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MYCL Promotes Ipsc-Like Colony Formation Via MYC Box 0 and 2 Domains

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MYCL Promotes Ipsc-Like Colony Formation Via MYC Box 0 and 2 Domains MYCL promotes iPSC-like colony formation via MYC Box 0 and 2 domains Chiaki Akifuji Department of Life Science Frontiers, Center for iPS cell Research and Application (CiRA), Kyoto University Mio Iwasaki Department of Life Science Frontiers, Center for iPS cell Research and Application (CiRA), Kyoto University Yuka Kawahara Department of Life Science Frontiers, Center for iPS cell Research and Application (CiRA), Kyoto University Chiho Sakurai Department of Life Science Frontiers, Center for iPS cell Research and Application (CiRA), Kyoto University Yusheng Cheng Department of Life Science Frontiers, Center for iPS cell Research and Application (CiRA), Kyoto University Takahiko Imai Department of Life Science Frontiers, Center for iPS cell Research and Application (CiRA), Kyoto University Masato Nakagawa ( [email protected] ) Department of Life Science Frontiers, Center for iPS cell Research and Application (CiRA), Kyoto University Research Article Keywords: mycl, reprogramming, ipsc, eciency, induced Posted Date: August 19th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-817686/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 MYCL promotes iPSC-like colony formation via MYC Box 0 and 2 domains 2 3 Authors 4 Chiaki Akifuji1, Mio Iwasaki1, Yuka Kawahara1, Chiho Sakurai1, Yusheng Cheng1, Takahiko 5 Imai1, and Masato Nakagawa1* 6 7 Affiliation 8 1 Department of Life Science Frontiers, Center for iPS cell Research and Application (CiRA), 9 Kyoto University, Kyoto 606-8507, Japan 10 11 *Correspondence: [email protected] 12 13 Abstract 14 Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell in 15 the body and thus have attractive regenerative medicine potential. Current iPSC generation 16 protocols, however, have low efficiency and show variable quality among clones. This 17 variability influences the efficiency and reproducibility of iPSC differentiation. Our previous 18 study reported that MYC proteins (c-MYC and MYCL) are important for the reprogramming 19 efficiency and germline transmission of iPSCs, but that MYCL can generate iPSC colonies more 20 efficiently than c-MYC. However, why c-MYC and MYCL cause different reprogramming 21 efficiencies is unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two 22 functional domains conserved in the MYC protein family, contribute to the phenotypic 23 difference and promote iPSC generation in MYCL-induced reprogramming. Proteome analysis 24 suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are 25 regulated by the MB0 domain, while RNA processes are regulated by the MB2 domain. These 26 findings provide a molecular explanation for why MYCL has higher reprogramming efficiency 27 than c-MYC. 28 29 Introduction 30 Induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells and have the 31 potential to differentiate into all three germ layers 1 2. The original reprogramming was done by 32 inducing four reprogramming factors, OCT3/4, SOX2, KLF4, and c-MYC (OSKM). iPSCs are 33 functionally equivalent to embryonic stem cells (ESCs) but ethically permissible for many types 34 of research that constrain the use of ESCs 3. Even so, the efficiency of their generation is poor, 35 and the cost is high 4. While new methods have improved iPSC generation, the efficiency 36 remains quite low or the manipulation is technically difficult 5. Furthermore, iPSC clones show 37 wide variety in their quality. 38 We have shown that excluding c-MYC from the reprogramming cocktail significantly 39 lowers the reprogramming and differentiation efficiencies 6. The MYC family consists of c- 40 MYC, MYCN, and MYCL in humans, and all are oncogenes 7. c-MYC was the first MYC gene 41 discovered in humans and has been a topic of cancer research ever since 8. Tumorigenesis 42 depends on high transformation activity derived from the N-terminus region of c-MYC protein 9. 43 Consequently, OSKM-based reprogramming may not be appropriate for the clinical application 44 of iPSCs. Accordingly, many groups have reported reprogramming methods that exclude c-MYC 45 overexpression, but at lower reprogramming efficiency and quality 5 6. Among these methods is 46 the substitution of c-MYC for MYCL 6. MYCL is about 30 amino acids shorter in the N- 47 terminus region than c-MYC and has lower transformation activity 9. We found that this 48 substitution can actually increase the number of iPSC colonies and the capacity for germline 49 transmission 6. Furthermore, fewer chimeric mice died by tumorigenesis after the transplantation 50 of MYCL-iPSCs, whereas the transplantation of c-MYC-iPSCs caused lethal tumorigenesis in 51 more than 50% of mice during two years of observation. Despite these observations, little is 52 known about the molecular function of MYCL and the different mechanisms between c-MYC 53 and MYCL to promote reprogramming. 