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Identification of Structural Elements of the Lysine Specific Demethylase 2B CxxC Domain Associated with Replicative Senescence Bypass in Primary Mouse Cells

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Abstract

Background

Lysine specific demethylase 2B, KDM2B, regulates genes that participate in cellular development, morphogenesis, differentiation and metabolism as a component of the polycomb repressive complex 1 (PRC1). The CxxC finger of KDM2B is responsible for the DNA binding capacity of this epigenetic regulator, acting as a sampling mechanism across chromatin for gene repression

Objectives

The molecular determinants of the CxxC—DNA interaction remain largely unknown, revealing a significant knowledge gap to be explored. Our goal was to elucidate the key residues of the CxxC domain that contribute to its function as well as to further elaborate on the significance of this domain in the KDM2B role

Methods

By using electrophoresis mobility swift assay, we identified structural elements of CxxC domain that participate in the DNA recognition. We created mouse embryonic fibroblasts overexpressing different truncated and point-mutated mouse KDM2B variants to examine the contribution of the KDM2B domains in replicative senescence bypass

Results

In this study, we show that only the CxxC finger is essential for the ability of mKDM2B to bypass replicative senescence in primary cells by ink4A-Arf-ink4B locus repression, and that this is mediated by specific interactions of residues R585, K608 and K616 with non-methylated CpG containing DNA

Conclusions

These results provide new structural insights into the molecular interactions of CxxC and could serve as a stepping-stone for developing domain-specific inhibitors for KDM2B.

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Abbreviations

PRC1:

Polycomb repressive complex 1

KDM2B:

Lysine specific demethylase 2B

MEFs:

Mouse embryonic fibroblasts

References

  1. Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature 403:41–45

    Article  PubMed  CAS  Google Scholar 

  2. Grant PA (2001) A tale of histone modifications. Genome Biol. https://doi.org/10.1186/gb-2001-2-4-reviews0003

    Article  PubMed  PubMed Central  Google Scholar 

  3. Berger SL (2007) The complex language of chromatin regulation during transcription. Nature 447:407–412

    Article  PubMed  CAS  Google Scholar 

  4. Lachner M, O'Sullivan RJ, Jenuwein T (2003) An epigenetic road map for histone lysine methylation. J Cell Sci 116:2117–2124

    Article  PubMed  CAS  Google Scholar 

  5. Caselle M, Di Cunto F, Provero P (2002) Correlating overrepresented upstream motifs to gene expression: a computational approach to regulatory element discovery in eukaryotes. BMC Bioinform 3:7

    Article  Google Scholar 

  6. Fischle W, Wang Y, Allis CD (2003) Histone and chromatin cross-talk. Curr Opin Cell Biol 15(2):172–183

    Article  PubMed  CAS  Google Scholar 

  7. Margueron R, Trojer P, Reinberg D (2005) The key to development: interpreting the histone code? Curr Opin Gen Dev 15(2):163–176

    Article  CAS  Google Scholar 

  8. Kouzarides T (2007) Chromatin modifications and their function. Cell 28:693–705

    Article  CAS  Google Scholar 

  9. Taverna SD, Li H, Ruthenburg AJ, Allis CD, Patel DJ (2007) How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat Struct Mol Biol 14(11):1025–1040

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Pfau R, Tzatsos A, Kampranis SC, Serebrennikova OB, Bear SE, Tsichlis PN (2008) Members of a family of JmjC domain-containing oncoproteins immortalize embryonic fibroblasts via a JmjC domain-dependent process. Proc Natl Acad Sci USA 105(6):1907–1912

    Article  PubMed  PubMed Central  Google Scholar 

  11. He J, Kallin EM, Tsukada Y, Zhang Y (2008) The H3K36 demethylase Jhdm1b/Kdm2b regulates cell proliferation and senescence through p15 (Ink4b). Nat Struct Mol Biol 15:1169–1175

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Tzatsos A, Pfau R, Kampranis SC, Tsichlis PN (2009) Ndy1/KDM2B immortalizes mouse embryonic fibroblasts by repressing the Ink4a/Arf locus. PNAS 106:2641–2646

