Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.
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Akiyama T, Xin L, Oda M, Sharov AA, Amano M, Piao Y, et al. Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells. DNA Res. 2015;22:307–18.
Amano T, Hirata T, Falco G, Monti M, Sharova LV, Amano M, et al. Zscan4 restores the developmental potency of embryonic stem cells. Nat Commun. 2013;4:1966.
Jiang J, Lv W, Ye X, Wang L, Zhang M, Yang H, et al. Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation. Cell Res. 2013;23:92–106.
Hisada K, Sánchez C, Endo TA, Endoh M, Román-Trufero M, Sharif J, et al. RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells. Mol Cell Biol. 2012;32:1139–49.
Falco G, Lee S-L, Stanghellini I, Bassey UC, Hamatani T, Ko MSH. Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells. Dev Biol. 2007;307:539–50.
Cerulo L, Tagliaferri D, Marotta P, Zoppoli P, Russo F, Mazio C, et al. Identification of a novel gene signature of ES cells self-renewal fluctuation through system-wide analysis. PLoS ONE 2014;9:e83235.
Dahl JA, Jung I, Aanes H, Greggains GD, Manaf A, Lerdrup M, et al. Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition. Nature. 2016;537:548–52.
Wu J, Greely HT, Jaenisch R, Nakauchi H, Rossant J, Belmonte JCI. Stem cells and interspecies chimaeras. Nature. 2016;540:51–9.
Hamatani T, Falco G, Carter MG, Akutsu H, Stagg CA, Sharov AA, et al. Age-associated alteration of gene expression patterns in mouse oocytes. Hum Mol Genet. 2004;13:2263–78.
Peaston AE, Evsikov AV, Graber JH, de Vries WN, Holbrook AE, Solter D, et al. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Dev Cell. 2004;7:597–606.
Evsikov AV, de Vries WN, Peaston AE, Radford EE, Fancher KS, Chen FH, et al. Systems biology of the 2-cell mouse embryo. Cytogenet Genome Res. 2004;105:240–50.
Kigami D, Minami N, Takayama H, Imai H. MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos. Biol Reprod. 2003;68:651–4.
Zhang W, Walker E, Tamplin OJ, Rossant J, Stanford WL, Hughes TR. Zfp206 regulates ES cell gene expression and differentiation. Nucleic Acids Res. 2006;34:4780–90.
Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D, et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature. 2012;487:57–63.
Hendrickson PG, Doráis JA, Grow EJ, Whiddon JL, Lim J-W, Wike CL, et al. Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons. Nat Genet. 2017;49:925–34.
De Iaco A, Planet E, Coluccio A, Verp S, Duc J, Trono D. DUX-family transcription factors regulate zygotic genome activation in placental mammals. Nat Genet. 2017;49:941–5.
Hackett JA, Surani MA. Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell. 2014;15:416–30.
Tagliaferri D, De Angelis MT, Russo NA, Marotta M, Ceccarelli M, Del Vecchio L, et al. Retinoic Acid Specifically Enhances Embryonic Stem Cell Metastate Marked by Zscan4. PLoS ONE 2016;11:e0147683.
Sharova LV, Sharov AA, Piao Y, Stagg CA, Amano T, Qian Y, et al. Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment. Vitr Cell Dev Biol Anim. 2016;52:616–24.
Heery DM, Kalkhoven E, Hoare S, Parker MG. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature. 1997;387:733–6.
McKenna NJ, O’Malley BW. Combinatorial control of gene expression by nuclear receptors and coregulators. Cell. 2002;108:465–74.
Epping MT, Wang L, Edel MJ, Carlée L, Hernandez M, Bernards R. The human tumor antigen PRAME is a dominant repressor of retinoic acid receptor signaling. Cell. 2005;122:835–47.
Napolitano G, Mazzocco A, Fraldi A, Majello B, Lania L. Functional inactivation of Cdk9 through oligomerization chain reaction. Oncogene. 2003;22:4882–8.
