Pro-neuronal activity of myod1 due to promiscuous binding to neuronal genes

ABSTRACT The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages—that is, neuronal and muscle identities, respectively. It is

unclear how these two basic helix–loop–helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts,

yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong

Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent.

Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that

chromatin access of on-target pioneer factors is primarily driven by the protein–DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription

factor binding requires specific silencing mechanisms to ensure lineage fidelity. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS H3K9ME SELECTIVELY BLOCKS TRANSCRIPTION FACTOR ACTIVITY AND ENSURES DIFFERENTIATED TISSUE

INTEGRITY Article Open access 04 November 2021 POLY(ADP-RIBOSE) POLYMERASE 1 (PARP1) RESTRAINS MYOD-DEPENDENT GENE EXPRESSION DURING MUSCLE DIFFERENTIATION Article Open access 15 September

2020 SKELETAL MUSCLE DIFFERENTIATION INDUCES WIDE-RANGING NUCLEOSOME REPOSITIONING IN MUSCLE GENE PROMOTERS Article Open access 24 April 2024 DATA AVAILABILITY ChIP–seq, RNA-seq and ATAC-seq

data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE126414. Previously published RNA-seq data for Figs. 1 and 2

were deposited in GSE43916 and GSE72121; micrococcal nuclease digestion with deep sequencing data for Extended Data Fig. 1d were deposited in GSE40896; ChIP–seq data for endogenous TF

expression in Extended Data Fig. 1f were deposited in GSE48336, GSE55840, GSE21621, GSE44824 and GSE24852; ChIP–seq data for TF overexpression in mESCs (Extended Data Fig. 1e) were deposited

in GSE97715; and ATAC-seq data for Fig. 2 were deposited in GSE101397. All other data supporting the findings of this study are available from the corresponding author on reasonable

request. CODE AVAILABILITY Specific code used for data analysis is available on request. REFERENCES * Vierbuchen, T. & Wernig, M. Direct lineage conversions: unnatural but useful? _Nat.

Biotechnol._ 29, 892–907 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Takahashi, K. & Yamanaka, S. A decade of transcription factor-mediated reprogramming to

pluripotency. _Nat. Rev. Mol. Cell Biol._ 17, 183–193 (2016). Article  CAS  PubMed  Google Scholar  * Xu, J. et al. Direct lineage reprogramming: strategies, mechanisms, and applications.

_Cell Stem Cell_ 16, 119–134 (2015). Article  CAS  PubMed  Google Scholar  * Gascón, S., Masserdotti, G., Russo, G. L. & Götz, M. Direct neuronal reprogramming: achievements, hurdles,

and new roads to success. _Cell Stem Cell_ 21, 18–34 (2017). Article  CAS  PubMed  Google Scholar  * Chen, Y., Yang, Z., Zhao, Z.-A. & Shen, Z. Direct reprogramming of fibroblasts into

cardiomyocytes. _Stem Cell Res. Ther._ 8, 118 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Di Stefano, B. et al. C/EBPa poises B cells for rapid reprogramming into induced

pluripotent stem cells. _Nature_ 506, 235–239 (2014). Article  CAS  PubMed  Google Scholar  * Zaret, K. S. & Mango, S. E. Pioneer transcription factors, chromatin dynamics, and cell

fate control. _Curr. Opin. Genet. Dev._ 37, 76–81 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell

reprogramming. _Genes Dev._ 28, 2679–2692 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Chanda, S. et al. Generation of induced neuronal cells by the single reprogramming

factor ASCL1. _Stem Cell Rep._ 3, 282–296 (2014). Article  CAS  Google Scholar  * Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. _Nature_

463, 1035–1041 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Marro, S. et al. Direct lineage conversion of terminally differentiated hepatocytes to functional neurons.

_Cell Stem Cell_ 9, 374–382 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Tanabe, K. et al. Transdifferentiation of human adult peripheral blood T cells into neurons.

_Proc. Natl Acad. Sci. USA_ 115, 6470–6475 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Karow, M. et al. Direct pericyte-to-neuron reprogramming via unfolding of a neural

stem cell-like program. _Nat. Neurosci._ 21, 932–940 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Chouchane, M. et al. Lineage reprogramming of astroglial cells from

different origins into distinct neuronal subtypes. _Stem Cell Rep._ 9, 162–176 (2017). Article  CAS  Google Scholar  * Wapinski, O. L. et al. Hierarchical mechanisms for direct reprogramming

of fibroblasts to neurons. _Cell_ 155, 621–635 (2013). Article  CAS  PubMed  Google Scholar  * Wapinski, O. L. et al. Rapid chromatin switch in the direct reprogramming of fibroblasts to

neurons. _Cell Rep._ 20, 3236–3247 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Castro, D. S. et al. A novel function of the proneural factor Ascl1 in progenitor

proliferation identified by genome-wide characterization of its targets. _Genes Dev._ 25, 930–945 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Davis, R. L., Weintraub, H.