    Article  PubMed  PubMed Central  Google Scholar 

  13. He J, Nguyen AT, Zhang Y (2011) KDM2b/JHDM1b, an H3K36me2-specific demethylase, is required for initiation and maintenance of acute myeloid leukemia. Blood 117:3869–3880

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Janzer A, Stamm K, Becker A, Zimmer A, Buettner R, Kirfel J (2012) The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology, and the metabolome of fibroblasts. J Biol Chem 287(37):30984–30992

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Kang JY, Kim JY, Kim KB, Park JW, Cho H, Hahm JY, Chae YC, Kim D, Kook H, Rhee S, Ha NC, Seo SB (2018) KDM2B is a histone H3K79 demethylase and induces transcriptional repression via SIRT1-mediated chromatin silencing. FASEB J. https://doi.org/10.1096/fj.201800242R

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kottakis F, Polytarchou C, Foltopoulou P, Sanidas I, Kampranis SC, Tsichlis PN (2011) FGF-2 regulates cell proliferation, migration, and angiogenesis through an NDY1/KDM2B-miR-101-EZH2 pathway. Mol Cell 43(2):285–298

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Vacík T, Lađinović D, Raška I (2018) KDM2A/B lysine demethylases and their alternative isoforms in development and disease. Nucleus 9(1):431–441

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Frescas D, Guardavaccaro D, Bassermann F, Koyama-Nasu R, Pagano M (2007) JHDM1B/FBXL10 is a nucleolar protein that represses transcription of ribosomal RNA genes. Nature 450:309–313

    Article  PubMed  CAS  Google Scholar 

  19. Blackledge NP, Farcas AM, Kondo T, King HW, McGouran JF, Hanssen LL, Ito S, Cooper S, Kondo K, Koseki Y, Ishikura T, Long HK, Sheahan TW, Brockdorff N, Kessler BM, Koseki H, Klose RJ (2014) Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation. Cell 157(6):1445–1459

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Zacharopoulou N, Tsapara A, Kallergi G, Schmid E, Alkahtani S, Alarifi S, Tsichlis PN, Kampranis S, Stournaras C (2018) The epigenetic factor KDM2B regulates EMT and small GTPases in colon tumor cells. Cell Physiol Biochem 47:368–377

    Article  PubMed  CAS  Google Scholar 

  21. Zacharopoulou N, Tsapara A, Kallergi G, Schmid E, Tsichlis PN, Kampranis SC, Stournaras C (2018) The epigenetic factor KDM2B regulates cell adhesion, small rho GTPases, actin cytoskeleton and migration in prostate cancer cells. Biochem Biophys Acta 1865(4):587–597

    Article  CAS  Google Scholar 

  22. Tzatsos A, Paskaleva P, Ferrari F, Deshpande V, Stoykova S, Contino G, Wong KK, Lan F, Trojer P, Park PJ, Bardeesy N (2013) KDM2B promotes pancreatic cancer via Polycomb-dependent and -independent transcriptional programs. J Clin Invest 123(2):727–739

    PubMed  PubMed Central  CAS  Google Scholar 

  23. Ueda T, Nagamachi A, Takubo K, Yamasaki N, Matsui H, Kanai A, Nakata Y, Ikeda K, Konuma T, Oda H, Wolff L, Honda Z, Wu X, Helin K, Iwama A, Suda T, Inaba T, Honda H (2015) Fbxl10 overexpression in murine hematopoietic stem cells induces leukemia involving metabolic activation and upregulation of Nsg2. Blood 125:3437–3446

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Tzatsos A, Paskaleva P, Lymperi S, Contino G, Stoykova S, Chen Z, Wong KK, Bardeesy N (2011) Lysine-specific demethylase 2B (KDM2B)-let-7-enhancer of zester homolog 2 (EZH2) pathway regulates cell cycle progression and senescence in primary cells. J Biol Chem 286(38):33061–33069

