MYCL promotes iPSC-like colony formation via 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 (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 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, , , 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 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 (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. 109 110 MYC Box 0 and 2 domains are crucial for colony formation during reprogramming. 111 Next, to identify which domains in the N-terminus of MYC proteins influence 112 reprogramming, we prepared domain deletion mutants (Fig. 2A, Supplementary Fig. S3A and 113 S3B) and applied EpiV reprogramming, because this method provided a clearer phenotype and 114 was easier to manipulate than the SeV method. The bHLHLZ domain in the C-terminus region is 115 a well-known binding domain of MAX, which is necessary for MYC proteins to bind to DNA 18. 116 Our previous report revealed that MYC mutants deficient of MAX-binding showed low 117 transformation activity and repressed the generation of iPSC-like colonies 6. That same study 118 also reported that c-MYC mutants deficient of transformation activity promoted the formation of 119 iPSC-like colonies compared with wild-type (WT). Together, the results suggested that MYC has 120 several functional domains that influence reprogramming. 121 The EpiV mutants were electroporated into HDFs with other reprogramming factors, 122 and the number of iPSC-like and non-iPSC-like colonies was counted (Fig. 2B and 123 Supplementary Fig. S4). c-MYC-ΔMB0 promoted the formation of iPSC-like colonies and 124 inhibited the formation of non-iPSC-like colonies compared to c-MYC-WT. We confirmed the 125 protein expression of the domain deletion mutants by western blotting (Supplementary Fig. 126 S5A). Notably, the expression level of c-MYC-ΔMB0 was quite low compared to c-MYC-WT. 127 Thus, to examine whether the phenotype of c-MYC-ΔMB0 is due to the expression level, we 128 investigated the effects of the expression level of c-MYC-WT on reprogramming 129 (Supplementary Fig. S5B and S5C). A lower expression of c-MYC-WT did not have a 130 significant effect on the formation of iPSC-like or non-iPSC-like colonies, thus confirming that 131 our observations were the result of the c-MYC-ΔMB0 function and not the expression level. On 132 the other hand, MYCL-ΔMB0, which had the same expression level as MYCL-WT 133 (Supplementary Fig. S6), showed almost no ability to form iPSC-like colonies (Fig. 2B). From 134 these results, we concluded that the MB0 domain has different functions in c-MYC and MYCL 135 for reprogramming and that c-MYC-ΔMB0 has a similar function as MYCL-WT. 136 Fig. 2B shows that c-MYC-ΔMB1 promoted iPSC-like colony formation like c-MYC- 137 ΔMB0, but it also led to the formation of non-iPSC-like colonies. The formation of iPSC-like 138 colonies by MYCL-ΔMB1 was about a quarter that by MYCL-WT. c-MYC-ΔMB2 did not 139 induce non-iPSC-like colonies but formed iPSC-like colonies like c-MYC-WT. MYCL-ΔMB2 140 showed little ability to form iPSC-like colonies, resembling MYCL-ΔMB0. c-MYC-ΔMB3a, - 141 ΔMB3b, and -ΔMB4 had similar colony-forming activities as c-MYC-WT. MYCL-ΔMB3b 142 showed the same reprogramming efficiency as MYCL-WT, but MYCL-ΔMB4 formed fewer 143 iPSC-like colonies. The ΔbHLHLZ mutants of both c-MYC and MYCL failed to induce colonies 144 and were therefore considered to have lost MYC function completely. 145 Next, we analyzed the effect of MYC-deletion mutants on the expression of TRA-1-60 146 and CD13 by flow cytometry 16 days after the start of reprogramming (Fig. 2C). Mutants that 147 increased the number of iPSC-like colonies also increased the expression of TRA-1-60, while 148 those that reduced the number of iPSC-like colonies also lowered TRA-1-60 expression (Fig. 2C 149 and Supplementary Fig. S7). c-MYC-WT showed little TRA-1-60 expression, whereas c-MYC- 150 ΔMB0 upregulated the expression. MYCL-ΔMB0, unlike MYCL-WT, failed to upregulate the 151 expression of TRA-1-60. From the results of the colony count and flow cytometry, we concluded 152 that the MB0 domain is important for the function of MYC in reprogramming and functions 153 differently between c-MYC and MYCL. 154 To analyze the function of the MB0 domain in more detail, we analyzed the expression of 155 TRA-1-60 and CD13 10 and 21 days after the start of reprogramming by flow cytometry (Fig. 156 2D). The expression of CD13 rapidly decreased by c-MYC-WT reprogramming, but slowly 157 decreased by c-MYC-ΔMB0 reprogramming. The rate of the CD13 expression by c-MYC- 158 ΔMB0 reprogramming was similar to that by MYCL-WT reprogramming. The effect of MYCL- 159 ΔMB0 on CD13 expression was smaller than that of MYCL-WT, c-MYC-WT, or c-MYC- 160 ΔMB0. On day 21, about 50% of cells were TRA-1-60 (+) by c-MYC-ΔMB0 or MYCL-WT

