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DNA methylation regulates long- range gene silencing of an X- linked homeobox gene cluster in a lineage- specific manner. Masaaki Oda. 1. Akiko Yamagiwa. Shinji Yamamoto. 3. Takao Nakayama. 1,4.
Akiko Tsumura. 1. Hiroshi Sasaki. 3. Kazuki Nakao. 2. En Li. Masaki Okano. 1,6. Laboratory for Mammalian Epigenetic Studies, Center for Developmental Biology, RIKEN, Kobe, Hyogo, 6. Japan. 2 Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN, Kobe, Hyogo, 6.
Japan. 3 Laboratory for Embryonic Induction, Center for Developmental Biology, RIKEN, Kobe, Hyogo, 6. Japan. 4 Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 9. Japan. 5 Epigenetics Program, Novartis Institute for Biomedical Research, Cambridge, Massachusetts 0. USA. Abstract. DNA methylation is a major epigenetic mechanism that has been suggested to control developmental gene regulation during embryogenesis.
In this report, we show that Cp. G islands associated with the X- linked homeobox. Rhox, which is highly expressed in the extraembryonic trophectoderm, are differentially methylated in a stage- and lineage- specific. Inactivation of both Dnmt.
Dnmt. 3b, DNA methyltransferases essential. DNA methylation, abolished the establishment of DNA methylation and the silencing of Rhox cluster genes in the embryo proper. The Dnmt. 3- dependent Cp. G- island methylation at the Rhox locus extended for a large genomic region (.
Complementation experiments using embryonic stem (ES) cells deficient in the DNA methyltransferases. Cp. G- island methylation by Dnmt. Dnmt. 3b was restricted within this large genomic region, and did not.
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Our results suggest that. DNA methylation plays important roles in both long- range gene silencing and lineage- specific silencing in embryogenesis. The direct modification of DNA by methylation plays important roles in repressing gene expression. Bird 2. 00. 2). DNA methylation marking can be interpreted in several ways, including the exclusion of transcriptional regulators at their. DNA- binding sites, or by the attraction of transcriptional repressor complexes through a group of proteins with methyl- Cp.
G- binding. activities (Jaenisch and Bird 2. DNA methylation profiles in mammals are reprogrammed and established during early embryogenesis through a highly orchestrated.
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Morgan et al. Following fertilization, a genome- wide and stepwise reduction of DNA methylation occurs during the cleavage cycles, causing. DNA methylation during embryogenesis. By the blastocyst stage, two rounds. ICM)/epiblast, the trophectoderm, and the primitive. Fig. Shortly after implantation, the ICM/epiblast- lineage cells undergo a wave of de novo methylation that establishes hypermethylated. These dynamic changes in DNA methylation. Morgan et al. DNA methylation states.
Line. 1 (dark gray), IAP element (light gray), and Igf. The graph indicates the percentage of total Cp. G sites that were methylated in the bisulfite- sequenced clones of. The DNA methylation patterns of bisulfite- sequenced clones for Igf. DMR2) are shown in Supplementary Figure 1. A. Dnmt. 1 associates with the replication foci and propagates DNA methylation profiles after DNA replication, probably by the. DNA (Bestor 1. 99.
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Leonhardt et al. The inactivation of mouse Dnmt. DNA methylation of the whole genome indiscriminately. Li et al. A Dnmt.
DNA methylation. is essential for vertebrate development (Li et al. Stancheva and Meehan 2. Rai et al. Dnmt. 3a and Dnmt. ES) cells, for the establishment of the embryonic hypermethylated genome in post- implantation development. DNA methylation imprints in the imprinted genes of male and female germ cells (Okano et al.
These functions indicate that Dnmt. Dnmt. 3b are physiological determinants for the DNA methylation profiles and dynamics. Long- range gene regulatory mechanisms have been shown to control the spatially and temporally coordinated expression or. Hox gene clusters (Verona et al.
West and Fraser 2. Recently, a new homeobox gene cluster on the mouse X chromosome has been identified, the Rhox gene cluster, which shows temporal- collinear expression in the reproductive and reproduction- associated tissues (Maclean et al.
In this study, we show that DNA methylation regulates cell lineage- specific silencing of the Rhox gene cluster in the post- implantation development of mice. The Cp. G islands associated with genes in a large genomic region.
Genetic analyses using knockout. Dnmt. 3a and Dnmt. Rhox region. Rescue experiments in which Dnmt. Dnmt. 3a, or Dnmt. ES cells suggested that Cp. G- island. hypermethylation was confined to the Rhox region and did not occur in the neighboring genomic regions, indicating the possibility of a specific boundary at the ends. In our previous study, we performed a conventional methylation analysis on these mutants by Southern.
Okano et al. Here, for a more quantitative analysis, we performed bisulfite sequencing and examined the DNA methylation status of tandem. LINE1 and IAP), and an imprinted gene (Igf.
