Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates

Abstract

Normal differentiation and induced reprogramming require the activation of target cell programs and silencing of donor cell programs1,2. In reprogramming, the same factors are often used to reprogram many different donor cell types3. As most developmental repressors, such as RE1-silencing transcription factor (REST) and Groucho (also known as TLE), are considered lineage-specific repressors4,5, it remains unclear how identical combinations of transcription factors can silence so many different donor programs. Distinct lineage repressors would have to be induced in different donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific transcription factor Myt1-like (Myt1l)6 exerts its pro-neuronal function by direct repression of many different somatic lineage programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b by binding to a previously uncharacterized N-terminal domain. In agreement with its repressive function, the genomic binding sites of Myt1l are similar in neurons and fibroblasts and are preferentially in an open chromatin configuration. The Notch signalling pathway is repressed by Myt1l through silencing of several members, including Hes1. Acute knockdown of Myt1l in the developing mouse brain mimicked a Notch gain-of-function phenotype, suggesting that Myt1l allows newborn neurons to escape Notch activation during normal development. Depletion of Myt1l in primary postmitotic neurons de-repressed non-neuronal programs and impaired neuronal gene expression and function, indicating that many somatic lineage programs are actively and persistently repressed by Myt1l to maintain neuronal identity. It is now tempting to speculate that similar ‘many-but-one’ lineage repressors exist for other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming additional cell types.

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Figure 1: Context-independent target chromatin access of Myt1l.
Figure 2: Myt1l target gene repression dominates induced neurogenesis.
Figure 3: Characterization of neurogenic and repressive Myt1l domains.
Figure 4: Myt1l represses Notch and Hes1 activity to promote neurogenesis.

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Acknowledgements

We acknowledge N. E. Davey for SID motif discovery, U. Litzenburger for initial in utero experiments and S. Marro and N. Yucel for primary cell preparations. We thank J. E. Johnson, T. Sudo, R. Kageyama, and T. Stearns for antibodies; G. Mantalas, B. Passarelli, M. Miranda and M. Nguyen for sequencing; and A. Stark and Wernig laboratory members for ideas and discussions. Support was provided by the German Research Foundation to M.M., an NCI training grant (#T32 CA09151), the DHHS and the Spectrum Child Health Research Institute to M.S.K, the Swedish Research Council and Swedish Government Initiative for Strategic Research Areas (StemTherapy) to H.A., the National Institutes of Health to L.M.S., T.C.S. and M.W. and the California Institute for Regenerative Medicine to M.W. T.C.S. is a Howard Hughes Medical Institute Investigator. M.W. is a New York Stem Cell Foundation-Robertson Investigator, a Tashia and John Morgridge Faculty Scholar at the Child Health Research Institute at Stanford, and a Howard Hughes Medical Institute Faculty Scholar.

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Authors

Contributions

M.M. was responsible for research design, execution, data analysis, and manuscript preparation. M.S.K. performed and designed the bioinformatics analysis and aided in manuscript preparation. S.C. and B.Z. performed the electrophysiological analysis. H.A. performed the NSC experiments and advised on research design and manuscript preparation. X.G., C.E.A. and S.D. performed in utero electroporation. N.P. aided in the biochemical interaction studies. S.D.G. performed the FACS analysis. T.V., B.M.W. and D.R.F. generated constructs. P.B. and L.M.S. performed the sequencing. K.R.N., A.J. and J.T. performed the SELEX. T.C.S. supported the research. M.W. was responsible for supervision and design of research, data interpretation, and manuscript preparation.

Corresponding author

Correspondence to Marius Wernig.

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Reviewer Information Nature thanks T. Graf and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Myt1l antibody design and characterization.

a, Schematic of mouse Myt1 family members mmMyt1 (Q8CFC2), mmMyt1l (P97500) and mmSt18 (A5LFV3) as well as human hsMyt1l homologue (Q9UL68). Highlighted are the nuclear localization signals (NLS), aspartic acid/glutamic acid-rich (Asp/Glu-rich), serine-rich (Ser-rich), Myt1 and coiled-coil domains and the CCHC-type zinc-fingers (ZF). Also shown is the predicted antigenicity and the conservation between the proteins generated using EpiC and T-Coffee, respectively. Based on these data, a fragment of mmMyt1l (amino acids 171–420) was used as an antigen for antibody induction in rabbits. The sequence identities among the antigen regions and the full-length proteins and their molecular masses are shown (right). bd, Western blots of MEF cells upon induction of Flag-tagged mmMyt1l (b), HEK293 cells upon transfection of Flag-tagged mmMyt1, mmMyt1l, St18, and untagged hsMyt1l (c), and E13.5 embryonic mouse whole brain lysate using preimmune serum and antibodies against Myt1l, Flag, and tubulin (d). e, Microscopy images of HEK293 cells upon transfection of Flag-tagged mmMyt1l followed by immunofluorescence using antibodies against Flag (red) and Myt1l (green). f, Microscopy images of a section from adult mouse cortex upon immunofluorescence using antibodies against NeuN (red) and Myt1l (green), and DAPI staining (blue). Scale bar, 10 μm. g, Myt1l antibody specifically marks mouse brain neurons in vivo. Immunofluorescence microscopy images of adult mouse brain cortex sections using antibodies against neuron-specific NeuN and Map2 or oligodendrocyte-specific Olig2 and Apc. Astrocytes (Gfap) and microglia (Iba1) are shown in red with Myt1l (green) and DAPI staining (blue). Note that Myt1l overlaps only with neuronal markers. Scale bar, 20 μm.