& Lassar, A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. _Cell_ 51, 987–1000 (1987). Article  CAS  PubMed  Google Scholar  * Choi, J. et al. MyoD

converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. _Proc. Natl Acad.

Sci. USA_ 87, 7988–7992 (1990). Article  CAS  PubMed  PubMed Central  Google Scholar  * Weintraub, H. et al. Activation of muscle-specific genes in pigment, nerve, fat, liver, and

fibroblast cell lines by forced expression of MyoD. _Proc. Natl Acad. Sci. USA_ 86, 5434–5438 (1989). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yao, Z. et al. Comparison of

endogenous and overexpressed MyoD shows enhanced binding of physiologically bound sites. _Skelet. Muscle_ 3, 8 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Berkes, C. A.

et al. Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential. _Mol. Cell_ 14, 465–477 (2004). Article  CAS  PubMed  Google

Scholar  * Maves, L. et al. Pbx homeodomain proteins direct Myod activity to promote fast-muscle differentiation. _Development_ 134, 3371–3382 (2007). Article  CAS  PubMed  Google Scholar  *

Fong, A. P. et al. Genetic and epigenetic determinants of neurogenesis and myogenesis. _Dev. Cell_ 22, 721–735 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Treutlein, B.

et al. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. _Nature_ 534, 391–395 (2016). * Teif, V. B. et al. Genome-wide nucleosome positioning during

embryonic stem cell development. _Nat. Struct. Mol. Biol._ 19, 1185–1192 (2012). Article  CAS  PubMed  Google Scholar  * Casey, B. H., Kollipara, R. K., Pozo, K. & Johnson, J. E.

Intrinsic DNA binding properties demonstrated for lineage-specifying basic helix-loop-helix transcription factors. _Genome Res._ 28, 484–496 (2018). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Borromeo, M. D. et al. A transcription factor network specifying inhibitory versus excitatory neurons in the dorsal spinal cord. _Development_ 141, 2803–2812 (2014).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Webb, A. E. et al. FOXO3 shares common targets with ASCL1 genome-wide and inhibits ASCL1-dependent neurogenesis. _Cell Rep._ 4,

477–491 (2013). Article  CAS  PubMed  Google Scholar  * Marinov, G. K., Kundaje, A., Park, P. J. & Wold, B. J. Large-scale quality analysis of published ChIP-seq data. _G3_ 4, 209–223

(2014). Article  PubMed  Google Scholar  * Mullen, A. C. et al. Master transcription factors determine cell-type-specific responses to TGF-β signaling. _Cell_ 147, 565–576 (2011). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Soleimani, V. D. et al. Snail regulates MyoD binding-site occupancy to direct enhancer switching and differentiation-specific transcription in

myogenesis. _Mol. Cell_ 47, 457–468 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime

cis-regulatory elements required for macrophage and B cell identities. _Mol. Cell_ 38, 576–589 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Davis, R. L. & Weintraub,

H. Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12. _Science_ 256, 1027–1030 (1992). Article  CAS  PubMed  Google Scholar  * Davis, R. L.,

Cheng, P. F., Lassar, A. B. & Weintraub, H. The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. _Cell_ 60, 733–746 (1990). Article  CAS  PubMed 

Google Scholar  * Ma, P. C., Rould, M. A., Weintraub, H. & Pabo, C. O. Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for

transcriptional activation. _Cell_ 77, 451–459 (1994). Article  CAS  PubMed  Google Scholar  * Weintraub, H. et al. Muscle-specific transcriptional activation by MyoD. _Genes Dev._ 5,

1377–1386 (1991). Article  CAS  PubMed  Google Scholar  * Nakada, Y., Hunsaker, T. L., Henke, R. M. & Johnson, J. E. Distinct domains within Mash1 and Math1 are required for function in

neuronal differentiation versus neuronal cell-type specification. _Development_ 131, 1319–1330 (2004). Article  CAS  PubMed  Google Scholar  * Chien, C. T., Hsiao, C. D., Jan, L. Y. &