1 54 MYC proteins have six MYC Box (MB) domains: MB0, 1, 2, 3a, 3b, and 4 in the N- 55 terminus and a basic helix-loop-helix leucine zipper (bHLHLZ) in the C-terminus 10. MYCL 56 does not have MB3a. The C-terminus of c-MYC and MYCL is essential in reprogramming due 57 to its binding with MAX protein, which allows MYC to access DNA 6 11. The N-terminus is 58 mainly known as a transactivation domain (TAD), which regulates the target gene, but its 59 function in reprogramming is less clear 12. We also revealed a c-MYC mutant of the N-terminus 60 region that shows low transformation activity increases colony formation during reprogramming 61 6. However, which of the MB domains in the N-terminus are crucial for reprogramming and how 62 this function depends on the transformation activity are not understood. In addition, MYC 63 proteins act as transcription factors upon interacting with several binding proteins 13. Although 64 MYCL-binding proteins are important for MYCL function, there are no reports about MYCL- 65 binding proteins during reprogramming. 66 In this study, we detected two types of colonies during reprogramming based on TRA-1- 67 60 expression, an established iPSC marker 14: iPSC-like colonies, which express TRA-1-60, and 68 non-iPSC-like colonies, which do not express TRA-1-60. Our analysis using domain deletion 69 mutants of MYC proteins revealed that the MB0 and MB2 domains are important for promoting 70 iPSC-like colonies. Moreover, we found the MB0 domain was functionally different between c- 71 MYC and MYCL. In the former case, it induced non-iPSC-like colonies by increasing nucleic 72 proteins related to transcription, but in the latter case, it induced iPSC-like colonies by increasing 73 the expression of cell adhesion-related proteins. We also found that deletion of the MB2 domain 74 in MYC proteins prevented colony formation and that MYCL could interact with RNA-binding 75 proteins (RBPs) via this domain. These results suggested that MYCL promotes reprogramming 76 by regulating RNA processing. 77 78 Results 79 MYCL promotes reprogramming more efficiently than c-MYC. 80 To compare the reprogramming phenotypes of MYCL and c-MYC, we used Sendai 81 virus (SeV)-based reprogramming (CytoTune®-iPS) and StemFit AK03N medium without 82 bFGF (Fig. 1A). The bFGF exclusion is based on unpublished data that showed it enhances 83 reprogramming efficiency. The SeV method has high reprogramming efficiency without genome 84 integration, and c-MYC and MYCL SeV kits are already available 15. We analyzed the 85 expression of TRA-1-60 by immunostaining from days 1 to 8 after infection (Fig. 1A). On day 8 86 of the reprogramming of human dermal fibroblasts (HDFs), we observed that c-MYC induced 87 greater morphological changes than MYCL, but some cell aggregations that looked like colonies 88 when using c-MYC did not express TRA-1-60 (Fig. 1B). Cell proliferation was highly increased 89 in HDFs infected with c-MYC compared to MYCL. On the other hand, the percentage of TRA- 90 1-60 (+) cells increased more in MYCL-infected HDFs 4 days after the infection (Fig. 1C). 91 We confirmed these reprogramming phenotypes using episomal vector (EpiV) 16 (Fig. 92 1D). Similar to the results with the SeV method, many colonies did not express TRA-1-60 in c- 93 MYC-transduced HDFs (Fig. 1E and Supplementary Fig. S1). The transduction of MYCL 94 caused a higher percentage of TRA-1-60 (+) cells and lower cell proliferation than the 95 transduction of c-MYC (Fig. 1F). The difference in the shape of the colonies between MYCL 96 and c-MYC was much clearer in EpiV reprogramming than SeV reprogramming. From these 97 results, we defined “iPSC-like” colonies as having ESC-like morphology with a flat and round 98 shape and a distinct edge that expresses TRA-1-60 and defined “non-iPSC-like” colonies as 99 having granulous cell aggregations with an irregular edge and not expressing TRA-1-60. We 100 counted the number of iPSC-like and non-iPSC-like colonies on day 21 and found that c-MYC 101 induced iPSC-like colonies as well as many non-iPSC-like colonies, but MYCL induced almost 102 only iPSC-like colonies and more of them than c-MYC (Fig. 1G and Supplementary Fig. S1). 103 Furthermore, we confirmed the expression of CD13 as a fibroblast marker, because its 104 expression decreased during the reprogramming 17. The percentage of CD13 (+) cells decreased 105 daily in HDFs transduced with c-MYC and MYCL, but the number of CD13 (-) cells rapidly 106 increased in c-MYC compared to MYCL (Fig. 1H and Supplementary Fig. S2). These results 2 107 suggested that MYCL promotes TRA-1-60 (+) cells more than c-MYC and that c-MYC 108 suppresses CD13 expression more than MYCL.
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