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MYCL promotes iPSC-like colony formation via MYC Box 0 and 2 domains

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Akifuji, Chiaki, Iwasaki, Mio, Kawahara, Yuka, Sakurai, Chiho, Cheng, Yu-Shen, Imai, Takahiko, Nakagawa, Masato·2021·Scientific Reports
DOI 10.1038/s41598-021-03260-5PMC PMC8688507

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Abstract

Human induced pluripotent stem cells (hiPSCs) can differentiate into cells of the three germ layers and are promising cell sources for regenerative medicine therapies. However, current protocols generate hiPSCs with low efficiency, and the generated iPSCs have variable differentiation capacity among different clones. Our previous study reported that MYC proteins (c-MYC and MYCL) are essential for reprogramming and germline transmission but that MYCL can generate hiPSC colonies more efficiently than c-MYC. The molecular underpinnings for the different reprogramming efficiencies between c-MYC and MYCL, however, are unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two functional domains conserved in the MYC protein family, contribute to the phenotypic differences and promote hiPSC generation in MYCL-induced reprogramming. Proteome analyses suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are regulated by the MB0 domain, while the MB2 domain regulates RNA processes. These findings provide a molecular explanation for why MYCL has higher reprogramming efficiency than c-MYC.

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Figure 1 MYCL promotes reprogramming more efficiently than c-MYC. (A) Schematic representation of HDF reprogramming with Sendai virus (SeV). HDFs were transduced with SeV carrying KLF4-OCT3/4-SOX2 (KOS), KLF4 (K), and c-MYC or MYCL on day 0. We used an MOI (multiplicity of infection) of 4.3 for each virus. StemFit AK03N without bFGF was used during the transduction and subsequent induction of iPSC-like colonies. We performed immunostaining of the reprogramming HDFs 1 to 7 days after the transduction and analyzed the results using ArrayScan. (B) Representative immunostaining images of reprogramming HDFs stained by anti-TRA-1-60 antibody (green) and Hoechst (blue) 7 days after the transduction. Scale bar, 300 μm. Ph, phase contrast. (C) Proliferation and expression of TRA-1-60 ( +) cells during reprogramming. HDFs were transduced with SeV, including c-MYC or MYCL, and immunostaining was performed from days 1 to 7. The number of total cells was counted as Hoechst-positive cells. Mean ± SD values are shown. n = 3, *p < 0.05 by paired t-test. (D) Schematic representation of HDF reprogramming with episomal plasmid vector (EpiP). HDFs were transduced with EpiP carrying SOX2, KLF4, OCT3/4-shp53, LIN28A, EBNA1, and c-MYC or MYCL. StemFit AK03N without bFGF was used during the transfection and subsequent induction of iPSC-like colonies. We performed flow cytometry of the reprogramming HDFs every three days from 1 to 19 days plus day 21 after the transduction. (E) Representative immunostaining images of reprogramming HDFs stained by anti-TRA-1-60 antibody (green) and Hoechst (blue) 21 days after the transduction. Scale bar, 300 μm. Ph, phase contrast. (F) Proliferation and expression of TRA-1-60 ( +) cells during reprogramming were analyzed by flow cytometry. HDFs were transduced with EpiP, including c-MYC or MYCL. Flow cytometry was performed every three days from days 1 to 19 days plus day 21. Mean ± SD for n = 3, *p < 0.05 and **p < 0.01 by paired t-test. (G) The number of iPSC-like and non-iPSC-like colonies derived from 1 × 105 HDFs transduced with EpiP including c-MYC or MYCL on day 21. Mean ± SD values are shown. n = 3, **p < 0.01 by unpaired t-test. (H) Percentage of CD13 ( +) cells during EpiP reprogramming determined by flow cytometry. Mean ± SD values are shown. n = 3, *p < 0.05 and **p < 0.01 by paired t-test.
Figure 1 MYCL promotes reprogramming more efficiently than c-MYC. (A) Schematic representation of HDF reprogramming with Sendai virus (SeV). HDFs were transduced with SeV carrying KLF4-OCT3/4-SOX2 (KOS), KLF4 (K), and c-MYC or MYCL on day 0. We used an MOI (multiplicity of infection) of 4.3 for each virus. StemFit AK03N without bFGF was used during the transduction and subsequent induction of iPSC-like colonies. We performed immunostaining of the reprogramming HDFs 1 to 7 days after the transduction and analyzed the results using ArrayScan. (B) Representative immunostaining images of reprogramming HDFs stained by anti-TRA-1-60 antibody (green) and Hoechst (blue) 7 days after the transduction. Scale bar, 300 μm. Ph, phase contrast. (C) Proliferation and expression of TRA-1-60 ( +) cells during