Somatic cell reprogramming is an ideal model for studying epigenetic regulation as it undergoes dramatic chromatin remodeling. However, a role for phosphorylation signaling in chromatin protein modifications for reprogramming remains unclear. Here, we identified mitogen-activated protein kinase kinase 6 (Mkk6) as a chromatin relaxer and found that it could significantly enhance reprogramming. The function of Mkk6 in heterochromatin loosening and reprogramming requires its kinase activity but does not depend on its best-known target, P38. We identified Gatad2b as a novel target of Mkk6 phosphorylation that acts downstream to elevate histone acetylation levels and loosen heterochromatin. As a result, Mkk6 over-expression facilitates binding of Sox2 and Klf4 to their targets and promotes pluripotency gene expression during reprogramming. Our studies not only reveal an Mkk phosphorylation mediated modulation of chromatin status in reprogramming, but also provide new rationales to further investigate and improve the cell fate determination processes.
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The Sequencing data reported in this paper has been deposited in the Genome Sequence Archive at the Beijing Institute of Genomics (BIG) Data Center, BIG, Chinese Academy of Sciences. The accession numbers for the ATAC-seq, ChIP-seq and RNA-seq data in this study are CRA005167, CRA005151 and CRA005159, which are publicly accessible at https://bigd.big.ac.cn/gsa.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–76.
Huangfu DW, Maehr R, Guo WJ, Eijkelenboom A, Snitow M, Chen AE, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008;26:795–7.
Liang G, Taranova O, Xia K, Zhang Y. Butyrate promotes induced pluripotent stem cell generation. J Biol Chem. 2010;285:25516–21.
Mattout A, Biran A, Meshorer E. Global epigenetic changes during somatic cell reprogramming to iPS cells. J Mol Cell Biol. 2011;3:341–50.
Ang YS, Tsai SY, Lee DF, Monk J, Su J, Ratnakumar K, et al. Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network. Cell. 2011;145:183–97.
Mansour AA, Gafni O, Weinberger L, Zviran A, Ayyash M, Rais Y, et al. The H3K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming. Nature. 2012;488:409–13.
Zhao W, Li Q, Ayers S, Gu Y, Shi Z, Zhu Q, et al. Jmjd3 Inhibits Reprogramming by Upregulating Expression of INK4a/Arf and Targeting PHF20 for Ubiquitination. Cell. 2013;152:1037–50.
Wang T, Chen K, Zeng X, Yang J, Wu Y, Shi X, et al. The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Cell Stem Cell. 2011;9:575–87.
Liang G, He J, Zhang Y. Kdm2b promotes induced pluripotent stem cell generation by facilitating gene activation early in reprogramming. Nat Cell Biol. 2012;14:457–66.
Chen J, Liu H, Liu J, Qi J, Wei B, Yang J, et al. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs. Nat Genet. 2013;45:34–42.
Apostolou E, Hochedlinger K. Chromatin dynamics during cellular reprogramming. Nature. 2013;502:462–71.
Singhal N, Graumann J, Wu G, Arauzo-Bravo MJ, Han DW, Greber B, et al. Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming. Cell. 2010;141:943–55.
Gaspar-Maia A, Alajem A, Polesso F, Sridharan R, Mason MJ, Heidersbach A, et al. Chd1 regulates open chromatin and pluripotency of embryonic stem cells. Nature. 2009;460:863–8.
Wang L, Du Y, Ward JM, Shimbo T, Lackford B, Zheng X, et al. INO80 facilitates pluripotency gene activation in embryonic stem cell self-renewal, reprogramming, and blastocyst development. Cell Stem Cell. 2014;14:575–91.
Luo M, Ling T, Xie W, Sun H, Zhou Y, Zhu Q, et al. NuRD blocks reprogramming of mouse somatic cells into pluripotent stem cells. Stem Cells. 2013;31:1278–86.
Rais Y, Zviran A, Geula S, Gafni O, Chomsky E, Viukov S, et al. Deterministic direct reprogramming of somatic cells to pluripotency. Nature. 2013;502:65–70.
dos Santos RL, Tosti L, Radzisheuskaya A, Caballero IM, Kaji K, Hendrich B, et al. MBD3/NuRD facilitates induction of pluripotency in a context-dependent manner. Cell Stem Cell. 2014;15:102–10.
Chen K, Long Q, Wang T, Zhao D, Zhou Y, Qi J, et al. Gadd45a is a heterochromatin relaxer that enhances iPS cell generation. EMBO Rep. 2016;17:1641–56.
