LETTER doi:10.1038/nature21722 Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates Moritz Mall1, Michael S. Kareta1†, Soham Chanda1,2, Henrik Ahlenius3, Nicholas Perotti1, Bo Zhou1,2, Sarah D. Grieder1, Xuecai Ge4†, Sienna Drake3, Cheen Euong Ang1, Brandon M. Walker1, Thomas Vierbuchen1†, Daniel R. Fuentes1, Philip Brennecke5†, Kazuhiro R. Nitta6†, Arttu Jolma6, Lars M. Steinmetz5,7, Jussi Taipale6,8, Thomas C. Südhof2 & Marius Wernig1 Normal differentiation and induced reprogramming require human Myt1l (Extended Data Fig. 1). Chromatin immunoprecipita- the activation of target cell programs and silencing of donor cell tion followed by DNA sequencing (ChIP–seq) of endogenous Myt1l programs1,2. In reprogramming, the same factors are often used to in fetal neurons (embryonic day (E) 13.5) and ectopic Myt1l in mouse reprogram many different donor cell types3. As most developmental embryonic fibroblasts (MEFs) two days after induction identified 3,325 repressors, such as RE1-silencing transcription factor (REST) and high-confidence Myt1l peaks that overlapped remarkably well between Groucho (also known as TLE), are considered lineage-specific neurons and MEFs (Fig. 1a, Extended Data Fig. 2, Supplementary repressors4,5, it remains unclear how identical combinations of Table 1). Thus, similar to the pioneer factor Ascl1, Myt1l can access transcription factors can silence so many different donor programs. the majority of its cognate DNA binding sites even in a distantly related Distinct lineage repressors would have to be induced in different cell type. However, unlike Ascl1 targets8, the chromatin at Myt1l donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific a Myt1l Myt1l b Myt1l Ascl1 Random Myt1l 6 Ascl1 + Brn2 endogenous transcription factor Myt1-like (Myt1l) exerts its pro-neuronal Closed 0.030 function by direct repression of many different somatic lineage k programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b 0.025 by binding to a previously uncharacterized N-terminal domain. In Reads per pea 0.020 agreement with its repressive function, the genomic binding sites of Open Myt1l are similar in neurons and fibroblasts and are preferentially in omatin-bound sites –1,000 0 1,000 an open chromatin configuration. The Notch signalling pathway is Summit distance (bp) repressed by Myt1l through silencing of several members, including 3 Myt1l Ascl1 Brn2 Myt1l chr c Hes1. Acute knockdown of Myt1l in the developing mouse brain 2 1 0.15 mimicked a Notch gain-of-function phenotype, suggesting that 0 Myt1l allows newborn neurons to escape Notch activation during Reprogramming broblasts Mouse brain 0.10 normal development. Depletion of Myt1l in primary postmitotic d e 5′ UTR (3%) Intron neurons de-repressed non-neuronal programs and impaired 3′ UTR (1%) Intergenic 0.05 neuronal gene expression and function, indicating that many Exon (4%) Promoter Reads per peak somatic lineage programs are actively and persistently repressed 0 0 0 0 23% 0 0 by Myt1l to maintain neuronal identity. It is now tempting to 41% –4 –200 2 400 –43 Summit distance (bp) speculate that similar ‘many-but-one’ lineage repressors exist for 32.1% of targets P = 1 × 10 28% other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming Figure 1 | Context-independent target chromatin access of Myt1l. additional cell types. a, Genome-wide occupancy profiles of endogenous Myt1l in E13.5 mouse The combination of the three transcription factors Ascl1, Brn2, and brains (n =​ 2) or overexpressed Myt1l with (n =​ 3) or without (n = 2) Ascl1 Myt1l has been shown to reprogram fibroblasts and other somatic cells and Brn2 (also known as Pou3f2) in MEFs two days after reprogramming. into induced neuronal (iN) cells7. Ascl1 acts as an ‘on-target’ pioneer Corresponding regions are displayed across all datasets ±​ 2 kb from summits. b, Chromatin accessibility based on micrococcal nuclease factor to activate the neuronal program, whereas access of Brn2 to the 25 chromatin is context-dependent and facilitates reprogramming later digestion combined with sequencing (MNase–seq) signal in MEFs shows 8 binding enrichment of Myt1l in open and Ascl1 in closed regions. c, Read on . Although Ascl1 alone is sufficient to generate iN cells, endoge- densities of Ascl1 and Brn2 chromatin binding8 shows minor overlap at nous Myt1l is induced during reprogramming and exogenous Myt1l Myt1l-bound regions. d, The Myt1l AAGTT core motif (green arrow) greatly improves the efficiency of reprogramming and the functional is significantly enriched at bound sites across datasets. P = 1 × 10−43, 9,10 maturity of the resulting iN cells . To investigate the role of Myt1l E = 9.6 × 10−3. e, Pie chart indicates enrichment of high-confidence in reprogramming, we first raised antibodies specific for mouse and Myt1l-bound sites at gene promoters. 