Extended Data Figure 2 Genome-wide chromatin binding of Myt1l.

a, b, ChIP of endogenous Myt1l from E13.5 mouse brain (a) or of wild-type Myt1l (b, left) and Myt1l200–623 (b, right) transgenes from MEF cell lysates 2 days after induction with or without Ascl1 and Brn2. Chromatin immunoprecipitates were analysed by western blotting with Myt1l, Brn2, and Ascl1 antibodies. Input, 0.3% of ChIP input; unbound, 0.3% of ChIP flow-through; ChIP, 3% of ChIP eluates. c, ChIP–seq genome-wide occupancy of endogenous Myt1l in E13.5 mouse brains (n = 2) or Myt1l and Myt1200–623 in MEFs two days after induction with (n = 3) or without (n = 2) Ascl1 and Brn2. A total of 6,911 peaks are sorted on the basis of intensity and corresponding genomic regions are displayed across all datasets; signal is displayed ± 2 kb from summits (see also Fig. 1). d, Chromatin reads for Myt1l, Ascl1 and Brn2 at Ascl1 (top) and Brn2 peaks (bottom)8. e, Chromatin reads of indicated histone marks in uninfected MEFs at the sites at which Myt1l is bound during reprogramming. Signal is displayed ± 2 kb from peak summit. f, Pearson correlation and clustering analysis of ChIP–seq samples highlight high binding overlap between different conditions. g, MA plots from DiffBind and corresponding Venn diagrams showing the distribution of Myt1l ChIP–seq peak intensities between indicated conditions; endogenous Myt1l in mouse brain versus overexpressed Myt1l in BAM MEFs (top), Myt1l overexpression alone versus in combination with Ascl1 and Brn2 (BAM) in MEFs (bottom left), and wild-type Myt1l versus Myt1l200–623 overexpression in MEFs (bottom right). Significantly different peaks are shown in colour and numbers are annotated. Peaks that are significantly changed by the experimental setup are highlighted red.

Extended Data Figure 3 Myt1l represses many genes but not the neuronal transcriptional network.

a, Heatmap of gene expression changes at promoter-bound Myt1l target genes during iN cell conversion of MEFs at the indicated time points based on RNA-seq show significant enrichment of Myt1l motifs at repressed genes (P = 6.85 × 10−6), n = 2 (ref. 8) (left) and inverse transcriptional effects upon Myt1l knockdown in primary hippocampal neurons (right). b, Mean expression of selected Myt1l target genes in MEFs upon induction of wild-type Myt1l together with Ascl1 for two days determined by quantitative real-time PCR show significant repression of canonical inhibitors of neurogenesis by Myt1l. Names and annotated functions of tested genes are indicated, expression levels were normalized to Ascl1-only induction and GAPDH expression, n = 4 biological replicates (with 2 technical replicates each). Error bars show s.e.m., pair wise fixed reallocation randomization test *P < 0.001 (ref. 51). c, Myt1l ChIP–seq profile at the Hes1 and Ncam1 promoter shows strong binding of endogenous Myt1l in E13.5 mouse brain and overexpressed wild-type Myt1l in MEFs two days after reprogramming; red bars mark multiple Myt1l AAGTTT motifs present in repressed Hes1 promoter and gene body. d, Overlap of Myt1l-bound target genes that are induced or repressed during conversion of MEFs into iN cells upon overexpression of Myt1l with or without Ascl1 and Brn2 and indicated cell type-specific expression signatures determined by GeneOverlap50. Odds ratio >2 represents strong association, P values are shown in brackets; n.s., not significant. e, Selected top gene ontology (GO) terms of Myt1l-targeted genes that are repressed (top) or induced (bottom) during reprogramming determined by PANTHER33. Enrichment scores and P values are shown. Highlighted are the terms ‘negative regulation of neuron differentiation’ (green) in the repressed cluster and ‘generation of neurons’ (red) in the induced cluster. Both analyses show a striking enrichment of repressed Myt1l target genes within several non-neuronal programs. Of note, several metabolic GO terms are among the upregulated target genes.