3 161 reprogramming, but far fewer by c-MYC-WT or MYCL-ΔMB0 reprogramming (Fig. 2D). 162 Additionally, c-MYC-ΔMB0 decreased cell proliferation compared to c-MYC-WT and showed 163 similar cell growth to MYCL-WT (Fig. 2E), suggesting c-MYC-ΔMB0 lost its transformation 164 activity. From these results, we concluded that the MB0 domain functions negatively in c-MYC 165 and positively in MYCL for reprogramming. 166 167 MYCL regulates cytoskeleton- and cell adhesion-related proteins during reprogramming 168 via the MB0 domain. 169 To confirm which are regulated by the MYCL MB0 domain in reprogramming, 170 we analyzed protein expressions during reprogramming, because it was reported that gene 171 expressions do not correlate well with protein expressions 19. We performed a comprehensive 172 analysis of expressed proteins during reprogramming induced by c-MYC and MYCL WT and 173 ΔMB0. We used reprogrammed HDFs 3, 5, and 7 days after the infection of SeV as samples for 174 mass spectrometry (MS). We also prepared HDFs electroporated with EpiV on day 10 as 175 samples for MS (Fig. 3A). There was more than a two-fold increase in the expression of 520 176 (SeV) and 128 (EpiV) proteins with MYCL-WT reprogramming compared to c-MYC-WT 177 reprogramming (Fig. 3B, groups (i) and (ii), respectively) and 183 (EpiV) proteins with c-MYC- 178 ΔMB0 reprogramming compared to c-MYC-WT reprogramming (Fig. 3B, group (iii)). We 179 identified 18 proteins common to the three groups (Fig. 3B, group (iv)). Then, we applied Gene 180 Ontology (GO) analysis using DAVID and detected enriched terms during reprogramming 20 21 181 (Fig. 3C and 3D, and Supplementary Table 1), finding cytoskeleton- and cell adhesion-related 182 proteins are involved in the promotion of reprogramming by MYCL-WT. The same analysis was 183 performed to identify proteins whose expression was up-regulated by c-MYC-WT and involved 184 in increasing the number of non-iPSC colonies (Supplementary Fig. S8A and Supplementary 185 Table 2). We found that c-MYC-WT regulates proteins involved in cell proliferation, such as the 186 cell cycle and DNA replication. Furthermore, to understand the function of MYCL in 187 reprogramming, MS analysis was applied to HDF samples electroporated with MYCL-WT, 188 MYCL-ΔMB0, or c-MYC-ΔMB0 (Supplementary Fig. S8B and Supplementary Table 3). GO 189 analysis indicated that these proteins were associated with cell adhesion and RNA processing. 190 We also compared phosphorylated proteins during SeV reprogramming with MYCL and 191 c-MYC. In total, there was more than a two-fold relative increase of 17 phosphorylated proteins 192 in MYCL-WT and 132 phosphorylated proteins for c-MYC-WT. GO analysis indicated that the 193 phosphorylated proteins increased by MYCL included cytoskeleton-related proteins and those 194 increased by c-MYC included transcription-related proteins (Supplementary Fig. S9A and S9B). 195 These results suggest that MYCL and c-MYC dynamically regulate protein function differently 196 during reprogramming. 197 198 MYCL regulates RNA processing-related proteins during reprogramming via the MB2 199 domain. 200 Our analysis also revealed that, along with the MYCL MB0 domain, the MYCL MB2 201 domain is important for reprogramming (Fig. 2B). It has been reported that the c-MYC MB2 202 domain is involved in the transformation activity of c-MYC and that tryptophan 135 within the 203 MB2 domain is necessary for this activity 9. MYCL also has a tryptophan residue within its MB2 204 domain but little transformation activity 22. We hypothesized that this domain in MYCL has 205 reprogramming function. We therefore produced a mutant in which tryptophan 96 was 206 substituted with glutamate (W96E). This tryptophan is equivalent to tryptophan 135 in c-MYC 207 (Fig. 4A and Supplementary Fig. S3B). We confirmed the expression of MYCL-W96E by 208 western blotting (Supplementary Fig. S10). Next, we examined the effect of MYCL-W96E for 209 reprogramming. HDFs were electroporated with reprogramming factors containing MYCL-WT 210 or -W96E. MYCL-W96E could not induce iPSC-like colonies, suggesting tryptophan 96 is 211 crucial for reprogramming (Fig. 4B and 4C). We thus hypothesized that the residue might be 212 important for MYCL to bind to other proteins and produced GST-fusion recombinant proteins 213 for the MYCL MB2 domain (Fig. 4A). GST-MYCL-MB2-WT or -W96E proteins were 214 immobilized on glutathione Sepharose, and affinity columns were prepared. Cell lysates were

