LIN28 coordinately promotes nucleolar/ ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

Abstract

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

Keywords

LIN28 / 2-cell-like program / nucleolar integrity / NCL/TRIM28 complex

References

[1] Baker CL, Pera MF (2018) Capturing totipotent stem cells . Cell Stem Cell 22:25–34

[2] Biggiogera M, Burki K, Kaufmann SH, Shaper JH, Gas N, Amalric F, Fakan S(1990) Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy . Development 110:1263–1270

[3] Birkedal U, Christensen-Dalsgaard M, Krogh N, Sabarinathan R, Gorodkin J, Nielsen H(2015) Profiling of ribose methylations in RNA by high-throughput sequencing . Angew Chem Int Ed Engl 54:451–455

[4] Bolger AM, Marc L, Bjoern U(2014) Trimmomatic: a flexible trimmer for Illumina sequence data . Bioinformatics 30:2114–2120

[5] Borsos M, Torres-Padilla ME(2016) Building up the nucleus: nuclear organization in the establishment of totipotency and pluripotency during mammalian development . Genes Dev 30:611–621

[6] Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI(2010) The nucleolus under stress . Mol Cell 40:216–227

[7] Cabili MN, Cole T, Loyal G, Magdalena K, Barbara TV, Aviv R, Rinn JL(2011) Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses . Genes Dev 25:1915

[8] Chen Z, Zhang Y(2019) Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development . Nat Genet 51:947–951

[9] Chen C, Liu W, Guo J, Liu Y, Liu X, Liu J, Dou X, Le R, Huang Y, Li C (2021) Nuclear m(6)A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos . Protein Cell 12:455–474

[10] Cho J, Chang H, Kwon SC, Kim B, Kim Y, Choe J, Ha M, Kim YK, Kim VN(2012) LIN28A is a suppressor of ER-associated translation in embryonic stem cells . Cell 151:765–777

[11] Daehwan K, Ben L, Salzberg SL(2015) HISAT: a fast spliced aligner with low memory requirements . Nat Methods 12:357–360

[12] Dai MS, Lu H(2004) Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5 . J Biol Chem 279:44475–44482

[13] Dai MS, Zeng SX, Jin Y, Sun XX, David L, Lu H(2004) Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition . Mol Cell Biol 24:7654–7668

[14] De Iaco A, Planet E, Coluccio A, Verp S, Duc J, Trono D(2017) DUX-family transcription factors regulate zygotic genome activation in placental mammals . Nat Genet 49:941–945

[15] Deng Q, Ramskold D, Reinius B, Sandberg R(2014) Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells . Science 343:193–196

[16] Eckersley-Maslin MA, Svensson V, Krueger C, Stubbs TM, Giehr P, Krueger F, Miragaia RJ, Kyriakopoulos C, Berrens RV, Milagre I (2016) MERVL/Zscan4 network activation results in transient genome-wide DNA demethylation of mESCs . Cell Rep 17:179–192

[17] Falahati H, Pelham-Webb B, Blythe S, Wieschaus E(2016) Nucleation by rRNA dictates the precision of nucleolus assembly . Curr Biol 26:277–285

[18] Feric M, Vaidya N, Harmon TS, Mitrea DM, Zhu L, Richardson TM, Kriwacki RW, Pappu RV, Brangwynne CP(2016) Coexisting liquid phases underlie nucleolar subcompartments . Cell 165:1686–1697

[19] Friedli M, Turelli P, Kapopoulou A, Rauwel B, Castro-Diaz N, Rowe HM, Ecco G, Unzu C, Planet E, Lombardo A (2014) Loss of transcriptional control over endogenous retroelements during reprogramming to pluripotency . Genome Res 24:1251–1259

[20] Fulka H, Aoki F(2016) Nucleolus precursor bodies and ribosome biogenesis in early mammalian embryos: old theories and new discoveries . Biol Reprod 94:143

[21] Ginisty H, Amalric F, Bouvet P(1998) Nucleolin functions in the first step of ribosomal RNA processing . EMBO J 17:1476–1486

[22] Golomb L, Volarevic S, Oren M(2014) p53 and ribosome biogenesis stress: the essentials . FEBS Lett 588:2571–2579

[23] Guallar D, Bi X, Pardavila JA, Huang X, Saenz C, Shi X, Zhou H, Faiola F, Ding J, Haruehanroengra P (2018) RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells . Nat Genet 50:443–451

[24] Guetg C, Santoro R(2012) Formation of nuclear heterochromatin: the nucleolar point of view . Epigenetics 7:811–814

[25] Guo M, Zhang Y, Zhou J, Bi Y, Xu J, Xu C, Kou X, Zhao Y, Li Y, Tu Z (2019) Precise temporal regulation of Dux is important for embryo development . Cell Res 29:956–959