DNA methyltransferase- deficient embryos. In normal embryogenesis, the methylated DNA of repetitive. Fig. 1. B, Blast; Morgan et al. Subsequently, extensive de novo methylation after implantation establishes a hypermethylation of these repetitive sequences. Fig. 1. B, Emb; Morgan et al. In contrast, the DNA methylation levels and allele- specific profiles of Igf. Fig. 1. B; Supplementary Fig.
We found that the DNA methylation levels of the repetitive sequences in the embryo proper of Dnmt. This indicates that DNA methylation of the repetitive sequences in the DKO embryo did not increase after implantation. The DNA methylation levels and allele- specific patterns. Igf. 2r were unaffected in the DKO embryo proper (Fig.
B; Supplementary Fig. In contrast, most of the DNA methylation in the repetitive sequences and Igf.
Dnmt. 1. 1. A), in agreement with this enzyme’s physiological function in maintenance methylation. Consistent with. our previous study (Okano et al. Dnmt. 3a and Dnmt. DNA methylation in the genome (de novo. DNA methylation during embryogenesis. Suppressive subtractive PCR screening resulted in the isolation of partial c.
DNA fragments of Rhox. Psx. 1), an X- linked homeobox gene highly expressed in the trophectodermal tissues in post- implantation development (Chun et al.
The Rhox. 6 gene is transcriptionally repressed during post- implantation development in the ICM/epiblast lineage, which gives rise to. Due to the extensive sequence similarity between Rhox. Rhox. 9 (also known as Psx. PCR primers for bisulfite sequencing amplified the Cp. G islands of both genes (Fig.
This analysis revealed that the Cp. G islands of both Rhox. Rhox. 9 were highly methylated in the embryonic day 9. E9. 5) embryo proper (Fig.
B; Supplementary Fig. C,D, Emb), in which Rhox.
Rhox. 9 are silenced, whereas the Cp. G islands in the same genes were largely hypomethylated in the trophectoderm tissues (Fig. B; Supplementary Fig. D, Troph), in which these genes are highly expressed (Fig.
The yolk sac, which consists of the extraembryonic mesoderm and extraembryonic visceral endoderm, showed a mixed DNA methylation. Fig. These results suggest that Rhox. Rhox. 9 are differentially methylated in a cell lineage- dependent manner. Regions around each transcription start site (arrows) meet the criteria of a Cp.
G island (filled gray boxes). PCR primers for bisulfite sequencing (white arrowheads). Cp. G sites (from . The PCR primers for Hpa. II- digestion PCR (black arrowheads) covered four Hpa. II sites. The numbering begins with.
A different set of PCR primers yielded similar results (Supplementary Fig. Genomic DNA digested with the Cp. G- methylation- sensitive restriction enzyme Hpa.
II were amplified. PCR using primers flanking the Hpa. II sites. Igf. 2r- DMR2 served as a control for a methylated locus (St. An arbitrarily selected genomic region lacking an Hpa. II site on mouse chromosome 1. Hpa. II) served as a loading control. Total RNA was isolated from the E9.
Emb), yolk sac (YS), and trophoblast (Troph). Gapdh was used as a control for equal RNA loading. PCR cycles were shown on the right. Total RNA was isolated from. E8. 5 male. Xist and IAP- type I are known to be derepressed in DKO embryos (male) and Dnmt.
Total RNA was isolated from E8. Using F1 hybrid embryos for Mus musculus domesticus (C5. BL/6) and Mus musculus molossinus (JF1), we found that Rhox. X inactivation in extraembryonic tissues (Supplementary Fig.
E; Takagi and Sasaki 1. However, we also found that the DNA methylation patterns of Rhox. Rhox. 9 were similar between males and females in the embryonic and extraembryonic tissues (Fig. B), and that these genes were similarly silenced in both the male and female embryo proper (Fig.
These findings indicate that the DNA methylation states of Rhox. Rhox. 9 are independent of X- chromosome inactivation. B; Supplementary Fig. We confirmed this hypomethylation of Rhox.
Rhox. 9 in the eight- cell embryo and the blastocyst, as well as the differential methylation in the embryo proper and the trophectoderm. Hpa. II- digestion PCR analysis (Fig. These results indicate that establishment of the embryonic lineage- specific Cp. G methylation occurs after implantation of. The DNA methylation of the Cp. G islands of Rhox.
Rhox. 9 in the embryo proper was almost completely lost in the DKO embryos, as was the case in the Dnmt. D; Supplementary Fig. However, the DNA methylation profiles in the same regions were unaffected in the Dnmt. These results suggest that, although the contribution of Dnmt. Dnmt. 3a and Dnmt. Cp. G- island methylation of Rhox.
Rhox. 9, and their functions in this process overlap. In addition, the DNA methylation states of Rhox. Rhox. 9 in the Dnmt.
We obtained consistent results using Hpa. II- digestion PCR analysis, which showed the Cp. G sites to be hypomethylated in the. DKO embryo proper (Fig. We did not observe clear signals for the Dnmt.
E), although we detected a significant amount of DNA methylation in these embryos by bisulfite sequencing (Fig.