Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

MAP2K6 remodels chromatin and facilitates reprogramming by activating Gatad2b-phosphorylation dependent heterochromatin loosening

Abstract

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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Mkk6 is a heterochromatin relaxer.
Fig. 2: Mkk6 enhances reprogramming.
Fig. 3: Mkk6 relaxes heterochromatin and enhances reprogramming through phosphorylation of Gatad2b.
Fig. 4: Mkk6 phosphorylates Gatad2b at S487 and T490.
Fig. 5: Mkk6 increases histone acetylation through phosphorylation of Gatad2b.
Fig. 6: Mkk6 facilitates the binding ability of Sox2 and Klf4 and promotes the expression of pluripotency genes.

Data availability

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.

References

  1. 1.

    Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–76.

    CAS  PubMed  Google Scholar 

  2. 2.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Liang G, Taranova O, Xia K, Zhang Y. Butyrate promotes induced pluripotent stem cell generation. J Biol Chem. 2010;285:25516–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Mattout A, Biran A, Meshorer E. Global epigenetic changes during somatic cell reprogramming to iPS cells. J Mol Cell Biol. 2011;3:341–50.

    PubMed  Google Scholar 

  5. 5.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    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.

    CAS  PubMed  Google Scholar 

  7. 7.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    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.

    CAS  PubMed  Google Scholar 

  9. 9.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    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.

    CAS  PubMed  Google Scholar 

  11. 11.

    Apostolou E, Hochedlinger K. Chromatin dynamics during cellular reprogramming. Nature. 2013;502:462–71.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    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.

    CAS  PubMed  Google Scholar 

  13. 13.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    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.

    CAS  PubMed  Google Scholar 

  16. 16.

    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.

    CAS  PubMed  Google Scholar 

  17. 17.

    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.

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    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.

    CAS  PubMed  Google Scholar 

  20. 20.

    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.

    CAS  PubMed  Google Scholar 

  21. 21.

    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.

    CAS  PubMed  Google Scholar 

  22. 22.

    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.

    CAS  Google Scholar 

  23. 23.

    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.

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    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.

    CAS  PubMed  Google Scholar 

  27. 27.

    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.

    CAS  PubMed  Google Scholar 

  28. 28.

    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.

    CAS  PubMed  Google Scholar 

  29. 29.

    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.

    CAS  PubMed  Google Scholar 

  30. 30.

    Li Z, Rana TM. A kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generation. Nat Commun. 2012;3:1085.

    PubMed  Google Scholar 

  31. 31.

    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.

    CAS  PubMed  Google Scholar 

  32. 32.

    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.

    CAS  PubMed  Google Scholar 

  33. 33.

    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.

    CAS  Google Scholar 

  34. 34.

    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.

    CAS  PubMed  Google Scholar 

  35. 35.

    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.

    CAS  PubMed  Google Scholar 

  36. 36.

    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.

    CAS  PubMed  Google Scholar 

  37. 37.

    Dhanasekaran N, Premkumar Reddy E. Signaling by dual specificity kinases. Oncogene. 1998;17:1447–55.

    CAS  PubMed  Google Scholar 

  38. 38.

    Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705.

    CAS  Google Scholar 

  39. 39.

    Rossetto D, Avvakumov N, Cote J. Histone phosphorylation: a chromatin modification involved in diverse nuclear events. Epigenetics. 2012;7:1098–108.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Klein AM, Zaganjor E, Cobb MH. Chromatin-tethered MAPKs. Curr Opin Cell Biol. 2013;25:272–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    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.

    CAS  PubMed  Google Scholar 

  42. 42.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    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.

    CAS  PubMed  Google Scholar 

  45. 45.

    Torchy MP, Hamiche A, Klaholz BP. Structure and function insights into the NuRD chromatin remodeling complex. Cell Mol Life Sci. 2015;72:2491–507.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    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.

    CAS  PubMed  Google Scholar 

  49. 49.

    Jaffer S, Goh P, Abbasian M, Nathwani AC. Mbd3 Promotes Reprogramming of Primary Human Fibroblasts. Int J Stem Cells. 2018;11:235–41.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    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.

    CAS  PubMed  Google Scholar 

  52. 52.

    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.

    CAS  PubMed  Google Scholar 

  53. 53.

    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.

    CAS  PubMed  Google Scholar 

  54. 54.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    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.

    CAS  PubMed  Google Scholar 

  56. 56.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Nelson JD, Denisenko O, Sova P, Bomsztyk K. Fast chromatin immunoprecipitation assay. Nucleic Acids Res. 2006;34:e2.

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    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.

    CAS  PubMed  Google Scholar 

  59. 59.

    Thevenaz P, Ruttimann UE, Unser M. A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process. 1998;7:27–41.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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).

Author information

Affiliations

Authors

Contributions

DP, XL, and KC supervised the project. DP, KC, and TW initiated wild-type and mutant Mkk6 effects on reprogramming efficiency. DP, XL, KC, GX, and DZ identified Mkk6’s function depends on not P38 but Gatad2b, a novel target which acts downstream to elevate histone acetylation levels, loosen heterochromatin and facilitate binding of Sox2 and Klf4 to their targets and promotes pluripotency gene expression during reprogramming. XL, KC, DZ, GX, ZL, and HY designed and performed iPSCs generation, ITRAQ, co-IP, western blot, RNA-seq, ChIP-qPCR, ChIP-seq, ATAC-seq, in vitro kinase assay, nuclease accessibility assay and salt extraction assay. LH, ZH, YL, JLu, and SL participated in plasmids construction, iPSCs generation, and the dependence of Mkk6 function on P38. YW, LL, JZ, JW, and BW participated in cell culture and ChIP-qPCR. QL and YZ participated in FRAP and HP1α immunofluorescence. JLiu and JC participated in chimeric mice and germline transmission mice generation. DP, XL, KC, GX, ZL, and DZ wrote the paper. All authors contributed to the writing and editing of the paper and approved the final paper.

Corresponding authors

Correspondence to Duanqing Pei, Xingguo Liu or Keshi Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethical approval

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.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by D Aberdam

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Search

Quick links