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Pro-neuronal activity of Myod1 due to promiscuous binding to neuronal genes

Nature Cell Biology volume 22, pages 401–411 (2020)Cite this article

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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.

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Fig. 1: Myod1 and Ascl1 bind similar targets in fibroblasts.
Fig. 2: Differential binding affinity of Ascl1 and Myod1 at shared sites confer lineage specificity.
Fig. 3: Ascl1 and Myod1 targeting is characterized by distinct DNA-binding affinity.
Fig. 4: C-terminal and DNA-binding domains confer reprogramming specificity of Myod1 and Ascl1.
Fig. 5: Characterization of iN cells from the Myod1(Ascl1 B+C) domain swap.
Fig. 6: Pro-neuronal activity of Myod1 by promiscuous binding to neuronal targets.

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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.

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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

  1. Qian Yi Lee

    Present address: Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore, Singapore

  2. Soham Chanda

    Present address: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA

  3. Michael S. Kareta

    Present address: Genetics and Genomics Group, Sanford Research, Sioux Falls, SD, USA

  4. These authors contributed equally: Qian Yi Lee, Moritz Mall.

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Qian Yi Lee & Cheen Euong Ang

  2. 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

  3. 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

  4. Hector Institute for Translational Brain Research (HITBR) gGmbH, Heidelberg, Germany

    Moritz Mall, Juan M. Adrian-Segarra & Sarah D. Grieder

  5. Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

    Moritz Mall, Juan M. Adrian-Segarra & Sarah D. Grieder

  6. 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

  7. Center for Personal Dynamic Regulomes and Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Orly L. Wapinski, Rui Li & Howard Y. Chang

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Contributions

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.

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Correspondence to Marius Wernig.

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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, Ct and dCt (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).

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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

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