Jan, Y. N. Neuronal type information encoded in the basic-helix-loop-helix domain of proneural genes. _Proc. Natl Acad. Sci. USA_ 93, 13239–13244 (1996). Article  CAS  PubMed  PubMed Central

  Google Scholar  * Mall, M. et al. Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates. _Nature_ 544, 245–249 (2017). Article  CAS  PubMed  Google Scholar  *

Hobert, O. & Kratsios, P. Neuronal identity control by terminal selectors in worms, flies, and chordates. _Curr. Opin. Neurobiol._ 56, 97–105 (2019). Article  CAS  PubMed  Google Scholar

  * Ptashne, M. Regulation of transcription: from lambda to eukaryotes. _Trends Biochem. Sci._ 30, 275–279 (2005). Article  CAS  PubMed  Google Scholar  * Chronis, C. et al. Cooperative

binding of transcription factors orchestrates reprogramming. _Cell_ 168, 442–459 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Soufi, A. et al. Pioneer transcription

factors target partial DNA motifs on nucleosomes to initiate reprogramming. _Cell_ 161, 555–568 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Soufi, A., Donahue, G. &

Zaret, K. S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. _Cell_ 151, 994–1004 (2012). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Luna-Zurita, L. et al. Complex interdependence regulates heterotypic transcription factor distribution and coordinates cardiogenesis. _Cell_ 164, 999–1014 (2016). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Metzakopian, E. et al. Genome-wide characterisation of Foxa1 binding sites reveals several mechanisms for regulating neuronal differentiation

in midbrain dopamine cells. _Development_ 142, 1315–1324 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hirai, H. et al. Radical acceleration of nuclear reprogramming by

chromatin remodeling with the transactivation domain of MyoD. _Stem Cells_ 29, 1349–1361 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Fong, A. P. et al. Conversion of MyoD to a

neurogenic factor: binding site specificity determines lineage. _Cell Rep._ 10, 1937–1946 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bailey, T. L. et al. MEME SUITE:

tools for motif discovery and searching. _Nucleic Acids Res._ 37, W202–W208 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ross-Innes, C. S. et al. Differential oestrogen

receptor binding is associated with clinical outcome in breast cancer. _Nature_ 481, 389–393 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Huang, D. W., Sherman, B. T.

& Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. _Nat. Protoc._ 4, 44–57 (2009). Article  CAS  Google Scholar  * Tucker, K.

L., Meyer, M. & Barde, Y.-A. Neurotrophins are required for nerve growth during development. _Nat. Neurosci._ 4, 29–37 (2001). Article  CAS  PubMed  Google Scholar  * Marro, S. &

Yang, N. in _Stem Cell Transcriptional Networks. Methods in Molecular Biology (Methods and Protocols)_ Vol. 1150 (ed. Kidder B.) 237–246 (Humana Press, 2014). * Boyer, L. A. et al. Core

transcriptional regulatory circuitry in human embryonic stem cells. _Cell_ 122, 947–56 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  * Langmead, B. & Salzberg, S. L. Fast

gapped-read alignment with Bowtie 2. _Nat. Methods_ 9, 357–359 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang, Y. et al. Model-based Analysis of ChIP-Seq (MACS).

_Genome Biol._ 9, R137 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, Q., Brown, J. B., Huang, H. & Bickel, P. J. Measuring reproducibility of high-throughput

experiments. _Ann. Appl. Stat._ 5, 1752–1779 (2011). Article  Google Scholar  * Schmieder, R. & Edwards, R. Quality control and preprocessing of metagenomic datasets. _Bioinformatics_

27, 863–864 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene

fusions. _Genome Biol._ 14, R36 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated

transcripts and isoform switching during cell differentiation. _Nat. Biotechnol._ 28, 511–515 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Buenrostro, J. D., Giresi, P.

G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome

position. _Nat. Methods_ 10, 1213–1218 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Pfaffl, M. W., Horgan, G. W. & Dempfle, L. Relative expression software tool (REST)

for group-wise comparison and statistical analysis of relative expression results in real-time PCR. _Nucleic Acids Res._ 30, e36 (2002). Article  PubMed  PubMed Central  Google Scholar  *

Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. _Mol. Syst. Biol._ 7, 539 (2011). Article  PubMed  PubMed Central 

Google Scholar  Download references ACKNOWLEDGEMENTS We thank the Stanford Functional Genomics Facility for sequencing (NIH award S10OD018220) and members of the Wernig and Chang lab for

their ideas and discussions. Support was provided by the National Science Scholarship from the Agency for Science, Technology and Research to Q.Y.L.; the Hector Foundation II and the

