npg Genomic imprinting and reprogramming Cell Research (2011) 21:466-473. 466 © 2011 IBCB, SIBS, CAS All rights reserved 1001-0602/11 $ 32.00 npg REVIEW www.nature.com/cr Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming Yufeng Li1, Hiroyuki Sasaki1 1Division of Epigenomics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Mai- dashi, Higashi-ku, Fukuoka 812-8582, Japan Genomic imprinting, an epigenetic gene-marking phenomenon that occurs in the germline, leads to parental-ori- gin-specific expression of a small subset of genes in mammals. Imprinting has a great impact on normal mammalian development, fetal growth, metabolism and adult behavior. The epigenetic imprints regarding the parental origin are established during male and female gametogenesis, passed to the zygote through fertilization, maintained throughout development and adult life, and erased in primordial germ cells before the new imprints are set. In this review, we focus on the recent discoveries on the mechanisms involved in the reprogramming and maintenance of the imprints. We also discuss the epigenetic changes that occur at imprinted loci in induced pluripotent stem cells. Keywords: genomic imprinting; reprogramming; DNA methylation; DNA demethylation; histone modification Cell Research (2011) 21:466-473. doi:10.1038/cr.2011.15; published online 1 February 2011 Introduction maternal allele [4]. To date, more than 100 imprinted genes have been identified in mice (http://www.mouse- In diploid organisms, the maternal and paternal alleles book.org/catalog.php?catalog=imprinting), and many of most autosomal genes are expressed at similar levels, of them are also imprinted in humans [5]. All imprinted and thus contribute equally to the phenotype. However, genes show either maternal-specific or paternal-specific in eutherian mammals (such as humans and mice) and mono-allelic expression, and their proper expression is marsupials, the parental alleles are not always function- essential for normal development, fetal growth, nutrient ally equivalent. This was first discovered in early 1980s metabolism and adult behavior. In humans, genetic and by embryological studies in mice: nuclear transfer ex- epigenetic disturbances in expression of the imprinted periments using pronuclear stage embryos showed that genes can cause well-known malformation disorders, reconstituted embryos with two maternal genomes and such as Prader-Willi syndrome, Angelman syndrome, no paternal complement and those with two paternal Beckwith-Wiedemann syndrome and Silver-Russell syn- genomes and no maternal complement never survive drome [5-7]. beyond mid-gestation. This suggested that the parental Most of the imprinted genes are found in clusters in genomes are functionally non-equivalent and marked or the genome, corresponding to the specific chromosomal imprinted differently during male and female gameto- segments identified by the above genetic studies. Such genesis [1, 2]. Almost at the same time, genetic experi- imprinted clusters often span hundreds to thousands of ments using chromosome translocations in mice showed kilobases. A given imprinted cluster can comprise both that specific chromosomal segments, but not the entire paternally and maternally expressed imprinted genes, genome, function differently depending on the parental some of which correspond to non-coding RNAs, and also origin [3]. Then, mouse Igf2r was identified as the first non-imprinted genes [8-10]. The clusters also contain imprinted gene in 1991: it was expressed only from the CpG-rich regions that are DNA-methylated only on one of the two parental chromosomes (differentially methy- lated regions, DMRs). At some DMRs, differential DNA methylation is also observed between sperm and oocytes, Correspondence: Hiroyuki Sasaki Fax: +81-92-642-6799 and therefore gametic in origin. These DMRs are called E-mail: [email protected] germline or gametic DMRs. In some cases, there is evi- Cell Research | Vol 21 No 3 | March 2011 Yufeng Li and Hiroyuki Sasaki npg 467 dence that the germline DMR functions as an imprinting control region, which controls the mono-allelic expres- sion of the imprinted genes and the methylation status of the other DMRs within the cluster [11]. Most of the germline DMRs are methylated in the female germline and only four DMRs (H19, Dlk1-Gtl2, Rasgrf1 and Zdbf2) are known to be methylated in the male germline [12, 13]. Importantly, mutations in the maintenance DNA methyltranferase DNMT1 disrupt the parental-origin- specific expression patterns of the imprinted genes in mouse embryos [14]. In addition to DNA methylation, other epigenetic modifications and factors, such as his- tone modifications, insulator proteins (such as CTCF) and long non-coding