1Department of Pathology and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA. 2Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA. 3Department of Clinical Sciences, Division of Neurology and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden. 4Department of Developmental Biology, Stanford University, Stanford, California 94305, USA. 5Department of Genetics, Stanford University, Stanford, California 94305, USA. 6Division of Functional Genomics and Systems Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden. 7Genome Biology Unit, European 8 Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany. Genome Scale Biology Program, University of Helsinki, 00014 Helsinki, Finland. †Present addresses: Genetics and Genomics Group, Sanford Research, Sioux Falls, SD 57104, USA (M.S.K.); Molecular and Cellular Biology, University of California Merced, Merced, CA 95343, USA (X.G.); Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA (T.V.); Leibniz-Institute for Molecular Pharmacology, 13125 Berlin, Germany (P.B.); Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan (K.R.N.). 13 APRIL 2017 | VOL 544 | NATURE | 245 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. RESEARCH LETTER s Target gene abMEF program downregulation c repression 1.0 0 Mock 2BAM days 2BAM day s13BAM days 22 day Enrichment scor Adam17 ) –0.2 Notch1 2 0.5 *** Notch2 –0.4 Notch3 0 Hes1 –0.4 Notch signalling Wnt5a ession ratio e Wnt9a –0.5 –0.8 Expr Fzd1 BAM 2 days (log Fzd5 –1.0 Fzd7 NES = –2.68, FDR = 0.0 log Tcf7l1 3 Wnt signalling ol 2 (FPKM+1) Ctnnb1 Myt1l ets 2 Genome rg 1 contr ta 0 Stat1 –1 Stat3 –2 d TUJ1/DAPI f Desmin/DAPI Yap1 –3 Smad3 WT Myt1l Myt1l Mock WT Myt1l oliferation Smad6 410–623 Pr + Gli2 n Klf6 + OFF MyoD Six5 Lbh Mock WT Myt1l Ascl1 EnR–Myt1l VP64–Myt1l 410–623 410–623 ON + Snai2 Olig1 FF Pax3 Ascl1 Ikzf2 FO Mafa Lineage specicatio OF Il6 e TUJ1/Tau–eGFP g Desmin/Myh MEFiN * * 100 ** 100 h MyoD Myt1l 75 d 75 Ascl1 170 Myt1l 50 esse 50 * 170 Myh Enhanced 25 Repr 25 * 55 Desmin iM induction (%) iN formation (%) * * 35 MyoD 0 0 l 3 MyoD 25 Ascl1 410–623 410–623 410–62 Myt1l 55 Tubulin Ascl1 + Ascl1 +WT Myt1 +Myt1l +EnR–Myt1l +VP64–Myt1l Figure 2 | Myt1l target gene repression dominates induced (Myt1l410–623) fused to a repressor (EnR) or activator (VP64); TUJ1 (red), neurogenesis. a, Repression of promoter-bound (transcription start DAPI staining (blue). e, Conversion efficiency of cells shown in d based site (TSS) − 5, +​ 2 kb) Myt1l targets (n =​ 1,798) dominates genome- on TUJ1-positive cells with neuronal morphology (black) or Tau–eGFP wide expression changes (n =​ 33,459) upon two days of reprogramming expression (white) show partial reprogramming using repressor fusion, (P = 2.78 × 10−12, two biological replicates each)8. b, GSEA identified MEF with many Tau–eGFP-positive cells without neuronal morphologies. signature among repressed Myt1l targets. NES, normalized enrichment f, Immunofluorescence of MEFs upon reprogramming for 14 days with score; FDR, false discovery rate. c, RNA-seq expression values of selected Ascl1 or MyoD without (mock) or with wild-type Myt1l; desmin (green), Myt1l targets at indicated time points during reprogramming, normalized DAPI staining (blue). g, Induced muscle (iM) conversion efficiency to the mock sample; n = 2 (ref. 8). Myt1l represses several Notch, Wnt and of cells shown in f based on either desmin (black) or Myh expression proliferation factors. Many lineage specifiers are bound and repressed (white) shows decreased muscle marker-positive cells upon addition of (ON→ OFF) or remain repressed (OFF→ OFF). FPKM, fragments per wild-type Myt1l. h, Western blot analysis of cells shown in f after 2 days kilobase of transcript per million mapped reads. d, Immunofluorescence of reprogramming using indicated antibodies (for gel source images see of iN cells derived from MEFs upon reprogramming for 14 days with Supplementary Fig. 1). d–g, Scale bar 50 μ m, n =​ 3, error bars show s.d., Ascl1 and wild-type (WT) Myt1l or a non-functional zinc-finger fragment t-test * P < 0.05. targets is preferentially open (nucleosome-free) in fibroblasts (Fig. 1b). Table 2). On average, Myt1l targets were significantly downregu- Hence, Myt1l appears to possess no pioneer factor activity, raising the lated in reprogramming fibroblasts, indicating that Myt1l may be question why the targets of a neuron-specific transcription factor are eas- a transcriptional repressor that functions to silence the fibroblast ily accessible in fibroblasts. As expected, there was little overlap between program during reprogramming. Indeed, gene set enrichment analysis the target sites of Myt1l and Ascl1, and the chromatin binding of Myt1l (GSEA) showed significant enrichment of MEF signature genes among was not strongly affected by Ascl1 and Brn2, indicating that these fac- the repressed Myt1l target genes (Fig.