Wang B, Wu L, Li D, Liu Y, Guo J, Li C, et al. Induction of Pluripotent Stem Cells from Mouse Embryonic Fibroblasts by Jdp2-Jhdm1b-Mkk6-Glis1-Nanog-Essrb-Sall4. Cell Rep. 2019;27:3473–85 e3475.
Kim SH, Kim MO, Cho YY, Yao K, Kim DJ, Jeong CH, et al. ERK1 phosphorylates Nanog to regulate protein stability and stem cell self-renewal. Stem Cell Res. 2014;13:1–11.
Simone C, Forcales SV, Hill DA, Imbalzano AN, Latella L, Puri PL. p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci. Nat Genet. 2004;36:738–43.
Cargnello M, Roux PP. Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases. Microbiol Mol Biol R. 2011;75:50–83.
Long Q, Qi J, Li W, Zhou Y, Chen K, Wu H, et al. Protocol for detecting chromatin dynamics and screening chromatin relaxer by FRAP assay. STAR Protoc. 2021;2:100706.
Meshorer E, Yellajoshula D, George E, Scambler PJ, Brown DT, Misteli T. Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev Cell. 2006;10:105–16.
Min X, Akella R, He H, Humphreys JM, Tsutakawa SE, Lee SJ, et al. The structure of the MAP2K MEK6 reveals an autoinhibitory dimer. Structure. 2009;17:96–104.
Chen K, Long Q, Xing G, Wang T, Wu Y, Li L, et al. Heterochromatin loosening by the Oct4 linker region facilitates Klf4 binding and iPSC reprogramming. EMBO J. 2020;39:e99165.
Moriguchi T, Kuroyanagi N, Yamaguchi K, Gotoh Y, Irie K, Kano T, et al. A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3. J Biol Chem. 1996;271:13675–9.
Stein B, Brady H, Yang MX, Young DB, Barbosa MS. Cloning and characterization of MEK6, a novel member of the mitogen-activated protein kinase kinase cascade. J Biol Chem. 1996;271:11427–33.
Remy G, Risco AM, Inesta-Vaquera FA, Gonzalez-Teran B, Sabio G, Davis RJ, et al. Differential activation of p38MAPK isoforms by MKK6 and MKK3. Cell Signal. 2010;22:660–7.
Li Z, Rana TM. A kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generation. Nat Commun. 2012;3:1085.
Xu X, Wang Q, Long Y, Zhang R, Wei X, Xing M, et al. Stress-mediated p38 activation promotes somatic cell reprogramming. Cell Res. 2013;23:131–41.
Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol. 2006;24:1285–92.
Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJ. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteom. 2005;4:873–86.
Unwin RD, Griffiths JR, Whetton AD. Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC-MS/MS. Nat Protoc. 2010;5:1574–82.
Yuan W, Wu T, Fu H, Dai C, Wu H, Liu N, et al. Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science. 2012;337:971–5.
Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 2001;22:153–83.
Dhanasekaran N, Premkumar Reddy E. Signaling by dual specificity kinases. Oncogene. 1998;17:1447–55.
Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705.
Rossetto D, Avvakumov N, Cote J. Histone phosphorylation: a chromatin modification involved in diverse nuclear events. Epigenetics. 2012;7:1098–108.
Klein AM, Zaganjor E, Cobb MH. Chromatin-tethered MAPKs. Curr Opin Cell Biol. 2013;25:272–7.
Bashir M, Parray AA, Baba RA, Bhat HF, Bhat SS, Mushtaq U, et al. beta-Amyloid-evoked apoptotic cell death is mediated through MKK6-p66shc pathway. Neuromolecular Med. 2014;16:137–49.
Feng Q, Cao R, Xia L, Erdjument-Bromage H, Tempst P, Zhang Y. Identification and functional characterization of the p66/p68 components of the MeCP1 complex. Mol Cell Biol. 2002;22:536–46.
Brackertz M, Gong Z, Leers J, Renkawitz R. p66alpha and p66beta of the Mi-2/NuRD complex mediate MBD2 and histone interaction. Nucleic Acids Res. 2006;34:397–406.
Brackertz M, Boeke J, Zhang R, Renkawitz R. Two highly related p66 proteins comprise a new family of potent transcriptional repressors interacting with MBD2 and MBD3. J Biol Chem. 2002;277:40958–66.