RNAs, are also involved in imprint- Figure 1 Life cycle of the genomic imprints. The paternal (blue) ing. and maternal imprints (red) are established in the germ-line The epigenetic modifications including DNA methyla- and maintained through fertilization and subsequent embryonic tion at the germline DMRs undergo dynamic reprogram- development. However, the imprints are erased in PGCs before ming during germ cell development but, on the other hand, the new imprints are set. The imprints need to be maintained during the extensive reprogramming that occurs in animal they are maintained and faithfully propagated throughout cloning and iPS cell generation (blue arrow). embryonic development [11, 15, 16]. The whole process is complex and regulated tightly. In this study, we review the recent discoveries on the mechanisms involved in the establishment, maintenance and erasure of the epigenetic tablished in the germline are transmitted to the zygote imprints. We also discuss the epigenetic changes ob- through fertilization and maintained faithfully throughout served at imprinted gene clusters in induced pluripoten- the development and adult life. Notably, the methylation tial stem (iPS) cells. imprints at the germline DMRs escape from the global epigenetic reprogramming that occurs in pre-implanta- Life cycle of the genomic imprints tion embryos [11, 16, 25]. The reprogramming at this stage includes the replacement of protamines by histones The life cycle of the genomic imprints in mammals in the paternal genome, active demethylation of the pa- is schematically shown in Figure 1. The cycle consists ternal genome [26] and subsequent passive demethyla- of three major steps: establishment, maintenance and tion of both parental genomes [27, 28]. After implanta- erasure, all of which are important for this biological tion, the differential methylation at the germline DMRs phenomenon. The establishment of the epigenetic im- has to survive another global epigenetic change, i.e., de prints occurs in male and female germ cells. In the male novo DNA methylation. While many genes including the germline, de novo DNA methylation of the four pater- pluripotency genes and germ-cell-specific genes become nally methylated germline DMRs occurs progressively highly methylated in early post-implantation embryos, in mitotically arrested (G1/G0) prospermatogonia (or the unmethylated allele of the DMR has to be protected gonocytes) after embryonic day 14.5 (E14.5). Then, the from this strong wave of de novo DNA methylation. In paternal methylation imprints become fully established fact, imprint maintenance is critical for the parental- in prospermatogonia by the neonatal stage [17-21]. In origin-specific mono-allelic expression of the imprinted the female germline, de novo DNA methylation initiates genes throughout development. asynchronously at different germline DMRs during the The last step of the imprint life cycle is the erasure of oocyte growth phase [22, 23]. Growing oocytes are at the the epigenetic imprints in primordial germ cells (PGCs): diplotene/dictyate stage of meiotic prophase I, and the this ensures the sex-dependent imprint establishment in maternal methylation imprints become fully established later stages of germ cell development described above. by the fully grown oocyte stage [22, 23]. The establish- PGCs are specified from the epiblast cells of early post- ment of the maternal methylation imprints is correlated implantation embryos. Then, PGCs proliferate actively, with the establishment of the functional imprints, which followed by migration to the genital ridge, the precursor was shown by the developmental potential of nuclear of the gonads, between E7.25 and E10.5. In this period, transferred bi-maternal embryos [24]. the genome of the PGCs undergoes epigenetic repro- The paternal and maternal epigenetic imprints es- gramming to restore pluripotency [25, 29, 30], but they www.cell-research.com | Cell Research 468 npg Genomic imprintingandreprogramming Table 1 Factors involved in the life cycle of the genomic imprints Step of the cycle Factor name Biochemical function Germ-line DMRs affected in knockout Reference Paternally methylated Maternally methylated DNMT3A de novo DNA methy- H19, Dlk1-Gtl2, Zdbf2, Rasgrf1 Snrpn, Peg1(Mest), Peg3, Igf2r [20, 21, 37-39] ltransferase DNMT3B Rasgrf1 [20, 38, 39] Establishment DNMT3L Cofactor of DNMT3A H19, Dlk1-Gtl2, Rasgrf1 Snrpn, Peg1, Peg3, Igf2r [20, 36, 38, 39 ,40] and DNMT3B KDM1B Histone H3K4 demethylase Peg1, Grb10, Zac1(Plagl1), Impact [45] ZFP57 KRAB zinc finger protein Snrpn [46] DNMT1 Maintenance DNA methy- H19, Rasgrf1 Igf2r, Peg3, Snrpn [14, 48-50] (DNMT1o ltransferase and DNMT1s) Maintenance ZFP57 KRAB zinc finger protein