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Kottakis F, Foltopoulou P, Sanidas I, Keller P, Wronski A, Dake BT, Ezell SA, Shen Z, Naber SP, Hinds PW, McNiel E, Kuperwasser C, Tsichlis PN (2014) NDY1/KDM2B functions as a master regulator of polycomb complexes and controls self-renewal of breast cancer stem cells. Cancer Res 74(14):3935–3946

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Ge R, Wang Z, Zeng Q, Xu X, Olumi AF (2011) F-box protein 10, an NF-κB-dependent anti-apoptotic protein, regulates TRAIL-induced apoptosis through modulating c-Fos/c-FLIP pathway. Cell Death Differ 18(7):1184–1195

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Yu X, Wang J, Wu J, Shi Y (2015) A systematic study of the cellular metabolic regulation of Jhdm1b in tumor cells. Mol Biosyst 11(7):1867–1875

    Article  PubMed  CAS  Google Scholar 

  28. Wang JJ, Dong R, Wang LP, Wang JS, Du J, Wang SL, Shan ZC, Fan ZP (2015) Histone demethylase KDM2B inhibits the chondrogenic differentiation potentials of stem cells from apical papilla. Int J Clin Exp Med 8(2):2165–2173

    PubMed  PubMed Central  CAS  Google Scholar 

  29. Polytarchou C, Pfau R, Hatziapostolou M, Tsichlis PN (2008) The JmjC domain histone demethylase Ndy1 regulates redox homeostasis and protects cells from oxidative stress. Mol Cell Biol 28:7451–7464

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Lu L, Gao Y, Zhang Z, Cao Q, Zhang X, Zou J, Cao Y (2015) Kdm2a/b lysine demethylases regulate canonical wnt signaling by modulating the stability of nuclear beta-catenin. Dev Cell 33:660–674

    Article  PubMed  CAS  Google Scholar 

  31. Zhou Z, Yang X, He J, Liu J, Wu F, Yu S, Liu Y, Lin R, Liu H, Cui Y, Zhou C, Wang X, Wu J, Cao S, Guo L, Lin L, Wang T, Peng X, Qiang B, Hutchins AP, Pei D, Chen J (2017) Kdm2b regulates somatic reprogramming through variant PRC1 complex-dependent function. Cell Rep 21(8):2160–2170

    Article  PubMed  CAS  Google Scholar 

  32. Li H, Lai P, Jia J, Song Y, Xia Q, Huang K, He N, Ping W, Chen J, Yang Z, Li J, Yao M, Dong X, Zhao J, Hou C, Esteban MA, Gao S, Pei D, Hutchins AP, Yao H (2017) RNA helicase DDX5 inhibits reprogramming to pluripotency by miRNA-based repression of RYBP and its PRC1-dependent and -independent functions. Cell Stem Cell 20:462–477

    Article  PubMed  CAS  Google Scholar 

  33. Wang T, Chen K, Zeng X, Yang J, Wu Y, Shi X, Qin B, Zeng L, Esteban MA, Pan G, Pei D (2011) The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Cell Stem Cell 9:575–587

    Article  PubMed  CAS  Google Scholar 

  34. He J, Shen L, Wan M, Taranova O, Wu H, Zhang Y (2013) Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes. Nat Cell Biol 15:373–384

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Sánchez C, Sánchez I, Demmers JA, Rodriguez P, Strouboulis J, Vidal M (2007) Proteomics analysis of Ring1B/Rnf2 interactors identifies a novel complex with the Fbxl10/Jhdm1B histone demethylase and the Bcl6 interacting corepressor. Mol Cell Proteom 6:820–834

    Article  CAS  Google Scholar 

  36. Farcas AM, Blackledge NP, Sudbery I, Long HK, McGouran JF, Rose NR, Lee S, Sims D, Cerase A, Sheahan TW, Koseki H, Brockdorff N, Ponting CP, Kessler BM, Klose RJ (2012) KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands. eLife 1:e00205