Iacovino M, Bosnakovski D, Fey H, Rux D, Bajwa G, Mahen E, et al. Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells. Stem Cells. 2011;29:1580–8.
Hamatani T, Carter MG, Sharov AA, Ko MS. Dynamics of global gene expression changes during mouse preimplantation development. Dev Cell. 2004;6:117–31.
Zeng F, Baldwin DA, Schultz RM. Transcript profiling during preimplantation mouse development. Dev Biol. 2004;272:483–96. Aug 15PubMed PMID: 15282163
Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, et al. NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Res. 2013;41:D991–5.
Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47.
Taiyun Wei and Viliam Simko R package “corrplot”: Visualization of a Correlation Matrix (Version 0.84). 2017. Available from: https://github.com/taiyun/corrplot
R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2017. https://www.R-project.org/
Ambrosio S, Di Palo G, Napolitano G, Amente S, Dellino GI, Faretta M, et al. Cell cycle-dependent resolution of DNA double-strand breaks. Oncotarget. 2016;7:4949–60.
Falco G, Stanghellini I, Ko MS. Use of Chuk as an internal standard suitable for quantitative RT-PCR in mouse preimplantation embryos. Reprod Biomed Online. 2006;13:394–403.
Al-Anee RS, Sulaiman GM, Al-Sammarrae KW, Napolitano G, Bagnati R, Lania L, et al. Chemical characterization, antioxidant and cytotoxic activities of the methanolic extract of Hymenocrater longiflorus grown in Iraq. Z Nat C. 2015;70:227–35.
Napolitano G, Amente S, Lavadera ML, Di Palo G, Ambrosio S, Lania L, et al. Sequence-specific double strand breaks trigger P-TEFb-dependent Rpb1-CTD hyperphosphorylation. Mutat Res. 2013;749:21–7.
Napolitano G, Amente S, Castiglia V, Gargano B, Ruda V, Darzacq X, et al. Caffeine prevents transcription inhibition and P-TEFb/7SK dissociation following UV-induced DNA damage. PLoS ONE 2010;5:e11245.
Probst AV, Okamoto I, Casanova M, El Marjou F, Le Baccon P, Almouzni G. A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development. Dev Cell. 2010;19:625–38.
Ambrosio S, Amente S, Napolitano G, Di Palo G, Lania L, Majello B. MYC impairs resolution of site-specific DNA double-strand breaks repair. Mutat Res. 2015;774:6–13.
Leitch HG, McEwen KR, Turp A, Encheva V, Carroll T, Grabole N, et al. Naive pluripotency is associated with global DNA hypomethylation. Nat Struct Mol Biol. 2013;20:311–6.
Habibi E, Brinkman AB, Arand J, Kroeze LI, Kerstens HHD, Matarese F, et al. Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell. 2013;13:360–9.
Eckersley-Maslin MA, Svensson V, Krueger C, Stubbs TM, Giehr P, Krueger F, et al. MERVL/Zscan4 Network Activation Results in Transient Genome-wide DNA Demethylation of mESCs. Cell Rep. 2016;17:179–92.
Ishiuchi T, Enriquez-Gasca R, Mizutani E, Bošković A, Ziegler-Birling C, Rodriguez-Terrones D, et al. Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly. Nat Struct Mol Biol. 2015;22:662–71.
Graf U, Casanova EA, Wyck S, Dalcher D, Gatti M, Vollenweider E, et al. Pramel7 mediates ground-state pluripotency through proteasomal-epigenetic combined pathways. Nat Cell Biol. 2017;19:763–73.
di Masi A, Leboffe L, De Marinis E, Pagano F, Cicconi L, Rochette-Egly C, et al. Retinoic acid receptors: from molecular mechanisms to cancer therapy. Mol Asp Med. 2015;41:1–115.
Itahana Y, Zhang J, Göke J, Vardy LA, Han R, Iwamoto K, et al. Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells. Sci Rep. 2016;6:28112.