4 215 applied to the column, and after washing, the bound proteins were eluted. We used the cell 216 lysates from reprogrammed HDFs, but since it was difficult to collect a large amount, we also 217 used cell lysates from human iPSCs. The reason for this choice is that many of the proteins 218 expressed in reprogrammed HDFs are likely to be highly expressed in human iPSCs as well. We 219 identified 31 candidate proteins that bind to the MB2 domain of MYCL-WT but not of MYCL- 220 W96E during reprogramming in the HDF lysates (Fig. 4D and Supplementary Table 4). Among 221 them, 25 proteins were also identified using hiPSC lysates and 23 were RNA-binding proteins 222 (RBPs). Six proteins were identified only in the reprogrammed HDFs lysates and included three 223 RBPs: HNRNPK, DDX17, and C1QBP. We confirmed the function of the 31 proteins using a 224 public database (https://www.nextprot.org/) 23 and found 16 of them are involved in RNA 225 processing. GO analysis using DAVID also showed that the 31 proteins were related to 226 controlling pre-mRNA splicing, capping, and polyadenylation, suggesting functions in mRNA 227 export, turnover, localization, and translation (Fig. 4E). These results suggested that MYCL 228 interacts with RBPs via its MB2 domain and promotes reprogramming by post-transcriptional 229 regulation. 230 231 Discussion 232 Here we described the molecular function of MYCL during reprogramming and 233 compared it to c-MYC function in reprogramming by focusing on MYC Box domains. We found 234 that the MB0 and MB2 domains are important for reprogramming and deleting either region 235 compromised the reprogramming ability of MYCL. Proteomic analysis revealed that MYCL 236 regulates the expression of cell adhesion-related proteins during reprogramming via the MB0 237 domain (Fig. 3C and 3D). We also found possibility that the same domain is regulated by post- 238 translational modifications (PTM), as discussed below. It is known that cell-substrate adhesion is 239 closely related to the mesenchymal-epithelial transition (MET) 24 and that MET occurs during the 240 reprogramming process 25 26 27. Furthermore, we identified that the MB2 domain is required for 241 MYCL to promote reprogramming by binding to RBPs, especially RNA processing-related 242 proteins (Fig. 4D). It has been reported that RBPs regulate MET through post-transcriptional 243 regulation. For example, heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates the 244 alternative splicing of Rac1 to control MET 28. From these findings, we propose that MYCL 245 regulates the RNA processing of cell adhesion-related genes transcribed by MYCL itself or other 246 genes. Therefore, we hypothesize that transcriptional and post-transcriptional regulation by 247 MYCL promotes MET, which in turn increases the efficiency of reprogramming and leads to the 248 production of higher quality iPSCs (Fig. 5). 249 Western blotting revealed that MYCL protein has a unique expression pattern 250 (Supplementary Fig. S6). The calculated molecular weight of MYCL is about 40 kDa (364 aa), 251 but it also showed three strong bands at around 60 kDa. We speculate that several amino acids in 252 MYCL are sites for PTM. Since the expression of MYCL-ΔMB0 showed a strong single band, 253 we speculate that the MYCL MB0 domain is the PTM site. Such a phenomenon was not 254 observed in c-MYC (Supplementary Fig. S5A). One possible type of relevant PTM is 255 phosphorylation. Phosphorylation is crucial for protein function. For example, RNA polymerase 256 Ⅱ (Pol Ⅱ) is required for transcription pauses in a promoter-proximal position during 257 transcription initiation. To initiate transcription, it is necessary that the C-terminal domain of Pol 258 Ⅱ be phosphorylated by P-TEFb 29. In addition, the phosphorylation of c-MYC on threonine 58 259 in the MB1 domain promotes c-MYC binding to F-box and WD repeat domain containing 7 260 (FBXW7), causing the ubiquitination of c-MYC, which triggers c-MYC degradation 30. 261 Similarly, MYCL might undergo phosphorylation to change its activity and interaction with 262 binding proteins. However, this hypothesis requires further study. 263 Comprehensive proteomic analysis suggested that the MYCL MB0 domain influences 264 the expression of cell adhesion-related proteins, and MYCL shows an up-regulation of 265 phosphorylated cytoskeletal proteins (Fig. 3C and 3D). Cell adhesion is mediated by adhesion

