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.Nature. 2016; 534: 391-395Crossref PubMed Scopus (269) Google Scholar). Day 13 was selected as the endpoint because 8 days following transduction no more iNs are induced, and the 13-day iNs have been shown to be functionally mature (Vierbuchen et al., 2010Vierbuchen T. Ostermeier A. Pang Z.P. Kokubu Y. Südhof T.C. Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors.Nature. 2010; 463: 1035-1041Crossref PubMed Scopus (2258) Google Scholar). MEFs were also profiled at the epigenomic level at the onset of transdifferentiation to define their initial chromatin configuration (Figure 1B). We evaluated the transdifferentiation potential of Kmt2a−/− and/or Kmt2b−/− MEFs in terms of both efficiency of iN generation and degree of their maturation. To distinguish between the effect of their loss on cell mortality and transdifferentiation, we harnessed the observation that already 1 day after doxycycline administration (day 3), the vast majority of cells are post-mitotic (Vierbuchen et al., 2010Vierbuchen T. Ostermeier A. Pang Z.P. Kokubu Y. Südhof T.C. Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors.Nature. 2010; 463: 1035-1041Crossref PubMed Scopus (2258) Google Scholar). To confidently ascribe any change in cell number to cell mortality rather than to cell proliferation, we selected cKO and control samples starting transdifferentiation from the empirically validated same number of cells on day 3. We found that loss of Kmt2b greatly impaired transdifferentiation efficiency in a manner independent of the mortality rate that accompanies transdifferentiation (Figures 2A and 2B ; Table S1). In contrast, for the loss of Kmt2a, transdifferentiation efficiency was correlated with cell viability (Figures S1C and S1D; Table S1), pointing to the former as a by-product of the latter. Next we sought to determine whether there is any redundancy between the two methylases by knocking out both enzymes. Kmt2a−/−Kmt2b−/− transdifferentiated MEFs showed both the highest cell death and the lowest conversion efficiency, indicating that KMT2A only partially compensates for the lack of KMT2B during transdifferentiation (Figures 2A and 2B; Table S1). Importantly, the combined loss of Kmt2a and Kmt2b did not affect MEF viability per se but was specifically triggered upon induction of transdifferentiation (Figures S1E and S1F). We confirmed these observations through FACS-based quantitation of PSA-NCAM+ cells. Indeed, both Kmt2a−/− and Kmt2b−/− MEFs showed a lower efficiency of iN induction with respect to controls (Figure 2C), but although, for Kmt2a−/− MEFs, this was entirely accounted for by the higher rate of cell death (Figures 2D, S1G and S1H), for Kmt2b−/− MEFs, it was independent of cell death and, thus, imputable to a specific requirement for KMT2B in transdifferentiation (Figures 2D, S1I and S1J). Moreover, although cell mortality in Kmt2a−/− transdifferentiating MEFs was higher with respect to Kmt2b−/− and controls (Figure 2D), the percentage of Kmt2a−/− PSA-NCAM+ cells overlapped or was even higher than in controls along the entire process (Figure 2E). This indicates that PSA-NCAM+ and PSA-NCAM− fractions are at least equally vulnerable to cell death across Kmt2a−/− and Kmt2a+/+. Finally, FACS analysis of the combined KO showed that, although the viability of Kmt2a−/−Kmt2b−/− MEFs was comparable with that of Kmt2a−/− and lower than that of controls (Figures 2D, S1K and S1L), their transdifferentiation efficiency was strongly reduced with respect both to control and the single cKO transdifferentiating MEFs (Figure 2C), underscoring that KMT2B is the main H3K4 trimethylase involved in direct cell conversion. Then we analyzed the morphology of the generated iNs to probe the role of either methylase on iN maturation. To this aim, we investigated neurite elongation as a defining hallmark of complete iN conversion, finding that, in Kmt2b−/− iNs, it was severely reduced both at 13 (Figures 2F, 2I, S2A, and S2B) and 21 days (Figure 2G) of transdifferentiation. On the contrary, and consistent with the unaltered efficiency of iN conversion, neurite length in Kmt2a−/− iNs was fully comparable with that of Kmt2a+/− (Figures 2F, 2G, 2H, S2C, and S2D). Finally, the few double cKO iNs only showed minimal neurite elongation (Figures 2F, 2J, and S2E). This demonstrates that KMT2B, besides playing a fundamental role in MEF-to-iN conversion, has also a specific effect on iN maturation. Given the role of KMT2B in H3K4me3 deposition at promoters (Denissov et al., 2014Denissov S. Hofemeister H. Marks H. Kranz A. Ciotta G. Singh S. Anastassiadis K. Stunnenberg H.G. Stewart A.F. Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant.Development. 2014; 141: 526-537Crossref PubMed Scopus (174) Google Scholar), its loss could impair the gene expression program underlying MEF-to-iN transition. To unveil the molecular basis of the dual phenotype observed upon loss of KMT2B (namely, less efficient iN conversion and defective iN maturation), we performed RNA sequencing (RNA-seq) on sorted PSA-NCAM+ and PSA-NCAM− Kmt2b−/− and control cells at day 13 of transdifferentiation. In particular, through the analysis of PSA-NCAM+ transcriptomes, we aimed to establish which genes responsible for transdifferentiation were differentially expressed in the absence of KMT2B and how Kmt2b−/− iNs differed from controls. Instead, through PSA-NCAM− transcriptomic profiling, we pursued mechanistic insight into the effect of Kmt2b loss on MEF-to-iN conversion. The first component identified by principal-component analysis (PCA) clearly distinguished genotypes in both PSA-NCAM populations (Figures S3A and S3B), indicating that Kmt2b status represents the largest source of variation in the datasets. Specifically, through the analysis of PSA-NCAM+ cells, we found 1,508 differentially expressed genes (DEGs) between Kmt2b−/− and control iNs (at false discovery rate [FDR] < 0.01 and with at least a 50% fold change), the majority of which were downregulated in the latter, consistent with the gene-activating function of KMT2B (Figure 3A). To define which stage of transdifferentiation was most affected by loss of KMT2B, we clustered day 13 DEGs between Kmt2b−/− and control iNs using K-means clustering according to their established expression pattern throughout transdifferentiation (Wapinski et al., 2013Wapinski O.L. Vierbuchen T. Qu K. Lee Q.Y. Chanda S. Fuentes D.R. Giresi P.G. Ng Y.H. Marro S. Neff N.F. et al.Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.Cell. 2013; 155: 621-635Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar; Figure 3B). Genes that are normally expressed on day 15 and downregulated on day 24 of transdifferentiation (clusters 2 and 3) were found to be already lowly expressed at 13 days in our Kmt2b−/− samples, further highlighting a defective MEF-to-iN transition. On the other hand, a high proportion of cluster 4, whose expression gradually increases throughout transdifferentiation, peaking at day 24, and that is enriched for synaptic genes and RE1-silencing transcription factor (REST) targets (4.79-fold enrichment, q-value [q] ∼ 1e−54 in neuronal progenitors), was downregulated in Kmt2b−/− iNs, further corroborating the defective neuronal fate acquisition in the absence of KMT2B. Recently, Treutlein et al., 2016Treutlein B. Lee Q.Y. Camp J.G. Mall M. Koh W. Shariati S.A. Sim S. Neff N.F. Skotheim J.M. Wernig M. Quake S.R. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.Nature. 2016; 534: 391-395Crossref PubMed Scopus (269) Google Scholar integrated the single-cell transcriptomes of Ascl1-only infected MEFs throughout transdifferentiation with those of 15-day BAM-infected iNs and identified the transcriptional regulators that most likely coordinate transdifferentiation progression, defining three subnetworks (i.e., MEF, initiation and maturation). We found that, in our PSA-NCAM+ dataset, the most disrupted subnetwork by loss of KMT2B was the maturation one, confirming at the molecular level the defective iN maturation phenotype we observed (Figure 3C). Indeed, the top 30 DEGs (Figures 3A and S3C) that have annotated function(s) included four key promoters of physiological neurite extension (Ppp1r9a, Myo16, Lrfn1, and Prka1b) and nine regulators of synapse formation, maturation, and function (Clstn3, Jph4, Syp, Sult4a1, Ptprn, Cadm3, Adcy1, Grin2b, and Ache). Together, these results show that the defective iN phenotype observed upon loss of KMT2B is determined by dysregulation of the transcriptional program sustaining iN maturation. To gain deeper insight into this phenomenon, we next compared the transcriptomes of our 13-day iNs with those of single cells undergoing MEF-to-iN transition (Treutlein et al., 2016Treutlein B. Lee Q.Y. Camp J.G. Mall M. Koh W. Shariati S.A. Sim S. Neff N.F. Skotheim J.M. Wernig M. Quake S.R. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.Nature. 