European Research Council to M.M.; the National Institutes of Health to T.C.S., H.Y.C. and M.W.; and the California Institute for Regenerative Medicine to H.Y.C. and M.W. H.Y.C. and T.C.S.

are Investigators of the Howard Hughes Medical Institute. M.W. is a Tashia and John Morgridge Faculty Scholar at the Child Health Research Institute at Stanford and a Howard Hughes Medical

Institute Faculty Scholar. AUTHOR INFORMATION Author notes * Qian Yi Lee Present address: Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology

and Research, Singapore, Singapore * Soham Chanda Present address: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA * Michael S. Kareta

Present address: Genetics and Genomics Group, Sanford Research, Sioux Falls, SD, USA * These authors contributed equally: Qian Yi Lee, Moritz Mall. AUTHORS AND AFFILIATIONS * Department of

Bioengineering, Stanford University, Stanford, CA, USA Qian Yi Lee & Cheen Euong Ang * Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, Stanford

University, Stanford, CA, USA Qian Yi Lee, Moritz Mall, Soham Chanda, Bo Zhou, Kylesh S. Sharma, Katie Schaukowitch, Sarah D. Grieder, Michael S. Kareta, Cheen Euong Ang & Marius Wernig

* Cell Fate Engineering and Disease Modeling Group, German Cancer Research Center (DKFZ) and DKFZ–ZMBH Alliance, Heidelberg, Germany Moritz Mall, Juan M. Adrian-Segarra & Sarah D.

Grieder * Hector Institute for Translational Brain Research (HITBR) gGmbH, Heidelberg, Germany Moritz Mall, Juan M. Adrian-Segarra & Sarah D. Grieder * Central Institute of Mental

Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Moritz Mall, Juan M. Adrian-Segarra & Sarah D. Grieder * Center for Personal Dynamic Regulomes, Department of

Genetics, Department of Dermatology, Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA Soham Chanda, Bo Zhou & Thomas C. Südhof * Center for Personal Dynamic

Regulomes and Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA Orly L. Wapinski, Rui Li & Howard Y. Chang Authors * Qian Yi Lee View author publications You can

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Conceptualization: Q.Y.L., M.M. and M.W. Methodology: Q.Y.L., M.M., H.Y.C. and M.W. Software: Q.Y.L. and M.S.K. Formal analysis: Q.Y.L., M.M., M.S.K., S.C., B.Z., K.S. and J.M.A.-S.

Investigation: Q.Y.L., M.M., S.C., B.Z., K.S.S., K.S., J.M.A.-S., S.D.G., M.S.K., O.L.W., C.E.A. and R.L. Writing of the original draft: Q.Y.L. and M.M. Writing, review and editing: Q.Y.L.,

M.M. and M.W. Funding acquisition: M.W. and H.Y.C. Resources: M.W. and H.Y.C. Supervision: T.C.S., H.Y.C. and M.W. CORRESPONDING AUTHOR Correspondence to Marius Wernig. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps

and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 BOTH ASCL1 AND MYOD1 BIND THEIR ENDOGENOUS BINDING SITES IN THE CLOSED CHROMATIN CONTEXT IN MEFS. A,B, Heatmap showing

FLAG ChIP-seq normalized read counts ±1 kb around peak summits for reproducible peaks from (A) FLAG–Ascl1 or (B) FLAG–Myod1 expressing MEFs 48 h after dox-induction. Cells expressing only

rtTA alone were used as a control, and the corresponding peaks from FLAG-ChIP of rtTA control samples were plotted next to Ascl1 or Myod1 samples to show specificity of FLAG antibody

pull-down. C, Distribution of Ascl1 (top) and Myod1 (bottom) peaks from (A-B). pro=promoter, enh=enhancer, gt=genetail, int=intergenic. D, Nucleosomal occupancy based on micrococcal nuclease

digestion combined with sequencing (MNase-seq) signal in MEFs26. Higher signal indicates increased protection from MNase digestion by nucleosome and thus decreased chromatin accessibility.