5 266 molecules such as integrins and cadherins, which function in the extracellular matrix (ECM) and 267 cell-cell adhesion and are important for cell communication and the regulation of fundamental 268 physiological processes such as tissue development and maintenance 31 32. Human PSCs have 269 unique focal adhesion localization, and appropriate adhesion to the ECM is required to regulate 270 reprogramming via MET and maintain pluripotency 33 34 35. Accordingly, our study supports 271 MYCL regulating cell-substrate adhesion through its MB0 domain to promote reprogramming. 272 In other words, MYCL might regulate proteins involved in cell adhesion and the cytoskeleton 273 directly or indirectly to cause MET and promote reprogramming. In c-MYC, loss of the MB0 274 domain has a positive effect on iPSC-like colony formation, suggesting that this domain has a 275 different function than in MYCL. This functional difference is somewhat surprising, since the 276 domain is well conserved (Supplementary Fig. S3B). We would like to clarify this point in the 277 future. 278 We also found that in addition to the MB0 domain, the MB2 domain has an important 279 function in MYCL-reprogramming (Fig. 2B and 2C). Deleting the MB2 domain completely 280 compromised the reprogramming ability of MYCL. In c-MYC, the MB2 domain has an 281 important function in transformation activity 13, and tryptophan 135 in the MB2 domain is 282 essential for this activity. The equivalent tryptophan residue in MYCL is tryptophan 96. MYCL 283 has little transformation activity, but we showed that W96E affects MYCL-reprogramming. To 284 further investigate the function, we sought interacting proteins by affinity column 285 chromatography. We found 31 proteins including 26 RBPs that interact with the MYCL MB2 286 domain (Fig. 4D and Supplementary Table 4). GO analysis suggested that some of the 31 287 proteins are involved in RNA processing (Supplementary Table 4). It has been reported that 288 altered RNA processing affects somatic cell reprogramming 36. Therefore, we hypothesize that 289 MYCL also promotes MET in reprogramming by regulating RNA processing via interactions 290 with RBPs at its MB2 domain in addition to PTM at its MB0 domain. 291 To conclude, we have demonstrated that MYCL promotes more efficient 292 reprogramming than c-MYC, regulates the expression of cell adhesion and cytoskeletal proteins, 293 and is involved in RNA processing via a single tryptophan residue in the MB2 domain. 294 Following these findings, we propose that MYCL causes MET by regulating the expression of 295 proteins involved in the promotion of reprogramming from the RNA-processing stage. Further 296 elucidation of the function of MYCL in reprogramming will improve the quality and efficiency 297 of iPSC generation. 298 299 Material and methods 300 Cell culture. HDFs (106-05f) were purchased from Cell Applications, Inc. HDFs were cultured 301 in DMEM (Nacalai Tesque) supplemented with 10% FBS (gibco) and 1% penicillin and 302 streptomycin (Pen/Strep; Invitrogen). The human iPSC clone 201B7 was used in this study 2. 303 iPSCs were cultivated on iMatrix-511 (Nippi)-coated (0.5 μg/cm2) cell culture plates with 304 StemFit AK03N (Ajinomoto) with bFGF and passaged via dissociation into single cells using 305 TrypLE Select (Life Technologies) on day 737. 306 307 Generation of iPSCs. A frozen stock of HDFs was thawed and cultured for 4 days, and then 1 × 308 105 cells were collected by trypsinization. With SeV HDFs were infected with the CytoTune®- 309 iPS 2.0 (c-MYC) or CytoTune®-iPS 2.0L (MYCL) Sendai Reprogramming Kit (ID Pharma). 310 With EpiV, HDFs were electroporated with 1.2 μg of plasmid mixtures with the Neon 311 Transfection System (Invitrogen). After that, cells were plated in a 6-well plate and cultured in 312 StemFit AK03N without bFGF (Ajinomoto) with iMatrix-511 (Nippi) of 0.25 μg/cm2 in SeV or 313 0.125 μg/cm2 in EpiV. The culture medium was changed the next day and every three days 314 thereafter. The colonies were counted 21 days after plating. 315 316 Episomal vector construction for deletion mutants of c-MYC and MYCL. We previously 317 generated pCXLE-c-MYC and -MYCL from human cDNAs encoding c-MYC and MYCL 318 amplified by PCR and cloned into pENR1A 16. Primers for the deletion mutants were designed 319 using the Primer Design tool for In-Fusion® (Takara) and inserted into pENTR1A. The switch