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Wu X, Johansen JV, Helin K (2013) Fbxl10/Kdm2b recruits polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation. Mol Cell 49:1134–1146

    Article  PubMed  CAS  Google Scholar 

  38. Wong SJ, Gearhart MD, Taylor AB, Nanyes DR, Ha DJ, Robinson AK, Artigas JA, Lee OJ, Demeler B, Hart PJ, Bardwell VJ, Kim CA (2016) KDM2B recruitment of the Polycomb group complex, PRC11, requires cooperation between PCGF1 and BCORL1. Structure 24(10):1795–1801

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Xu C, Liu K, Lei M, Yang A, Li Y, Hughes TR, Min J (2018) DNA sequence recognition of human CXXC domains and their structural determinants. Structure 26:85–95

    Article  PubMed  CAS  Google Scholar 

  40. Long HK, Blackledge NP, Klose RJ (2013) ZF-CxxC domain-containing proteins, CpG islands and the chromatin connection. Biochem Soc Trans 41:727–740

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Koyama-Nasu R, David G, Tanese N (2007) The F-box protein Fbl10 is a novel transcriptional repressor of c-Jun. Nat Cell Biol 9:1074–1080

    Article  PubMed  CAS  Google Scholar 

  42. Boulard M, Edwards JR, Bestor TH (2015) FBXL10 protects Polycomb-bound genes from hypermethylation. Nat Genet 47(5):479–485

    Article  PubMed  CAS  Google Scholar 

  43. Liang G, He J, Zhang Y (2012) Kdm2b promotes induced pluripotent stem cell generation by facilitating gene activation early in reprogramming. Nat Cell Biol 14:457–466

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Zhou JC, Blackledge NP, Farcas AM, Klose RJ (2012) Recognition of CpG island chromatin by KDM2A requires direct and specific interaction with linker DNA. Mol Cell Biol 32(2):479–489

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Ignea C, Pontini M, Maffei ME, Makris AM, Kampranis SC (2014) Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase. ACS Synth Biol 3:298–306

    Article  PubMed  CAS  Google Scholar 

  46. Blackledge NP, Zhou JC, Tolstorukov MY, Farcas AM, Park PJ, Klose RJ (2010) CpG islands recruit a histone H3 lysine 36 demethylase. Mol Cell 38(2):179–190

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Cierpicki T, Risner LE, Grembecka J, Lukasik SM, Popovic R, Omonkowska M, Shultis DD, Zeleznik-Le NJ, Bushweller JH (2010) Structure of the MLL CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia. Nat Struct Mol Biol 17:62–68

    Article  PubMed  CAS  Google Scholar 

  48. Kampranis SC, Tsichlis PN (2009) Histone demethylases and cancer. Adv Cancer Res 102:103–169

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Yan M, Yang X, Wang H, Shao Q (2018) The critical role of histone lysine demethylase KDM2B in cancer. Am J Transl Res 10(8):2222–2233

    PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We thank Prof. Kriton Kalantidis (IMBB-FORTH) and Dr. Ioannis Vlatakis (Department of Biology, University of Crete) for their help with the EMSA experiments.

Funding

This research was funded by a State Scholarship Foundation (IKY) Partnership Agreement Program (ΕΣΠΑ 2014-20) that was co-financed by the European Social Fund (ESF), grant no. 2017-050-0504-10806. Work in PNT’s laboratory was supported by grant R01 CA109747 from the National Cancer Institute. These two funding sources did not overlap.

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EED, MA and SCK carried out experiments; SCK, PNT and CT designed experiments; EED, MA, and SCK analyzed data; EED, PNT and SCK wrote and revised the manuscript.

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Correspondence to Eleftherios E. Deiktakis.

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Deiktakis, E.E., Abrams, M., Tsapara, A. et al. Identification of Structural Elements of the Lysine Specific Demethylase 2B CxxC Domain Associated with Replicative Senescence Bypass in Primary Mouse Cells. Protein J 39, 232–239 (2020). https://doi.org/10.1007/s10930-020-09895-z

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