Torchy MP, Hamiche A, Klaholz BP. Structure and function insights into the NuRD chromatin remodeling complex. Cell Mol Life Sci. 2015;72:2491–507.
Mor N, Rais Y, Sheban D, Peles S, Aguilera-Castrejon A, Zviran A, et al. Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency. Cell Stem Cell. 2018;23:412–25.
Fidalgo M, Faiola F, Pereira CF, Ding J, Saunders A, Gingold J, et al. Zfp281 mediates Nanog autorepression through recruitment of the NuRD complex and inhibits somatic cell reprogramming. Proc Natl Acad Sci USA. 2012;109:16202–7.
Wang S, Xia P, Ye B, Huang G, Liu J, Fan Z. Transient activation of autophagy via Sox2-mediated suppression of mTOR is an important early step in reprogramming to pluripotency. Cell Stem Cell. 2013;13:617–25.
Jaffer S, Goh P, Abbasian M, Nathwani AC. Mbd3 Promotes Reprogramming of Primary Human Fibroblasts. Int J Stem Cells. 2018;11:235–41.
Sakurai K, Talukdar I, Patil VS, Dang J, Li Z, Chang KY, et al. Kinome-wide functional analysis highlights the role of cytoskeletal remodeling in somatic cell reprogramming. Cell Stem Cell. 2014;14:523–34.
Vazquez-Martin A, Vellon L, Quiros PM, Cufi S, Ruiz de Galarreta E, Oliveras-Ferraros C, et al. Activation of AMP-activated protein kinase (AMPK) provides a metabolic barrier to reprogramming somatic cells into stem cells. Cell Cycle. 2012;11:974–89.
Yao K, Ki MO, Chen H, Cho YY, Kim SH, Yu DH, et al. JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity. Stem Cell Res. 2014;12:139–52.
Tang Y, Luo Y, Jiang Z, Ma Y, Lin CJ, Kim C, et al. Jak/Stat3 signaling promotes somatic cell reprogramming by epigenetic regulation. Stem Cells. 2012;30:2645–56.
Wu Y, Chen K, Xing G, Li L, Ma B, Hu Z, et al. Phospholipid remodeling is critical for stem cell pluripotency by facilitating mesenchymal-to-epithelial transition. Sci Adv. 2019;5:eaax7525.
Esteban MA, Wang T, Qin B, Yang J, Qin D, Cai J, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell. 2010;6:71–79.
Chen J, Liu J, Chen Y, Yang J, Chen J, Liu H, et al. Rational optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells with ultra-high efficiency and fast kinetics. Cell Res. 2011;21:884–94.
Nelson JD, Denisenko O, Sova P, Bomsztyk K. Fast chromatin immunoprecipitation assay. Nucleic Acids Res. 2006;34:e2.
Li L, Chen K, Wang T, Wu Y, Xing G, Chen M, et al. Glis1 facilitates induction of pluripotency via an epigenome-metabolome-epigenome signalling cascade. Nat Metab. 2020;2:882–92.
Thevenaz P, Ruttimann UE, Unser M. A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process. 1998;7:27–41.
We thank all the members in the labs of Prof DP and Prof XL. This work was financially supported by the National Key Research and Development Program of China (2018YFA0107100), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030505), the National Key Research and Development Program of China (2017YFA0106300, 2017YFA0102900, 2017YFA0504100, 2019YFA09004500), the National Natural Science Foundation projects of China (32025010, 31900614, 31970709, 81901275, 32070729, 32100619, 32170747, 31830060), the Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-SMC001), and International Cooperation Program, CAS (154144KYSB20200006), Guangdong Province Science and Technology Program (2020B1212060052, 2018A030313825, 2018GZR110103002, 2020A1515011200, 2020A1515010919, 2020A1515011410, 2021A1515012513), Guangzhou Science and Technology Program (201807010067, 202002030277, 202102020827, 202102080066), Open Research Program of Key Laboratory of Regenerative Biology, CAS (KLRB201907, KLRB202014), and CAS Youth Innovation Promotion Association (to YW).
The authors declare no competing interests.
All the cells were obtained with approval from the ethics committee of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (GIBH). All the animals were handled according to approved Institutional Animal Care and Use Committee protocols of the GIBH.
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Xing, G., Liu, Z., Huang, L. et al. MAP2K6 remodels chromatin and facilitates reprogramming by activating Gatad2b-phosphorylation dependent heterochromatin loosening. Cell Death Differ (2021). https://doi.org/10.1038/s41418-021-00902-z