6 320 from pENTR1A to pCXLE was done using the Gateway system (Invitrogen). The primers used 321 are listed in Supplementary Table 5. 322 323 Immunostaining. The cells were fixed with 4% formaldehyde (Wako) for 20 min at room 324 temperature. Then, the fixed cells were treated with PBS (Nacalai Tesque) containing 0.5% 325 Triton X-100 (Wako) and 3% bovine serum albumin (BSA, Nacalai Tesque) for 20 min at room 326 temperature for permeabilization. The cells were incubated with primary antibodies diluted in 327 PBS containing 3% BSA at 4℃ overnight. After washing with PBS, the cells were incubated 328 with fluorescence-conjugated secondary antibodies for 1 h at room temperature. Nuclei were 329 visualized with Hoechst 33342 (Thermo Fisher Scientific). Anti-TRA-1-60 (1:500, 560071, BD 330 Pharmingen) and Alexa 488-conjugated goat anti-mouse IgG, IgM (H+L) (1:250, A10680, 331 Invitrogen) were used as the antibodies. 332 333 Imaging and quantification. Stained cells were imaged using a BZ-9000 imaging system 334 (KEYENCE) or ArrayScan™High-Content Systems (Thermo Fisher Scientific). HCS StudioTM 335 2.0 Cell Analysis Software (Thermo Fisher Scientific) was used to quantify cell counts and 336 signal intensities. The Cellomics BioApplication system (Thermo Fisher Scientific) was 337 programmed to capture and analyze 25 images per well. The total cell number was detected by 338 Hoechst 33342 staining. The number of TRA-1-60 (+) cells was calculated as the number of 339 TRA-1-60 (+) cells among Hoechst (+) cells. The proportion of TRA-1-60 (+) cells was 340 calculated by dividing this number by the total cell number. 341 342 Flow cytometry. Transduced cells were harvested with 0.25% trypsin/1 mM EDTA each day 343 after the transduction for the analysis. At least 5 × 104 cells were stained with the following 344 antibodies in FACS buffer (2% FBS, 0.36% glucose (Nacalai Tesque), 50 μg/μL Pen/Strep in 345 PBS) for 30 min at room temperature: BV510-conjugated anti-TRA-1-60 (1:40, 563188, BD 346 Biosciences) and PE-Cy7-conjugated anti-CD13 (1:40, 561599, BD Biosciences) antibodies. The 347 analysis was performed using MACSQuant® Analyzers (Miltenyi Biotec). Negative controls 348 used a mixture of HDFs without any EpiV transduction and reprogrammed HDFs induced with 349 EpiV including c-MYC or MYCL. Isotype means mixed HDFs staining with the isotype control 350 of anti-TRA-1-60 and -CD13 antibodies. 351 352 SDS-PAGE. Cells were lysed with SDS sample buffer (0.125 M Tris-base, 0.96 M glycine, and 353 17.3 mM SDS) containing 3-mercaptoethanol (Wako). Samples were applied and separated in an 354 8% polyacrylamide gel composed of 30% (w/v)-Acrylamide/Bis Mixed Solution (29:1) (Nacalai 355 Tesque) and Separating Gel Buffer Solution (4x) for SDS-PAGE (Nacalai Tesque). 356 357 Western Blotting. Proteins on an SDS-PAGE gel were transferred to a PVDF membrane 358 (Immobilon-P, Millipore) and probed with the following antibodies using an iBind Flex system 359 (Invitrogen): anti-human MYCL (1:250, AF4050, R&D), anti-human c-MYC (1:250, D84C12, 360 CST), and anti-β-actin (1:1000, A5441, SIGMA) antibodies. 361 362 Preparation of recombinant proteins and affinity purification (AP). The MB2 region of 363 MYCL-WT or -W96E was cloned into pGEX-6P-1 (GE Life Science). The plasmids were 364 transformed into BL21 E.coli (DE3) competent cells. The fusion proteins, GST-MYCL-WT- 365 MB2 and GST-MYCL-W96E-MB2, were induced by treatment with 0.5 mM IPTG for 4 h at 366 37˚C. The proteins were purified using glutathione Sepharose beads (GE Healthcare). Human 367 iPSC lysates or reprogrammed HDFs lysates were lysed in RIPA buffer (20 mM Tris/HCl (pH 368 7.6), 1% NP-40, 0.1% SDS, 150 mM NaCl, and protease inhibitors) and then centrifuged. Cell 369 lysates (supernatant) were transferred into a column (BIO-RAD) packed with beads conjugated 370 to GST-protein. After washing, binding proteins were eluted in lysis buffer (12 mM sodium 371 deoxycholate, 12 mM sodium lauroyl sarcosinate, and 100 mM Tris-HCl (pH9.0)) for MS 372 analysis. The iPSC lysates were prepared 6 days after passaging in two 10-cm dishes (n=1) and