[26] Hendrickson PG, Dorais JA, Grow EJ, Whiddon JL, Lim JW, Wike CL, Weaver BD, Pflueger C, Emery BR, Wilcox AL (2017)Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons . Nat Genet 49:925–934

[27] Heo I, Joo C, Kim Y-K, Ha M, Yoon M-J, Cho J, Yeom K-H, Han J, Kim VN(2009) TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation . Cell 138:696–708

[28] Hung SS, Wong RC, Sharov AA, Nakatake Y, Yu H, Ko MS(2013) Repression of global protein synthesis by Eif1a-like genes that are expressed specifically in the two-cell embryos and the transient Zscan4-positive state of embryonic stem cells . DNA Res 20:391–402

[29] Ishiuchi T, Enriquez-Gasca R, Mizutani E, Boskovic A, Ziegler-Birling C, Rodriguez-Terrones D, Wakayama T, Vaquerizas JM, Torres-Padilla ME(2015) Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly . Nat Struct Mol Biol 22:662–671

[30] Jia W, Yao Z, Zhao J, Guan Q, Gao L(2017) New perspectives of physiological and pathological functions of nucleolin (NCL) . Life Sci 186:1–10

[31] Jukam D, Shariati SAM, Skotheim JM(2017) Zygotic genome activation in vertebrates . Dev Cell 42:316–332

[32] Kim SK, Lee H, Han K, Kim SC, Choi Y, Park SW, Bak G, Lee Y, Choi JK, Kim TK (2014) SET7/9 methylation of the pluripotency factor LIN28A is a nucleolar localization mechanism that blocks let-7 biogenesis in human ESCs . Cell Stem Cell 15:735–749

[33] Kiss T(2002) Small nucleolar RNAs: an abundant group of noncoding RNAs with diverse cellular functions . Cell 109:145–148

[34] Langdon EM, Gladfelter AS(2018) A new lens for RNA localization: liquid-liquid phase separation . Annu Rev Microbiol 72:255–271

[35] Li M, He Y, Dubois W, Wu X, Shi J, Huang J(2012) Distinct regulatory mechanisms and functions for p53-activated and p53-repressed DNA damage response genes in embryonic stem cells . Mol Cell 46:30–42

[36] Lohrum MA, Ludwig RL, Kubbutat MH, Hanlon M, Vousden KH(2003) Regulation of HDM2 activity by the ribosomal protein L11 . Cancer Cell 3:577–587

[37] Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D, Firth A, Singer O, Trono D, Pfaff SL (2012) Embryonic stem cell potency fluctuates with endogenous retrovirus activity . Nature 487:57–63

[38] Maksakova IA, Thompson PJ, Goyal P, Jones SJ, Singh PB, Karimi MM, Lorincz MC(2013) Distinct roles of KAP1, HP1 and G9a/ GLP in silencing of the two-cell-specific retrotransposon MERVL in mouse ES cells . Epigen Chromatin 6:15

[39] Marchand V, Blanloeil-Oillo F, Helm M, Motorin Y(2016) Illuminabased RiboMethSeq approach for mapping of 2’-O-Me residues in RNA . Nucleic Acids Res 44:

[40] Matsui T, Leung D, Miyashita H, Maksakova IA, Miyachi H, Kimura H, Tachibana M, Lorincz MC, Shinkai Y(2010) Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET . Nature 464:927–931

[41] Messerschmidt DM, de Vries W, Ito M, Solter D, Ferguson-Smith A, Knowles BB(2012) Trim28 is required for epigenetic stability during mouse oocyte to embryo transition . Science 335:1499–1502

[42] Mihaela P, Pertea GM, Antonescu CM, Tsung-Cheng C, Mendell JT, Salzberg SL(2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads . Nat Biotechnol 33:290–295

[43] Mongelard F, Bouvet P(2007) Nucleolin: a multiFACeTed protein . Trends Cell Biol 17:80–86

[44] Nam Y, Chen C, Gregory RI, Chou JJ, Sliz P(2011) Molecular basis for interaction of let-7 microRNAs with Lin28 . Cell 147:1080–1091

[45] Peaston AE, Evsikov AV, Graber JH, de Vries WN, Holbrook AE, Solter D, Knowles BB(2004) Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos . Dev Cell 7:597–606

[46] Percharde M, Lin CJ, Yin Y, Guan J, Peixoto GA, Bulut-Karslioglu A, Biechele S, Huang B, Shen X, Ramalho-Santos M (2018) A LINE1-nucleolin partnership regulates early development and ESC identity . Cell 174(391–405):