Extended Data Figure 4 Myt1l blocks muscle differentiation in primary myoblasts.

a, Representative micrographs of muscle cells derived from primary myoblasts upon differentiation for 4 days with with rtTA alone (mock) or in combination with wild-type Myt1l followed by immunofluorescence using antibodies against Myh (green), Myt1l (red) and DAPI staining (blue); scale bar, 100 μm. b, Muscle differentiation efficiency of cells shown in a highlight the repressive effect of Myt1l expression (+) on Myh-induction compared to Myt1l-negative cells (−). n = 3, error bars show s.e.m., t-test *P < 0.005. c, Western blot of muscle cells shown in a using indicated antibodies shows reduction of several muscle markers upon Myt1l overexpression.

Extended Data Figure 5 Truncation screen identifies minimal neurogenic domains of Myt1l.

a, Schematic of Flag- and NLS-tagged Myt1l truncation proteins including amino acid positions. Ability to enhance neurogenic conversion together with Ascl1 is indicated by (+), minimal active truncation Myt1l200–623 is boxed red (see also Fig. 3). Myt1l truncations with partial or without enhanced conversion activity are indicated with (+/−) and (–), respectively. b, Representative micrographs of iN cells derived from MEFs upon reprogramming for 14 days with Ascl1 together with the indicated transgenes followed by immunofluorescence using antibodies against TUJ1 (red) and DAPI staining (blue); scale bar, 50 μm. cg, Electrophysiological characterization of iN cells derived in b upon maturation for 21 days on mouse glia. c, Representative action potential (AP) traces of iN cells upon reprogramming with Ascl1 together with indicated Myt1l truncation. Pie charts indicate fraction of cells firing single (grey), multiple (white), or no (black) action potentials. d, Mean number of action potentials fired plotted with respect to pulse amplitude measured at −60 mV holding potential. e, Mean resting membrane-potential (Vrest). f, g, Mean membrane resistance (Rm; f) and capacitance (Cm; g) measured at −70 mV holding potential. Dotted line indicates intrinsic properties of Ascl1 + GFP cells; n = 3 biological replicates (total number of individual cells measured indicated), error bars show s.e.m., t-test *P < 0.05. h, Microscopy images showing nuclear localization of all tested Myt1l truncations 2 days after induction in MEFs by immunofluorescence using antibodies against Flag (grey) and DAPI staining (blue), scale bar 10 μm. i, Western blot analysis of HEK293 cells 2 days after transfection with the indicated transgenes confirms protein integrity using antibodies against Flag and tubulin.

Extended Data Figure 6 Characterization of mouse and human iN cells generated with Ascl1 and Myt1l.

a, Microscopy images of iN cells derived from MEFs upon reprogramming for 21 days on mouse glia by overexpression of Ascl1 together with either wild-type Myt1l or Myt1l200–623 followed by immunofluorescence using antibodies against Map2 (red) and synapsin (green) or NeuN (red) and Tau–eGFP (green), scale bar 10 μm. b, Synaptic recordings of Tau–eGFP-positive mouse iN cells shown in a. c, d, Spontaneous and evoked EPSCs were recorded at a holding potential of −70 mV (blue) and blocked by the addition of CNQX (red), indicating that the excitatory nature of the resulting induced neurons is mediated through AMPA-type receptors (AMPARs). e, Immunofluorescence images of iN cells derived from human embryonic fibroblasts upon reprogramming for 6 weeks by overexpression of GFP, Ascl1, Ngn2 and Brn2 together with either wild-type Myt1l or Myt1l200–623 and co-culture with primary cortical mouse neurons using antibodies against synapsin (red) and GFP (green); scale bar,10 μm. f, Synaptic recordings of GFP-positive human iN cells shown in e. g, h, Spontaneous and evoked EPSCs were recorded at a holding potential of −70 mV, indicating synaptic competence of the resulting induced human neurons. n = 4 cells (fraction of active cells indicated).