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10 516 *p<0.05 by unpaired t-test. 517 D. Schematic representation of HDF reprogramming with episomal vector (EpiV). HDFs were 518 electroporated with EpiV carrying SOX2, KLF4, OCT3/4-shp53, LIN28A, EBNA1, and c- 519 MYC or MYCL. StemFit AK03N without bFGF was used during the infection and subsequent 520 induction of iPSC-like colonies. We performed flow cytometry of the reprogrammed HDFs 521 every three days from 1 to 19 days plus day 21 after the electroporation. 522 E. Representative immunostaining images of reprogrammed HDFs stained by anti-TRA-1-60 523 antibody (green) and Hoechst (blue) 21 days after the electroporation. Scale bars, 300 μm. Ph, 524 phase contrast. 525 F. Proliferation and expression of TRA-1-60 (+) cells during reprogramming were analyzed by 526 flow cytometry. HDFs were electroporated with EpiV including c-MYC or MYCL. Flow 527 cytometry was performed every three days from days 1 to 19 days plus day 21. Mean (SD) for 528 n=3, *p<0.05 and ***p<0.001 by unpaired t-test. 529 G. The number of iPSC-like and non-iPSC-like colonies derived from 1×105 HDFs induced with 530 EpiV including c-MYC or MYCL on day 21. Mean (SD) for n=3, **p<0.01 by unpaired t-test. 531 H. Percentage of CD13 (+) cells during EpiV reprogramming determined by flow cytometry. 532 Mean (SD) for n=3, *p<0.05, **p<0.01, and ****p<0.0001 by unpaired t-test. 533 534 Figure 2 MYC Box 0 and 2 domains are crucial for colony formation during 535 reprogramming. 536 A. Schematic representation of WT c-MYC and MYCL protein. Black boxes show important 537 domains for MYC function, including MB0, 1, 2, 3a (c-MYC only), 3b, 4, and basic-helix- 538 loop-helix leucine zipper motif (bHLHLZ). The percentage of common amino acids in each 539 MYC box domain between MYCL and c-MYC is shown (Identity%). The numbers on the right 540 indicate amino acid lengths. 541 B. Number of iPSC-like and non-iPSC-like colonies induced with EpiV including c-MYC- 542 WT/mutants (left) or MYCL-WT/mutants (right) on day 21. Mean (SD) for n=3, *p<0.05 by 543 one-way factor repeated measures ANOVA and Dunnett’s test vs. WT. 544 C. Expression of TRA-1-60 (+) HDFs and CD13 (+) HDFs induced with EpiV including c-MYC- 545 WT/mutants (left) or MYCL-WT/mutants (right) on day 16. Mean (SD) for n=3, *p<0.05 and 546 **p<0.01 by one-way factor repeated measures ANOVA and Dunnett’s test vs. WT. 547 D. Representative flow cytometry images for TRA-1-60 and CD13 for HDFs induced with EpiV 548 including c-MYC-WT/ΔMB0 or MYCL-WT/ΔMB0 10 and 21 days after the induction. 549 Numbers indicate the expression percentage of each quadrant. 550 E. Proliferation of HDFs induced with EpiV including c-MYC-WT/ΔMB0 or MYCL-WT/ΔMB0 551 10 and 21 days later. Mean (SD) for n=3, *p<0.05 by unpaired t-test. The number of cells was 552 counted by Cell Counter model R1 (OLYMPUS). 553 554 Figure 3 MYCL regulates cytoskeleton- and cell adhesion-related proteins during 555 reprogramming via the MB0 domain. 556 A. Schematic of the mass spectrometry (MS) and GO analysis (DAVID). 557 B. Venn diagram of up-regulated proteins during iPSC-like colony formation. 558 C. Molecular functions from the GO analysis of the four groups in (B). 559 D. KEGG pathways from the GO analysis of the four groups in (B). 560 561 Figure 4 MYCL regulates RNA processing-related proteins during reprogramming via 562 the MB2 domain. 563 A. W96 and W135 in the MB2 domain of MYCL and c-MYC, respectively. The structure with 564 the recombinant protein of the MB2 domain of MYCL-WT/W96E is shown below. The 565 numbers on the right indicate amino acid lengths. 566 B. The number of iPSC-like and non-iPSC-like colonies derived from 1×105 HDFs induced with 567 EpiV including MYCL-WT or MYCL-W96E on day 21. Mean (SD) for n=3, *p<0.05 by 568 unpaired t-test. 569 C. Representative images of reprogrammed HDFs 21 days after the induction of EpiV including

11 570 MYCL-WT or MYCL-W96E. Scale bars, 100 μm. 571 D. Venn diagram of enriched proteins between reprogrammed HDFs and hiPSCs by AP-MS. A 572 list of the 25 commonly enriched proteins is shown below. Blue indicates RBP (23 in total). 573 E. Molecular function from the GO analysis of the 25 commonly identified proteins in D. 574 575 Figure 5 Model of the reprogramming process by MYCL. 576 MYCL promotes iPSC-like colonies via its MB0 and MB2 domains. The MB0 domain 577 regulates the expression of cell-adhesion proteins, possibly via post-translational modifications 578 (PTM). The MB2 domain regulates RNA processing by interacting with RNA-binding proteins 579 (RBP). We speculate that MYCL promotes reprogramming through the synergistic effect of 580 these two mechanisms.