reprogramming. HDFs were transduced with SeV, including c-MYC or MYCL, and immunostaining was performed from days 1 to 7. The number of total cells was counted as Hoechst-positive cells. Mean ± SD values are shown. n = 3, *p < 0.05 by paired t-test. (D) Schematic representation of HDF reprogramming with episomal plasmid vector (EpiP). HDFs were transduced with EpiP carrying SOX2, KLF4, OCT3/4-shp53, LIN28A, EBNA1, and c-MYC or MYCL. StemFit AK03N without bFGF was used during the transfection and subsequent induction of iPSC-like colonies. We performed flow cytometry of the reprogramming HDFs every three days from 1 to 19 days plus day 21 after the transduction. (E) Representative immunostaining images of reprogramming HDFs stained by anti-TRA-1-60 antibody (green) and Hoechst (blue) 21 days after the transduction. Scale bar, 300 μm. Ph, phase contrast. (F) Proliferation and expression of TRA-1-60 ( +) cells during reprogramming were analyzed by flow cytometry. HDFs were transduced with EpiP, including c-MYC or MYCL. Flow cytometry was performed every three days from days 1 to 19 days plus day 21. Mean ± SD for n = 3, *p < 0.05 and **p < 0.01 by paired t-test. (G) The number of iPSC-like and non-iPSC-like colonies derived from 1 × 105 HDFs transduced with EpiP including c-MYC or MYCL on day 21. Mean ± SD values are shown. n = 3, **p < 0.01 by unpaired t-test. (H) Percentage of CD13 ( +) cells during EpiP reprogramming determined by flow cytometry. Mean ± SD values are shown. n = 3, *p < 0.05 and **p < 0.01 by paired t-test.CC BY · PubMed Central
Figure 2 MYC Box 0 and 2 domains are crucial for colony formation during reprogramming. (A) Schematic representation of WT c-MYC and MYCL protein. Black boxes show important domains for MYC function, including MB0, 1, 2, 3a (c-MYC only), 3b, 4, and basic-helix-loop-helix leucine zipper motif (bHLHLZ). The percentage of common amino acids in each MYC box domain between MYCL and c-MYC is shown (Identity%). The numbers on the right indicate amino acid lengths. (B) Number of iPSC-like and non-iPSC-like colonies transduced with EpiP including c-MYC-WT/mutants (left) or MYCL-WT/mutants (right) on day 21. Mean ± SD values are shown. n = 3, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by ordinary one-way ANOVA and Dunnett’s test vs. WT. (C) Expression of TRA-1-60 ( +) HDFs and CD13 ( +) HDFs transduced with EpiP including c-MYC-WT/mutants (left) or MYCL-WT/mutants (right) on day 16. Mean ± SD values are shown. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by ordinary one-way ANOVA and Dunnett’s test vs. WT. (D) Representative flow cytometry images for TRA-1-60 and CD13 for HDFs transduced with EpiP including c-MYC-WT/ΔMB0 or MYCL-WT/ΔMB0 10 and 21 days after the transduction. Numbers indicate the expression percentage of each quadrant. (E) Proliferation of HDFs transduced with EpiP including c-MYC-WT/ΔMB0 or MYCL-WT/ΔMB0 10 and 21 days later. Mean ± SD values are shown. n = 3, ***p < 0.001 by ordinary one-way ANOVA and Dunnett’s test vs. c-MYC-WT. The number of cells was counted using a Cell Counter model R1 (OLYMPUS).
Figure 2 MYC Box 0 and 2 domains are crucial for colony formation during reprogramming. (A) Schematic representation of WT c-MYC and MYCL protein. Black boxes show important domains for MYC function, including MB0, 1, 2, 3a (c-MYC only), 3b, 4, and basic-helix-loop-helix leucine zipper motif (bHLHLZ). The percentage of common amino acids in each MYC box domain between MYCL and c-MYC is shown (Identity%). The numbers on the right indicate amino acid lengths. (B) Number of iPSC-like and non-iPSC-like colonies transduced with EpiP including c-MYC-WT/mutants (left) or MYCL-WT/mutants (right) on day 21. Mean ± SD values are shown. n = 3, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by ordinary one-way ANOVA and Dunnett’s test vs. WT. (C) Expression of TRA-1-60 ( +) HDFs and CD13 ( +) HDFs transduced with EpiP including c-MYC-WT/mutants (left) or MYCL-WT/mutants (right) on day 16. Mean ± SD values are shown. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by ordinary one-way ANOVA and Dunnett’s test vs. WT. (D) Representative flow cytometry images for TRA-1-60 and CD13 for HDFs transduced with EpiP including c-MYC-WT/ΔMB0 or MYCL-WT/ΔMB0 10 and 21 days after the transduction. Numbers indicate the expression percentage of each quadrant. (E) Proliferation of HDFs transduced with EpiP including c-MYC-WT/ΔMB0 or MYCL-WT/ΔMB0 10 and 21 days later. Mean ± SD values are shown. n = 3, ***p < 0.001 by ordinary one-way ANOVA and Dunnett’s test vs. c-MYC-WT. The number of cells was counted using a Cell Counter model R1 (OLYMPUS).CC BY · PubMed Central