Liu M, Iavarone A, Freedman LP. Transcriptional activation of the humanp21(WAF1/CIP1) gene by retinoic acid receptor. Correlation with retinoid induction of U937 cell differentiation. J Biol Chem. 1996;271:31723–8.
Pan G, Tian S, Nie J, Yang C, Ruotti V, Wei H, et al. Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell Stem Cell. 2007;1:299–312.
Sachs M, Onodera C, Blaschke K, Ebata KT, Song JS, Ramalho-Santos M. Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo. Cell Rep. 2013;3:1777–84.
Harikumar A, Meshorer E. Chromatin remodeling and bivalent histone modifications in embryonic stem cells. EMBO Rep. 2015;16:1609–19.
Gherardi S, Bovolenta M, Passarelli C, Falzarano MS, Pigini P, Scotton C, et al. Transcriptional and epigenetic analyses of the DMD locus reveal novel cis‑acting DNA elements that govern muscle dystrophin expression. Biochim Biophys Acta. 2017;1860:1138–47.
Heery DM, Hoare S, Hussain S, Parker MG, Sheppard H. Core LXXLL motif sequences in CREB-binding protein, SRC1, and RIP140 define affinity and selectivity for steroid and retinoid receptors. J Biol Chem. 2001;276:6695–702.
Plevin MJ, Mills MM, Ikura M. The LxxLL motif: a multifunctional binding sequence in transcriptional regulation. Trends Biochem Sci. 2005;30:66–9.
Guo R, Ye X, Yang J, Zhou Z, Tian C, Wang H, et al. Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells. Nat Commun. 2018;9:2620. 5
Gal-Yam EN, Egger G, Iniguez L, Holster H, Einarsson S, Zhang X, et al. Frequent switching of Polycomb repressive marks and DNA hypermethylation in the PC3 prostate cancer cell line. Proc Natl Acad Sci USA. 2008;105:12979–84.
De Carvalho DD, Mello BP, Pereira WO, Amarante-Mendes GP. PRAME/EZH2-mediated regulation of TRAIL: a new target for cancer therapy. Curr Mol Med. 2013;13:296–304.
Yu JSL, Cui W. Proliferation, survival and metabolism: the role of PI3K/AKT/ mTORsignalling in pluripotency and cell fate determination. Development. 2016;143:3050–60.
Storm MP, Kumpfmueller B, Thompson B, Kolde R, Vilo J, Hummel O, et al. Characterization of the phosphoinositide 3-kinase-dependent transcriptome in murine embryonic stem cells: identification of novel regulators of pluripotency. Stem Cells. 2009;27:764–75.
Singh AM, Bechard M, Smith K, Dalton S. Reconciling the different roles of Gsk3β in “naïve” and “primed” pluripotent stem cells. Cell Cycle. 2012;16:2991–299. 11
Paling NRD, Wheadon H, Bone HK, Welham MJ. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J Biol Chem. 2004;279:48063–70.
Niwa H, Ogawa K, Shimosato D, Adachi K. A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature. 2009;460:118–22.
Wray J, Kalkan T, Gomez-Lopez S, Eckardt D, Cook A, Kemler R, et al. Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation. Nat Cell Biol. 2011;13:838–45.
Vivo M, Fontana R, Ranieri M, Capasso G, Angrisano T, Pollice A, et al. p14ARF interacts with the focal adhesion kinase and protects cells from anoikis. Oncogene. 2017;36:4913–28.
We thank Dr. D. Antonini for valuable discussion, Dr. Pietro Zoppoli for supporting statistical analysis, Dr. Floriana Della Ragione for the kind gift of the LNA probe and Prof. Enrico Avvedimento for the kind gift of the anti-5mC antibody. This work was supported by Biogem, Istituto di Biologia e Genetica Molecolare, Via Camporeale, Ariano Irpino (AV), STAR Linea 1, 2014 (University of Naples Federico II). InterOmics 2017 “PROPAGA” (IEOS, CNR) to GF.
Conflict of interest
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Edited by Y. Shi