2016; 534: 391-395Crossref PubMed Scopus (269) Google Scholar). Strikingly, Kmt2b−/− PSA-NCAM+ iNs showed a higher correlation with the transcriptomes of earlier stages of transdifferentiation, providing the molecular basis for our observation that the few Kmt2b−/− MEFs that manage to convert to iNs fail to undergo complete neuronal maturation (Figures S3D and S3E). Furthermore, these maturation-defective iNs also present an increased correlation with myocyte gene expression, suggesting a concomitant failure to fully suppress alternative fates (Figures S3D and S3E). Thus, to confirm whether Kmt2b−/− MEFs, which transdifferentiated less efficiently than controls, switched toward the myocyte fate, we compared Kmt2b−/− and control PSA-NCAM− 13-day transcriptomes and found that upregulated genes in Kmt2b−/− PSA-NCAM− cells were significantly enriched for targets bound by ASCL1 in the first phases of transdifferentiation (Wapinski et al., 2013Wapinski O.L. Vierbuchen T. Qu K. Lee Q.Y. Chanda S. Fuentes D.R. Giresi P.G. Ng Y.H. Marro S. Neff N.F. et al.Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.Cell. 2013; 155: 621-635Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar; Figure 3D). Although these ASCL1-bound genes have been identified in MEFs transduced either with this TF alone (3.9-fold, p ∼ 2e−7) or with all BAM factors (3-fold, p ∼ 2e−10), a large fraction is then rapidly repressed by BRN2 and MYT1L in BAM-transduced MEFs (Figure 3D), preventing the shift toward myocytes (Treutlein et al., 2016Treutlein B. Lee Q.Y. Camp J.G. Mall M. Koh W. Shariati S.A. Sim S. Neff N.F. Skotheim J.M. Wernig M. Quake S.R. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.Nature. 2016; 534: 391-395Crossref PubMed Scopus (269) Google Scholar). Therefore, the enrichment for such genes in Kmt2b−/− PSA-NCAM− cells indicates that loss of KMT2B prevents their expected silencing, rerouting conversion toward alternative fates. Consistent with this observation, the top gene ontology (GO) enrichments of upregulated genes in Kmt2b−/− PSA-NCAM− cells were all related to myocyte fate acquisition and function (Figure 3E). Moreover, among these genes, we scored a 35-fold increase in MyoD1, a fundamental driver of myogenic differentiation (Pinney et al., 1988Pinney D.F. Pearson-White S.H. Konieczny S.F. Latham K.E. Emerson Jr., C.P. Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway.Cell. 1988; 53: 781-793Abstract Full Text PDF PubMed Scopus (139) Google Scholar; Figure 3F). Because we observed a very high correlation in fold changes between the transcriptomes (correlation [cor] ∼ 0.8, p ∼ 2e−16) of PSA-NCAM+ and PSA-NCAM− fractions (Figure 4A), we hypothesized that much of the impairment in Kmt2b−/− transcriptomic resetting occurred early during MEF-to-iN transition. Thus, we analyzed the genes that changed in the same direction in both PSA-NCAM fractions and whose level of expression is regulated during transdifferentiation (Wapinski et al., 2013Wapinski O.L. Vierbuchen T. Qu K. Lee Q.Y. Chanda S. Fuentes D.R. Giresi P.G. Ng Y.H. Marro S. Neff N.F. et al.Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.Cell. 2013; 155: 621-635Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Indeed, most of the genes downregulated in the absence of KMT2B in both fractions are upregulated in the first phases of transdifferentiation (Figure 4B), and among them, we found the genes of the maturation subnetwork, Zfp612, Arnt2, and Lass4. In particular, Arnt2 and Zfp612 are upregulated or remain expressed only when MEFs are transduced with either Brn2 or Myt1l or all BAM factors but not with Ascl1 alone. Hence, in the absence of KMT2B, these genes could not be activated despite the presence of Brn2 and Myt1l. Furthermore, one of the main direct targets of ASCL1, the repressor ZFP238 (Wapinski et al., 2013Wapinski O.L. Vierbuchen T. Qu K. Lee Q.Y. Chanda S. Fuentes D.R. Giresi P.G. Ng Y.H. Marro S. Neff N.F. et al.Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.Cell. 2013; 155: 621-635Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar), was deregulated both in PSA-NCAM+ and PSA-NCAM− Kmt2b−/− fractions with respect to controls (Figure 4B), underscoring that the loss of KMT2B disrupts the dynamic interplay among BAM factors on their targets. Together, the combined transcriptomic analysis of PSA-NCAM+ and PSA-NCAM− cKO and control cells demonstrates that, upon transdifferentiation, Kmt2b−/− MEFs undergo an early massive dysregulation of the transcriptional program enabling conversion. This, on one side, leads to a lower transdifferentiation efficiency and a substantial shift toward a myocytic fate; on the other," @default.
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• W2897051414 date "2018-10-01" @default.
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• W2897051414 title "KMT2B Is Selectively Required for Neuronal Transdifferentiation, and Its Loss Exposes Dystonia Candidate Genes" @default.
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• W2897051414 doi "https://doi.org/10.1016/j.celrep.2018.09.067" @default.
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