Both Ascl1 (purple) and Myod1(magenta) bind to closed regions compared to the randomized control (orange). E, Heatmap showing ChIP-seq normalized read counts ±1 kb around summits of merged

peaks from FLAG ChIP in FLAG-Ascl1 expressing MEFs (48 h post induction), Ascl1 ChIP in Ascl1 expressing mESC (24 h post induction)27, FLAG ChIP in FLAG-Ascl1 expressing mESCs (48 h post

induction), FLAG ChIPs in FLAG-Myod1 expressing mESCs (24 h27 and 48 h post induction), and FLAG ChIP in rtTA expressing mESC as a negative control. Heatmap was sorted by signal in 48 h mESC

FLAG-Ascl1 ChIP-seq. F, Heatmap showing ChIP-seq normalized read counts ±1 kb around summits of merged peaks from FLAG ChIP in FLAG-Ascl1 expressing MEFs (48 h post induction), endogenous

Ascl1 ChIP in E12.5 neural tubes28 and adult neural progenitor cells29, FLAG ChIP in FLAG-Myod1 expressing MEFs (48 h post induction), and endogenous Myod1 ChIP in differentiating C2C12

cells30,31 and skeletal myoblasts32, sorted by the combined FLAG-Ascl1 ChIP-seq signal. G, Gene ontology terms for clusters of significantly changing genes defined in Fig. 1d using DAVID

v6.852. Source data EXTENDED DATA FIG. 2 DIFFERENTIAL BINDING AFFINITIES OF ASCL1 AND MYOD1 MEDITATE DISTINCT TRANSCRIPTION FACTOR ACTIONS. A,B, Average normalized read counts of all peaks

for (A) H3K27ac ChIP and (B) ATAC-seq in no-virus control (novir), Ascl1 (A1) and Myod1 (MD) samples 48 hr post-TF-induction, separated into Ascl1-enriched, Myod1-enriched and common peaks

(from Fig. 2a). C, Gene ontology of nearest genes associated with a FLAG ChIP-seq peak that fall within the gene promoter or enhancer at Ascl1-enriched, Myod1-enriched and common peaks (from

Fig. 2a) using DAVID v6.852. (D–G) Genome browser tracks showing FLAG ChIP signals and corresponding H3K27ac, ATAC-seq and RNA-seq signals for neurogenic genes, (D) Sox11 and (F) Syn2, and

myogenic genes (E) Klhl41 and (G) Myog. Source data EXTENDED DATA FIG. 3 MOTIF ENRICHMENT AT DIFFERENTIALLY BOUND SITES. A, Reverse search of known motifs at FLAG ChIP peaks, as defined in

Fig. 2a, that are associated with significantly changing genes (fold-change ≥ 1.2, p ≤ 0.05) using HOMER33. Ascl1-enriched and common sites mainly contain bHLH motifs, while Myod1-enriched

sites also contain homeodomains and other motifs. HOMER uses ZOOPS scoring (zero or one occurrence per sequence) coupled with hypergeometric enrichment calculations to determine

significance. (B-C) Examples of (B) bHLH and (C) homeobox motifs that are enriched in differentially bound sites from Fig. 3. EXTENDED DATA FIG. 4 BASIC DOMAIN AND C-TERMINUS OF ASCL1 ARE

SUFFICIENT TO CONFER NEURONAL REPROGRAMMING ABILITY. A-B, Representative immunofluorescence images of cells reprogrammed using indicated (A) Ascl1 and (B) Myod1 domain swap constructs, as

summarized in Fig. 4a, 14 days post TF induction stained with TUJ1 (green), DES (red) and DAPI (blue) (top) or with MAP2 (green), MYH (red) and DAPI (blue) (bottom). Repeated 3 times with

similar results. Scale bar: 100μm. C, Representative immunofluorescence images of cells reprogrammed using Myod1(Ascl1 B+C) domain swaps with various truncations on the Ascl1 C-terminus

stained with TUJ1 (green), MAP2 (red) and DAPI staining (blue). The full-length Ascl1 C-terminus has 66 amino acids and we tested truncations containing 15, 7 or 0 amino acids for their

effect. While reprogramming efficiency is reduced in the C-terminal truncations only 7 amino acids were sufficient to induced Tuj1+/Map2+ neurons. Removing the entire C-terminus abolished

reprogramming activity completely. Repeated 3 times with similar results. Scale bar: 200μm. D, Sequence alignment of mouse Ascl1 (purple) and Myod1 (magenta) using Clustal Omega64 with

highlighted N- and C-terminus as well as basic (B), helix 1 (H1), loop (L), and helix 2 (H2) domains. EXTENDED DATA FIG. 5 FUSING PBX1B TO ASCL1 CONTAINING THE BASIC REGION OF MYOD1 ENHANCES

INDUCTION OF A SUBSET OF MYOD1 TARGET GENES. A, Representative immunofluorescence images of cells 14 days post TF induction using the indicated Ascl1, Ascl1-Pbx1b fusion, Ascl1 (Myod1

B)-Pbx1b fusion or Myod1 constructs, stained with TUJ1 (green), DES (red) and DAPI staining (blue) (top) or stained with MAP2 (green), MYH (red) and DAPI staining (blue) (bottom). While all

constructs containing Ascl1 (Myod1 B) induced the muscle markers DES and MYH, fusion to Pbx1b did not greatly alter the switch to a muscle cell fate. Repeated 3 times with similar results.