Extended Data Figure 7 Zinc-fingers are essential for neurogenic function of Myt1l.

a, Schematic of Myt1l zinc-finger 2–3 point and deletion mutants. The ability to enhance neurogenic conversion together with Ascl1 is indicated by (+), non-functional mutants are indicated with (−) (see also Fig. 3). b, Sequence alignments and conservation of CCHC-type zinc-fingers from Myt1l; cysteine and histidine residues that can coordinate Zn(II) are highlighted in purple, non-coordinating mutated histidines are shown in green. c, Representative immunofluorescence of iN cells derived from MEFs upon reprogramming for 14 days with Ascl1 and the indicated transgenes; TUJ1 (red), DAPI staining (blue), scale bar, 50 μm. dh, Electrophysiological characterization of iN cells derived in c upon maturation for 21 days on mouse glia. d, Representative action potential (AP) traces of iN cells generated with indicated transgenes; pie charts indicate fraction of cells firing single (grey), multiple (white), or no (black) action potentials. e, Mean number of action potentials fired plotted with respect to pulse amplitude measured at −60 mV holding potential. f, Mean resting membrane potential (Vrest). g, h, Mean membrane resistance (Rm; g) and capacitance (Cm; h) measured at −70 mV holding potential. Dotted line indicates intrinsic properties of Ascl1 + wild-type Myt1l or Ascl1 + Myt1l200–912 cells; n = 3 biological replicates (total number of individual cells measured indicated), error bars show s.e.m., t-test *P < 0.05.

Extended Data Figure 8 Sin3b binds Myt1l via N-terminal SID domains and is essential for reprogramming.

a, Schematic of FLAG and NLS-tagged Myt1l truncations and GST-tagged Myt1l fusion proteins. Putative SIN3 interaction domains (SIDs) are highlighted (see also Fig. 3). Ability and inability to interact biochemically with Sin3b are indicated by (+) and (−), respectively. b, GST bait loading after pull down was controlled by Ponceau staining of the western blot membrane. Input, 0.2% of pull down input; pull down lanes, 20% of pull down eluates. c, ChIP–seq tracks of Sin3b, HDAC1, and Myt1l show binding at the Hes1 promoter 2 days after MEF reprogramming with Ascl1, Brn2 and wild-type Myt1l. Vertical bars mark Myt1l AAGTT motifs. d, Multiple sequence alignments of the highly conserved putative SIDs within minimal functional region of Myt1l from selected eukaryotic species. The alignment was generated using T-Coffee and putative SID regions are shown above the alignment. e, Western blot of iN cells derived from MEFs upon reprogramming for 2 days with Ascl1 and wild-type Myt1l together with either control or Sin3b-targeting shRNA constructs using indicated antibodies. f, Representative micrographs of iN cells derived in e upon reprogramming for 14 days followed by immunofluorescence using antibodies against TUJ1 (red) and DAPI staining (blue); scale bar, 50 μm. g, Conversion efficiency of cells shown in f based on TUJ1-positive cells with neuronal morphology highlight the deleterious effect of Sin3b knockdown on iN cell formation. n = 3, error bars show s.e.m., t-test *P < 0.005.

Extended Data Figure 9 Myt1l acts upstream of Hes1 to repress Notch signalling and stabilize Ascl1.

a, Immunofluorescence of iN cells quantified in Fig. 4a derived from MEFs upon reprogramming for 7 days with Ascl1 and wild-type Myt1l, NICD or a combination; TUJ1 (red), Tau–eGFP (green), DAPI staining (blue); scale bar, 50 μm. b, Neurogenic conversion efficiency of MEF cells upon reprogramming for 7 days with Ascl1 together with either wild-type Myt1l (n = 6), Hes1 (n = 3) or a combination of indicated transgenes or upon treatment with DAPT (10 μM) (n = 3) based on Tau–eGFP induction determined by flow cytometry. Dotted line indicates mean conversion efficiency of Ascl1 + Myt1l cells; error bars show s.d., t-test *P < 0.05. c, Western blot analysis of cells shown in a and b after 2 days of reprogramming and mouse NSCs using indicated antibodies shows no striking induction of the neural stem-cell markers nestin, Pax6 (arrowhead) or Sox1 in any condition but strong reduction of Ascl1 upon Hes1 overexpression. d, Mean expression levels of endogenous and exogenous (overexpressed) Ascl1 transcripts in MEFs upon overexpression of Ascl1 and Hes1 with or without wild-type Myt1l for 2 days determined by quantitative real time PCR show significant repression of both endogenous and exogenous Ascl1 by Hes1 overexpression independent of Myt1l. Expression levels were normalized to Ascl1 only induction and GAPDH expression. n = 4 biological replicates (with 4 technical replicates each), error bars show s.e.m., pair-wise fixed reallocation randomization test *P < 0.001 (ref. 51). e, Western blot analysis of MEF cells upon induction of Ascl1 together with GFP, wild-type Myt1l or Myt1l200–623 after 0, 2, 5 and 7 days of reprogramming using antibodies against Myt1l, Ascl1, GFP and tubulin shows no striking induction of full-length Myt1l upon overexpression of minimal fragment but stabilization of Ascl1 levels. f, Immunofluorescence of neurons quantified in Fig. 4c derived from mouse NSCs upon differentiation for 7 days with rtTA alone (mock) or in combination with Myt1l200–623; TUJ1 (red), Myt1l (green), DAPI staining (blue), scale bar 50 μm. Of note, all neurons formed in the control condition expressed endogenous Myt1l.