12 Fig. 1

Sendai virus C A Day 50,00050000 KOS c-MYC Infection 1 2 3 4 5 6 7 8 + or 40,00040000 KLF4 StemFit AK03N without bFGF MYCL 30,00030000 HDFs Immunostaining MOI:4.3 20,00020000

B Ph TRA-1-60 Hoechst Cell number 10,00010000 c-MYC 0 MYCL

50 ay1 ay2 ay3 ay4 ay5 ay6 ay7 c-MYC 40 y 8 300 µm 300 µm 300 µm ✱ 30 20 ✱ MYCL

TRA-1-60 % 10

300 µm 300 µm 300 µm 0 day1 day2 day3 day4 day5 day6 day7

D Episomal vector F Electroporation Day KLF4 4,000,0004000000 c-MYC ✱ SOX2 1 4 7 10 13 16 19 21 OCT3/4- ✱ or shp53 + StemFit AK03N without bFGF 3,000,0003000000 EBNA1 ✱ MYCL HDFs LIN28A Flow cytometry ✱✱✱ 2,000,0002000000 ✱

1,000,0001000000 ✱ E Ph TRA-1-60 Hoechst Cell number 0 c-MYC 60 MYCL y 10y 13y 16y 19y 21y ay1 ay4 ay7 ✱ c-MYC 40

20 TRA-1-60 % MYCL ✱ ✱ 0 day1 day4 day7 day10 day13 day16 day19 day21

G H c-MYC MYCL ✱ ✱ ✱✱✱✱ c-MYC 100 ✱✱ ✱✱ ✱ MYCL 500 75 ✱ 400 c-MYC 50 300 MYCL CD13 % 25 200 100 0 Colony number

0 day1 day4 day7 iPSC-like Non-iPSC-like day10 day13 day16 day19 day21 Fig. 2

A MYC Box domain 0 1 2 3a 3b 4 bHLHLZ c-MYC 439 aa

MYCL 364 aa

Identity% 60 84 71 91 100 46

B c-MYC MYCL 400 400

300 300

✱ 200 200 iPSC-like 100 iPSC-like 100 ✱ colonynumber colonynumber ✱ ✱ ✱ 0 0 600 600

400 400

✱ 200 200

✱

✱ Non-iPSC-like Non-iPSC-like ✱ colonynumber colonynumber 0 0 WT WT MB4 MB0 MB1 MB4 MB2 MB0 MB1 MB2 MB3a MB3b Δ MB3b Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ bHLHLZ bHLHLZ Δ Δ

C 15 c-MYC 15 MYCL

10 10

5 5

✱ ✱ TRA-1-60 % TRA-1-60 % ✱

0 0 ✱ ✱ ✱ ✱ 100 100 ✱ ✱✱ 75 75

50 50 CD13 % CD13 % 25 25

0 0 WT WT MB0 MB1 MB2 MB4 MB0 MB1 MB2 MB4 MB3b MB3a MB3b Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ bHLHLZ bHLHLZ Δ Δ D c-MYC MYCL E WT ΔMB0 WT ΔMB0 3,000,0003000000 88 0.06 99 0.6 99 1 99 1 3 3 3 3 10 10 10 10 10 10

2 2 2 2 10 10 10 10 10 10

1 1 1 1 10 10 10 10 10 10 2,000,0002000000 D10 Y4-A :: PE-Cy7-A Y4-A :: PE-Cy7-A Y4-A :: PE-Cy7-A Y4-A :: PE-Cy7-A

0 0 0 0 10 10 10 10 10 10

-1 -1 -1 -1 10 10 10 10 10 10 3 -1 0 1 2 3 -1 0 1 2 3 -1 0 1 2 3 -1 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 41 12 0 0.6 0 0.5 5E-3 0.1 0 1,000,0001000000 CD13 24 1 16 9 20 7 94 3 ✱ 3 3 3 ✱ 10 3 ✱ 10 10 10 10 10 Cell number ✱

2 2 10 2 2 10 10 10 10 10

1 1 10 1 1 0 10 10 10 10 10 D21 Y4-A :: PE-Cy7-A Y4-A :: PE-Cy7-A Y4-A :: PE-Cy7-A Y4-A :: PE-Cy7-A day 10 day 21 0 0 0 10 10 10 0 c-MYC- MB0 10 10 10 ∆