Scale bar: 400 µm. B, Mean expression levels of muscle and neuronal marker genes of cells in A determined by qRT-PCR. Fusion of Pbx1b to Ascl1 or Ascl1 (Myod1 B) does not enhance the

expression of muscle markers Des or Myh3. n = 3 biologically independent samples, error bars = SEM, two-tailed Student’s t-test * p < 0.05; ** p < 0.01; *** p < 0.001. C, Mean

expression levels of potential Myod1 and Pbx1b target genes in cells from (A) determined by qRT-PCR. A subset of these genes (Myog, Cdh15, Dysf) increased expression in the Pbx1b-Ascl1

(Myod1 B) condition compared to Ascl1 (Myod1 B) alone (top row). n = 3 biologically independent samples, error bars = SEM, two-tailed Student’s t-test * p < 0.05; ** p < 0.01; *** p

< 0.001. Source data EXTENDED DATA FIG. 6 PRO-NEURONAL ACTIVITY OF MYOD1 UPON CO-EXPRESSION OF MULTI-LINEAGE REPRESSOR MYT1L. A, Representative immunofluorescence images of reprogrammed

cells with MAP2 (red), MYH (green) and DAPI (blue) 14 days after TF-induction. Scale bar: 100 µm. B, Single cell quantification of MAP2+ cells with neuronal morphology (left) and MYH+ cells

(right) in A. n = 3 biologically independent samples. C, Normalized expression levels of neuronal and muscle marker genes 14 days post TF induction, determined by qRT-PCR. n = 3 biologically

independent samples. D, Western blot of reprogramming MEFs in A, two days post TF induction. E, Representative immunofluorescence images of MEFs reprogrammed with either Ascl1+Myt1l+Gfp or

Myod1+Myt1l+Gfp for six weeks on primary mouse glia. Scale bar: 50μm. F, Quantification of the fraction of TUJ1+/GFP+ neurons found in E that are vGAT or VGLUT positive. Violin plots

indicate the median and interquartile range. n = 4 technical replicates. (G,J) Representative immunofluorescence images of reprogrammed MEFs 14 days post Ascl1 (G) or Myod1 (J) induction

together with either Gfp, Myt1l full length, or a non-functional Myt1l (410-623) truncation that contains two DNA binding zinc fingers fused with either an engrailed repressor (EnR) domain

or a VP64-activator (VP64) domain. Scale bar: 100 µm. (H,K) Single cell quantification of TUJ1+ cells with neuronal morphology (green) and DES+ cells (red) in G,J upon addition of Myt1l full

length or Myt1l-EnR fusion constructs to either Ascl1 (H) or Myod1 (K) alone. n = 3 biologically independent samples. (I,L) Western blot of reprogramming MEFs in G,J two days post-induction

of Ascl1 (I) or Myod1 (L) in combination with indicated constructs. All immunofluorescence and western blots were repeated 3 times with similar results, except ED Fig. 6e, which was only

repeated twice; Unprocessed blots ED Fig. 6; For all bar graphs, error bars = SEM, two-tailed Student’s t-test, * p < 0.05, ** p < 0.01; *** p < 0.001. Source data SUPPLEMENTARY

INFORMATION REPORTING SUMMARY SUPPLEMENTARY VIDEO 1 Movie of twitching induced muscle cells using Myod1 or Ascl1(Myod1 B+C). SUPPLEMENTARY TABLES Supplementary Table 1. Quantitative

real-time polymerase chain reaction (qRT-PCR) primers. Supplementary Table 2. List of antibodies. Supplementary Table 3. (1) cDNA sequences for domain swap proteins. (2) cDNA sequences for

