Control of ground-state pluripotency by allelic regulation of Nanog.
Pluripotency is established through genome-wide reprogramming during mammalian pre-implantation development, resulting in the formation of the naive epiblast. Reprogramming involves both the resetting of epigenetic marks and the activation of pluripotent-cell-specific genes such as Nanog and Oct4 (also known as Pou5f1). The tight regulation of these genes is crucial for reprogramming, but the mechanisms that regulate their expression in vivo have not been uncovered. Here we show that Nanog--but not Oct4--is monoallelically expressed in early pre-implantation embryos. Nanog then undergoes a progressive switch to biallelic expression during the transition towards ground-state pluripotency in the naive epiblast of the late blastocyst. Embryonic stem (ES) cells grown in leukaemia inhibitory factor (LIF) and serum express Nanog mainly monoallelically and show asynchronous replication of the Nanog locus, a feature of monoallelically expressed genes, but ES cells activate both alleles when cultured under 2i conditions, which mimic the pluripotent ground state in vitro. Live-cell imaging with reporter ES cells confirmed the allelic expression of Nanog and revealed allelic switching. The allelic expression of Nanog is regulated through the fibroblast growth factor-extracellular signal-regulated kinase signalling pathway, and it is accompanied by chromatin changes at the proximal promoter but occurs independently of DNA methylation. Nanog-heterozygous blastocysts have fewer inner-cell-mass derivatives and delayed primitive endoderm formation, indicating a role for the biallelic expression of Nanog in the timely maturation of the inner cell mass into a fully reprogrammed pluripotent epiblast. We suggest that the tight regulation of Nanog dose at the chromosome level is necessary for the acquisition of ground-state pluripotency during development. Our data highlight an unexpected role for allelic expression in controlling the dose of pluripotency factors in vivo, adding an extra level to the regulation of reprogramming.
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM U964, Université de Strasbourg, F-67404 Illkirch, Cité Universitaire de Strasbourg, France.
Yusuke
Y
Miyanari
Maria-Elena
ME
Torres-Padilla
D000483:Alleles; D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D053624:Blastocyst Inner Cell Mass / Q000166:cytology / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D065150:Cellular Reprogramming / Q000235:genetics*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004261:DNA Replication; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D018392:Genomic Imprinting; D005855:Germ Layers / Q000166:cytology / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008297:Male; D056892:Mediator Complex / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D000097722:Cohesins
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D018398:Homeodomain Proteins; D053605:Leukemia Inhibitory Factor; D056892:Mediator Complex; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D000097722:Cohesins; C475540:Nanog protein, mouse; C494174:Pou5f1 protein, mouse