-1 -1 10 -1 -1 10 10 10 10 10 3 -1 0 1 2 3 -1 0 1 2 3 3 -1 0 1 2 3 -1 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 c-MYC-WT MYCL-WT 70 5 18 57 25 48 2 1 c-MYC- MB0 MYCL- MB0 TRA-1-60 ∆ ∆ Fig. 3 A Sendai virus ・Infection (-) Day 3, 5, 7 ・KOSK+c-MYC ・KOSK+MYCL GO analysis Mass (i) MYCL-WT > c-MYC-WT (SeV) Episomal vector spectrometry (day 3 ∪ day 5 ∪ day 7) (Oshp53SKLE + ) Day 10 (ii) MYCL-WT > c-MYC-WT (EpiV) ・c-MYC-WT/ΔMB0 (iii) c-MYC-ΔMB0 > c-MYC-WT (EpiV) ・MYCL-WT/ΔMB0 (iv) (i) ∩ (ii) ∩ (iii)

B MYCL-WT > c-MYC-WT (SeV) (i) 520

(iv)18 c-MYC-ΔMB0 > c-MYC-WT (EpiV) (iii) 183 MYCL-WT > c-MYC-WT (EpiV) (ii) 128

C GO analysis D GO analysis Molecular function KEGG pathway Group Group Focal adhesion (i) protein binding (i) ECM- interaction integrin binding Proteoglycans in cancer extracellular matrix structural constituent Amoebiasis collagen binding Platelet activation double-stranded RNA binding PI3K-Akt signaling pathway platelet-derived growth factor binding Thyroid hormone signaling pathway proteoglycan binding Bacterial invasion of epithelial cells laminin binding Leukocyte transendothelial migration thyroid binding Protein digestion and absorption GTPase activity 0.0 3.3 6.5 9.8 13.0 0.0 2.3 4.5 6.8 9.0 (ii) actin binding (ii) ECM-receptor interaction extracellular matrix structural constituent Amoebiasis actin filament binding Focal adhesion protein homodimerization activity PI3K-Akt signaling pathway structural constituent of muscle calcium-dependent protein binding Hypertrophic cardiomyopathy (HCM) glycoprotein binding Dilated cardiomyopathy integrin binding Small cell lung cancer S100 protein binding Toxoplasmosis insulin-like growth factor binding 0.0 1.0 2.0 3.0 4.0 0.0 1.8 3.5 5.3 7.0

(iii) protein binding (iii) ECM-receptor interaction actin binding Focal adhesion calcium-dependent protein binding integrin binding Hypertrophic cardiomyopathy (HCM) structural constituent of cytoskeleton structural constituent of muscle Dilated cardiomyopathy structural molecule activity Leukocyte transendothelial migration actin filament binding collagen binding involved in cell-matrix adhesion PI3K-Akt signaling pathway protease binding 0.0 0.8 1.6 2.4 3.2 (iv) 0.0 1.3 2.5 3.8 5.0 (iv) structural constituent of muscle ECM-receptor interaction

actin binding Focal adhesion

structural constituent of cytoskeleton PI3K-Akt signaling pathway

actin filament binding Cardiac muscle contraction

structural molecule activity Hypertrophic cardiomyopathy (HCM)

calcium-dependent protein binding Dilated cardiomyopathy

0.0 0.8 1.6 2.4 3.2 0.0 0.7 1.3 2.0 2.6 -log10(P-value) -log10(P-value) Fig. 4

A MYC Box domain 0 1 2 3a 3b 4 bHLHLZ W135 c-MYC 439 aa

MYCL 364 aa W96

MYCL-MB2-WT/W96E GST 44 96 135

BC

40 MYCL-WT MYCL-W96E

30

20

10 ✱ Day21

0 iPSC-like colony number WT W96E MYCL

D E GO analysis Molecular function Reprogrammed hiPSCs (60) HDFs (31) poly(A) RNA binding RNA binding nucleotide binding 6 25 35 structural constituent of ribosome nucleic acid binding poly(A) binding protein binding mRNA 3'-UTR binding poly(G) binding telomeric DNA binding HNRNP0 HNRNPAB NCL HNRNPU FAU rRNA binding HNRNPA1 RPL37A RPL22 HNRNPD RPL23A

HNRNPDL CIRBP RPS24 RPS7 HNRNPA2B1 0E+00 2E+01 3E+01 HNRNPA3 KHDRBS1 RPS4X ALYREF DDX5 -log10(P-value) RPL31 SYNCRIP HNRNPR MCFD2 PTGES3 Fig. 5

Transcription

Cell adhesion -related gene

processing RNA PTM

MYCL MB0 MB2 MET

RBP

ing RNA process

iPSC-like Post-transcriptional regulation colony Supplementary Files

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Supplementaryinformationandguresv2.pdf