Myod1 (Ascl1 B+C) truncation proteins. (3) cDNA sequences for Pbx-fusion proteins. SOURCE DATA SOURCE DATA FIG. 1 c, Normalized read counts for Ascl1 and Myod1 ChIP-seq, ±1 kb around merged

peak summits, sorted by MEF_flagAscl1_r1 ChIP-seq signal. Reads for each sample are binned into 100 windows of 20 bp each. d, Left, log2[FPKM] of all differentially expressed genes (fold

change ≥ 2, FDR ≤ 0.05, with respect to rtTA control) 2 d after TF induction. Right, scaled _Z_-scores for log2[FPKM] values on the left. SOURCE DATA FIG. 1 Unprocessed western blots

(bottom). SOURCE DATA FIG. 2 a, (1) log2[FC] of Ascl1/Myod1 RPKM values summed from +/-100 bp around merged peak summits. Peaks are ordered first by FDR ≤ 0.05 cut-off, then by log2[FC] of

RPKM. Peak classification was performed using FDR and fold changes calculated using Diffbind 2.12.0 (using DEseq2 for differential binding analysis). Peaks are classified into Ascl1-enriched

(FDR ≤ 0.05, FC ≥ 2), Myod1-enriched (FDR ≤ 0.05, FC ≤ −2) and common (all other peaks). (2) log2[FC] of quantile normalized RPKM values for H3K27ac ChIP-seq, summed from ±500 bp around

merged peak summits, over average of MEF controls. (3) log2[FC] of quantile normalized RPKM values for ATAC-seq, summed from ±100 bp around merged peak summits, over MEF control. (4)

log2[FC] of RPKM values of nearest gene with respect to the rtTA controls. FLAG ChIP peaks were associated with a nearest gene, taking only genes with a peak that falls within the promoter

or enhancer regions (between 20 kb upstream and 2 kb downstream from the TSS). c, Average normalized read counts of H3K27ac ChIP-seq in no-virus control, Ascl1 and Myod1 conditions at

Ascl1-enriched, Myod1-enriched and common peaks, including only peaks that are associated with significantly changing genes (fold change ≥ 1.2, _P_ ≤ 0.05). Counts are taken ±1 kb from

merged peak summits, binned into 100 windows. d, Average normalized read counts of ATAC-seq in no-virus control, Ascl1 and Myod1 conditions at Ascl1-enriched, Myod1-enriched and common

peaks, including only peaks that are associated with significantly changing genes (fold change ≥ 1.2, _P_ ≤ 0.05). Counts are taken ±1 kb from merged peak summits, binned into 100 windows.

SOURCE DATA FIG. 4 c, Left, counts of Tuj1+ or Des+ cells per 20× image taken, ten images per biological replicate. Middle, average of Tuj1+ or Des+ cells per 10× field of view in each

biological replicate. Right, mean and s.e.m. of Tuj1+ or Des+ cells in Ascl1, Myod1 or Myod1(Ascl1 B+C) conditions, and pairwise _t_-tests for all three conditions. d, Left, _C_t and d_C_t

(with respect to _Gapdh_) of each individual biological replicate. Right, normalized (% _Gapdh_) expression of _Des_, _Myh_, _Map2_ and _Tuj1_ in no virus (control) and Ascl1, Myod1 and

Myod1(Ascl1 B+C) conditions at 2 d and 14 d post TF induction. SOURCE DATA FIG. 4 Unprocessed western blots (bottom). SOURCE DATA FIG. 5 a–c, Electrophysiological characterization of induced

neuronal cells at 13–14 d post dox induction. a, Fraction of cells firing success and failure action potentials at the 85-pA pulse. b, AP threshold. c, AP height. d–f, Electrophysiological

characterization of induced neuronal cells at 22–24 d post dox induction. d, Fraction of cells firing single or multiple action potentials at 85-pA pulse. e, AP threshold. f, Mean number of

action potentials fired, plotted with respect to pulse amplitude measured at −60 mV holding potential. g, Normalized RPKM values of FLAG–Ascl1, FLAG–Myod1 (Ascl1 B+C) and FLAG–Myod1 FLAG

ChIP-seq. Reproducible peaks from all three conditions were merged. RPKM was calculated based on tag counts within ±100 bp of each peak summit and then normalized. Peaks were sorted by the

fold change between FLAG–Ascl1 and FLAG–Myod1 conditions. SOURCE DATA FIG. 6 b, Average number of indicated marker+ cells counted in 10*10× fields of view in MEFs upon TF induction.