[47] Picelli S, Faridani OR, Bjorklund AK, Winberg G, Sagasser S, Sandberg R(2014) Full-length RNA-seq from single cells using Smart-seq2 . Nat Protoc 9:171–181

[48] Piskounova E, Polytarchou C, Thornton JE, LaPierre RJ, Pothoulakis C, Hagan JP, Iliopoulos D, Gregory RI(2011) Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms . Cell 147:1066–1079

[49] Rodriguez-Terrones D, Torres-Padilla ME(2018) Nimble and ready to mingle: transposon outbursts of early development . Trends Genet 34:806–820

[50] Rubbi CP, Milner J(2003) Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses . EMBO J 22:6068–6077

[51] Shinoda G, Shyh-Chang N, Soysa TYD, Zhu H, Seligson MT, Shah SP, Abo-Sido N, Yabuuchi A, Hagan JP, Gregory RI (2013) Fetal deficiency of Lin28 programs life-long aberrations in growth and glucose metabolism . Stem Cells 31:1563–1573

[52] Shyh-Chang N, Daley GQ(2013) Lin28: primal regulator of growth and metabolism in stem cells . Cell Stem Cell 12:395–406

[53] Tollervey D, Lehtonen H, Jansen R, Kern H, Hurt EC(1993) Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly . Cell 72:443–457

[54] Viswanathan SR, Daley GQ(2010) Lin28: a microRNA regulator with a macro role . Cell 140:445–449

[55] Viswanathan SR, Daley GQ, Gregory RI(2008) Selective blockade of microRNA processing by Lin28 . Science 320:97–100

[56] Walter M, Teissandier A, Perez-Palacios R, Bourc’his D(2016) An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells . Elife 5

[57] Wang C, Liu X, Gao Y, Yang L, Li C, Liu W, Chen C, Kou X, Zhao Y, Chen J (2018) Reprogramming of H3K9me3-dependent heterochromatin during mammalian embryo development . Nat Cell Biol 20:620–631

[58] Whiddon JL, Langford AT, Wong CJ, Zhong JW, Tapscott SJ(2017) Conservation and innovation in the DUX4-family gene network . Nat Genet 49:935–940

[59] Wilbert ML, Huelga SC, Kapeli K, Stark TJ, Liang TY, Chen SX, Yan BY, Nathanson JL, Hutt KR, Lovci MT (2012) LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance . Mol Cell 48:195–206

[60] Xu B, Zhang K, Huang Y(2009) Lin28 modulates cell growth and associates with a subset of cell cycle regulator mRNAs in mouse embryonic stem cells . RNA 15:357–361

[61] Yang BX, El Farran CA, Guo HC, Yu T, Fang HT, Wang HF, Schlesinger S, Seah YF, Goh GY, Neo SP (2015) Systematic identification of factors for provirus silencing in embryonic stem cells . Cell 163:230–245

[62] Yang K, Wang M, Zhao Y, Sun X, Yang Y, Li X, Zhou A, Chu H, Zhou H, Xu J (2016) A redox mechanism underlying nucleolar stress sensing by nucleophosmin . Nat Commun 7:13599

[63] Yang K, Yang J, Yi J(2018) Nucleolar stress: hallmarks, sensing mechanism and diseases . Cell Stress 2:125–140

[64] Yang F, Huang X, Zang R, Chen J, Fidalgo M, Sanchez-Priego C, Yang J, Caichen A, Ma F, Macfarlan T (2020) DUX-miR-344-ZMYM2-mediated activation of MERVL LTRs induces a totipotent 2C-like state . Cell Stem Cell 26:234–250.e237

[65] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R (2007) Induced pluripotent stem cell lines derived from human somatic cells . Science 318:1917–1920

[66] Zeng Y, Yao B, Shin J, Lin L, Kim N, Song Q, Liu S, Su Y, Guo JU, Huang L (2016) Lin28A binds active promoters and recruits Tet1 to regulate gene expression . Mol Cell 61:153–160

[67] Zhang Y, Lu H(2009) Signaling to p53: ribosomal proteins find their way . Cancer Cell 16:369–377

[68] Zhang J, Ratanasirintrawoot S, Chandrasekaran S, Wu Z, Ficarro SB, Yu C, Ross CA, Cacchiarelli D, Xia Q, Seligson M (2016) LIN28 regulates stem cell metabolism and conversion to primed pluripotency . Cell Stem Cell 19:66–80

[69] Zilionis R, Nainys J, Veres A, Savova V, Zemmour D, Klein AM, Mazutis L(2016) Single-cell barcoding and sequencing using droplet microfluidics . Nat Protoc 12:44–73

RIGHTS & PERMISSIONS

The Author(s)