Extended Data Figure 10 Myt1l maintains neuronal identity by repression of non-neuronal programs.

a, Myt1l knockdown in P0 mouse primary hippocampal neuronal cultures impairs neuronal maturation and maintenance. Cells were infected with shRNA-expressing lentivirus on third day of in vitro culture and analysed 11 days later by quantitative western blot using indicated antibodies. While tubulin served as loading control, several neuronal markers were severely downregulated by Myt1l depletion. Representative western blot images are shown, n = 5, error bars show s.e.m., t-test *P < 0.05. bf, Electrophysiological characterization of Myt1l knockdown neurons derived in a. b, Representative action potential (AP) traces of hippocampal neurons upon indicated knockdown; pie charts indicate fraction of cells firing single (grey), multiple (white), or no (black) action potentials at the 90 pA pulse. c, Mean number of action potentials fired, plotted with respect to pulse amplitude measured at −60 mV holding potential. d, Mean resting membrane-potential (Vrest). e, f, Mean membrane resistance (Rm; e) and capacitance (Cm; f) measured at −70 mV holding potential. Dotted line indicates intrinsic properties upon control shRNA treatment. n = 5 biological replicates (total number of individual cells measured indicated), error bars show s.e.m., t-test *P < 0.05. g, Myt1l knockdown in P0 mouse primary hippocampal neuronal cultures induces non-neuronal gene expression programs. Overlap of Myt1l-bound target genes that are induced or repressed upon knock down of Myt1l in primary hippocampal neurons and indicated cell type-specific expression signatures determined by GeneOverlap50. Odds ratio >2 represents strong association, P values are shown; n.s., not significant. h, Relative number of Myt1l and REST DNA binding motifs at cell type specific-genes highlight depletion of Myt1l and enrichment of REST motifs at neuronal genes (t-test *P < 0.005). i, RNA–seq analysis of genes shown in a, confirm decreased expression of neuronal genes upon Myt1l depletion. In addition several Notch and Wnt signalling factors that are direct targets of Myt1l are de-repressed (see also Fig. 2c). In addition, transcription of several non-neuronal lineage specifiers is induced compared to the control. Shown are gene expression values of cells treated as in a based on RNA–seq; fold change is represented in logarithmic scale normalized to the control shRNA-treated sample, n = 2. j, Selected top gene ontology (GO) terms of Myt1l-targeted genes that are repressed (top) or induced (bottom) upon knock down in primary hippocampal neurons determined by PANTHER33. Enrichment scores and P values are shown. Highlighted are the terms ‘generation of neurons’ (green) in the repressed cluster and ‘negative regulation of neurogenesis’ (red) in the induced cluster. In addition this analysis highlights induction of several non-neuronal gene expression programs upon Myt1l depletion.

Supplementary information

Supplementary Information

Full scan western blots for all gel data. (PDF 2392 kb)

Supplementary Table 1

The parameters and identities of Myt1l chromatin binding peaks based on chromatin immunoprecipitation sequencing experiments in the manuscript. (XLSX 1410 kb)

Supplementary Table 2

Gene expression changes at Myt1l target genes based on RNA sequencing experiments in the manuscript. (XLSX 1994 kb)

Supplementary Table 3

Table comprising the genes used to specify the cell type specific gene expression signatures used for the GSEA analysis in Figure 2 and the gene overlap and motif analysis in Extended Data Figure 3 and 10. (XLSX 143 kb)

Supplementary Table 4

This table indicates the DNA primers and plasmid constructs used and generated in this study. (XLSX 38 kb)

Supplementary Table 5

Detailed results of the SELEX experiments shown in Figure 2. (XLSX 28 kb)

Supplementary Table 6

This table indicates the DNA sequences of shRNA oligos, quantitative PCR and mutagenesis DNA primers used in this study. (XLSX 46 kb)

Supplementary Table 7

The antibodies and their working dilutions as used in this study. (XLSX 34 kb)

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Mall, M., Kareta, M., Chanda, S. et al. Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates. Nature 544, 245–249 (2017). https://doi.org/10.1038/nature21722

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