Individual replicates on the top, mean and s.e.m. on the bottom. c, AP firing traces for Ascl1+Gfp or Myod1+Myt1l. _n_ = 8 different cells for both conditions. d, AP threshold (14 d after TF

induction). e, AP height (14 d after TF induction). f, Normalized RPKM values of FLAG–Ascl1, FLAG–Myod1+Myt1l and FLAG–Myod1 FLAG ChIP-seq. Reproducible peaks from all three conditions were

merged and normalized RPKM was calculated based on tag counts within ±100 bp of each peak summit. Peaks were sorted by the fold change between FLAG–Ascl1 and FLAG–Myod1 conditions. SOURCE

DATA EXTENDED DATA FIG. 1 a, FLAG ChIP-seq normalized read counts ±1 kb around peak summits for reproducible peaks from FLAG–Ascl1- or rtTA-expressing cells 48 h after dox induction. Reads

for each sample are binned into 100 windows of 20 bp each. b, FLAG ChIP-seq normalized read counts ±1 kb around peak summits for reproducible peaks from FLAG–Myod1- or rtTA-expressing cells

48 h after dox induction. Reads for each sample are binned into 100 windows of 20 bp each. d, Nucleosomal occupancy based on MNase-seq signal in MEFs. e, ChIP-seq normalized read counts ±1 

kb around summits of merged peaks from FLAG ChIP in FLAG–Ascl1-expressing MEFs (48 h post induction), Ascl1 ChIP in Ascl1-expressing mESC (24 h post induction), FLAG ChIP in

FLAG–Ascl1-expressing mESCs (48 h post induction), FLAG ChIPs in FLAG–Myod1-expressing mESCs (24 h and 48 h post induction) and FLAG ChIP in rtTA-expressing mESCs as a negative control.

Reads for each sample are binned into 100 windows of 20 bp each and peaks are sorted by signal in 48 h mESC FLAG–Ascl1 ChIP-seq. f, ChIP–seq normalized read counts ±1 kb around summits of

merged peaks from FLAG ChIP in FLAG–Ascl1-expressing MEFs (48 h post induction), endogenous Ascl1 ChIP in E12.5 neural tubes and adult neural progenitor cells, FLAG ChIP in

FLAG–Myod1-expressing MEFs (48 h post induction) and endogenous Myod1 ChIP in differentiating C2C12 cells and skeletal myoblasts, sorted by the combined FLAG–Ascl1 ChIP-seq signal. Reads for

each sample are binned into 100 windows of 20 bp each. SOURCE DATA EXTENDED DATA FIG. 2 a, Average normalized read counts of all peaks for H3K27ac ChIP in no-virus control, Ascl1 and Myod1

conditions at Ascl1-enriched, Myod1-enriched and common peaks, ±1 kb from merged peak summits, binned into 100 windows. b, Average normalized read counts of all peaks for ATAC-seq in

no-virus control, Ascl1 and Myod1 conditions at Ascl1-enriched, Myod1-enriched and common peaks, ±1 kb from merged peak summits, binned into 100 windows. SOURCE DATA EXTENDED DATA FIG. 5

b,c, Expression levels of muscle and neuronal markers (b), and potential Myod1+Pbx target genes (c) in MEFs upon TF induction. Individual replicates on the left, mean and s.e.m. on the

right. SOURCE DATA EXTENDED DATA FIG. 6 b, Average number of indicated marker+ cells counted in 10 × 10× fields of view in MEFs upon TF induction. Individual replicates on the top, mean and

s.e.m. on the bottom. c, Expression levels of indicated muscle and neuronal marker genes in MEFs upon TF induction. Individual replicates on the top, mean, s.d. and s.e.m. on the bottom. f,

vGlut or vGat costaining of randomly selected GFP+Tuj1+ cells with neuronal morphology upon indicated TF induction in MEFS. Replicates on the left, mean and percentage on the right. h, Ascl1

 + Myt1l full-length/fusion constructs. k, Myod1 + Myt1l full-length/fusion constructs. Average number of indicated marker+ cells counted in 10 × 10× fields of view in MEFs upon TF

induction. Individual replicates on the top, mean and s.e.m. on the bottom. SOURCE DATA EXTENDED DATA FIG. 6 Unprocessed western blots (bottom). RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Lee, Q.Y., Mall, M., Chanda, S. _et al._ Pro-neuronal activity of Myod1 due to promiscuous binding to neuronal genes. _Nat Cell Biol_ 22,

401–411 (2020). https://doi.org/10.1038/s41556-020-0490-3 Download citation * Received: 17 March 2019 * Accepted: 18 February 2020 * Published: 30 March 2020 * Issue Date: April 2020 * DOI:

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