Figure 3 MYCL regulates cytoskeleton- and cell adhesion-related proteins during reprogramming via the MB0 domain. (A) Schematic of the mass spectrometry (MS) and GO analysis (DAVID). (B) Venn diagram of upregulated proteins during iPSC-like colony formation. (C) Molecular functions from the GO analysis of the four groups in (B). (D) KEGG pathways from the GO analysis of the four groups in (B).
Figure 3 MYCL regulates cytoskeleton- and cell adhesion-related proteins during reprogramming via the MB0 domain. (A) Schematic of the mass spectrometry (MS) and GO analysis (DAVID). (B) Venn diagram of upregulated proteins during iPSC-like colony formation. (C) Molecular functions from the GO analysis of the four groups in (B). (D) KEGG pathways from the GO analysis of the four groups in (B).CC BY · PubMed Central
Figure 4 MYCL regulates RNA processing-related proteins during reprogramming via the MB2 domain. (A) W96 and W135 in the MB2 domain of MYCL and c-MYC, respectively. The structure with the recombinant protein of the MB2 domain of MYCL-WT/W96E is shown below. The numbers on the right indicate amino acid lengths. (B) The number of iPSC-like and non-iPSC-like colonies derived from 1 × 105 HDFs transduced with EpiP including MYCL-WT or MYCL-W96E on day 21. Mean ± SD values are shown. n = 3, *p < 0.05 by unpaired t-test. (C) Representative images of reprogramming HDFs 21 days after the transduction of EpiP, including MYCL-WT or MYCL-W96E. Scale bars, 100 μm. (D) Venn diagram of enriched proteins between reprogramming HDFs and hiPSCs by AP-MS. A list of the 25 commonly enriched proteins is shown below. Blue indicates RBP (23 in total). (E) Molecular function from the GO analysis of the 25 commonly identified proteins in (D).
Figure 4 MYCL regulates RNA processing-related proteins during reprogramming via the MB2 domain. (A) W96 and W135 in the MB2 domain of MYCL and c-MYC, respectively. The structure with the recombinant protein of the MB2 domain of MYCL-WT/W96E is shown below. The numbers on the right indicate amino acid lengths. (B) The number of iPSC-like and non-iPSC-like colonies derived from 1 × 105 HDFs transduced with EpiP including MYCL-WT or MYCL-W96E on day 21. Mean ± SD values are shown. n = 3, *p < 0.05 by unpaired t-test. (C) Representative images of reprogramming HDFs 21 days after the transduction of EpiP, including MYCL-WT or MYCL-W96E. Scale bars, 100 μm. (D) Venn diagram of enriched proteins between reprogramming HDFs and hiPSCs by AP-MS. A list of the 25 commonly enriched proteins is shown below. Blue indicates RBP (23 in total). (E) Molecular function from the GO analysis of the 25 commonly identified proteins in (D).CC BY · PubMed Central
Figure 5 Model of the reprogramming process by MYCL. MYCL promotes iPSC-like colonies via its MB0 and MB2 domains. The MB0 domain regulates the expression of cell-adhesion proteins, possibly via post-translational modifications (PTM). The MB2 domain regulates RNA processing by interacting with RNA-binding proteins (RBP). We speculate that MYCL promotes reprogramming through the synergistic effects of these two mechanisms.
Figure 5 Model of the reprogramming process by MYCL. MYCL promotes iPSC-like colonies via its MB0 and MB2 domains. The MB0 domain regulates the expression of cell-adhesion proteins, possibly via post-translational modifications (PTM). The MB2 domain regulates RNA processing by interacting with RNA-binding proteins (RBP). We speculate that MYCL promotes reprogramming through the synergistic effects of these two mechanisms.CC BY · PubMed Central

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Metadata

Authors
Akifuji, C., Iwasaki, M., Kawahara, Y., Sakurai, C., Cheng, Y.-S., Imai, T., & Nakagawa, M.
Affiliations
Kyoto University (JP)
Corresponding author
Nakagawa, Masato
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2021-12-20
Volume
11
Issue
1
Pages
24254-24254
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-021-03260-5
PMC
PMC8688507
OpenAlex
W4226025092
Cited by
3
References
4
Citations by year
2025: 1 · 2024: 1 · 2023: 1
FWCI
0.16
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Japan Agency for Medical Research and Development (JP21bm0104001)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
Keywords
MYCL, induced pluripotent stem cells, reprogramming efficiency, c-Myc, cell adhesion, cytoskeletal proteins, RNA processing, proteomics
MeSH terms
Protein Domains, Cell Adhesion, Cell Differentiation, Cells, Cultured, Flow Cytometry, Humans, Mutation, Phenotype, Recombinant Proteins, Proto-Oncogene Proteins c-myc, Gene Deletion, Cell Culture Techniques, Proteome, Proteomics, Induced Pluripotent Stem Cells, Cellular Reprogramming

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