The Role of N-?-acetyltransferase 10 Protein in DNA Methylation and Genomic Imprinting.
ABSTRACT: Genomic imprinting is an allelic gene expression phenomenon primarily controlled by allele-specific DNA methylation at the imprinting control region (ICR), but the underlying mechanism remains largely unclear. N-?-acetyltransferase 10 protein (Naa10p) catalyzes N-?-acetylation of nascent proteins, and mutation of human Naa10p is linked to severe developmental delays. Here we report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorders, and maternal effect lethality, phenotypes commonly observed in defective genomic imprinting. Genome-wide analyses further revealed global DNA hypomethylation and enriched dysregulation of imprinted genes in Naa10p-knockout embryos and embryonic stem cells. Mechanistically, Naa10p facilitates binding of DNA methyltransferase 1 (Dnmt1) to DNA substrates, including the ICRs of the imprinted allele during S phase. Moreover, the lethal Ogden syndrome-associated mutation of human Naa10p disrupts its binding to the ICR of H19 and Dnmt1 recruitment. Our study thus links Naa10p mutation-associated Ogden syndrome to defective DNA methylation and genomic imprinting.
Project description:Genomic imprinting is an allelic gene expression phenomenon primarily controlled by allele-specific DNA methylation at the imprinting control region (ICR). How allele-specific DNA methylation at ICR is regulated is largely unknown. Mammalian N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins. Mutation of human Naa10p is linked to severe developmental defects and mental retardation, including the Ogden syndrome. Here we report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorder and maternal-effect lethality, phenotypes commonly observed in defective genomic imprinting. Genome-wide analyses reveal global DNA hypomethylation and enriched dysregulation of imprinted genes in Naa10p-null embryos and ES cells. Naa10p regulates global DNA methylation by stimulating DNA methyltransferase 1 (Dnmt1) activity, while maintaining imprinted allele methylation by binding to GCXGXG and recruits Dnmt1. Clinical mutation of Naa10p disrupts its H19-ICR binding and Dnmt1 recruitment. Our study thus links Naa10p mutation-associated human diseases to defective DNA methylation and genomic imprinting. Overall design: In this data set, we include RRBS data of both WT and Naa10-KO mESCs
Project description:Genomic imprinting is an allelic gene expression phenomenon primarily controlled by allele-specific DNA methylation at the imprinting control region (ICR), of which the underlying mechanism remains largely unclear. Mammalian N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins. Mutation of human Naa10p is linked to severe developmental delays. Here we report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorder and maternaleffect lethality, phenotypes commonly observed in defective genomic imprinting. Genome-wide analyses further revealed global DNA hypomethylation and enriched dysregulation of imprinted genes in Naa10p-knockout embryos and ES cells. Mechanistically, Naa10p facilitates the binding of DNA methyltransferase 1 (Dnmt1) to DNA substrates and recruits Dnmt1 to ICRs of the imprinted allele during S phase. Moreover, the clinical lethal Ogden syndrome-associated mutation of Naa10p disrupts its binding to H19-ICR and Dnmt1 recruitment. Our study thus links Naa10p mutationassociated human disease to defective DNA methylation and genomic imprinting. Overall design: Examination of Naa10p binding sites in WT mouse embryonic stem (ES) cells using Naa10-KO ES cells as control
Project description:Imprinted genes are expressed from one parental allele and regulated by differential DNA methylation at imprinting control regions (ICRs). ICRs are reprogrammed in the germline through erasure and re-establishment of DNA methylation. Although much is known about DNA methylation establishment, DNA demethylation is less well understood. Recently, the Ten-Eleven Translocation proteins (TET1-3) have been shown to initiate DNA demethylation, with Tet1-/- mice exhibiting aberrant levels of imprinted gene expression and ICR methylation. Nevertheless, the role of TET1 in demethylating ICRs in the female germline and in controlling allele-specific expression remains unknown. Here, we examined ICR-specific DNA methylation in Tet1-/- germ cells and ascertained whether abnormal ICR methylation impacted imprinted gene expression in F1 hybrid somatic tissues derived from Tet1-/- eggs or sperm. We show that Tet1 deficiency is associated with hypermethylation of a subset of ICRs in germ cells. Moreover, ICRs with defective germline reprogramming exhibit aberrant DNA methylation and biallelic expression of linked imprinted genes in somatic tissues. Thus, we define a discrete set of genomic regions that require TET1 for germline reprogramming and discuss mechanisms for stochastic imprinting defects.
Project description:We report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorder and maternal-effect lethality, phenotypes commonly observed in defective genomic imprinting. Genome-wide analyses further reveal enriched dysregulation of imprinted genes in Naa10p-knockout ES cells. Overall design: mRNA profiles of 2 biological ESC replicates of each WT and Naa10-KO group
Project description:Rasgrf1 is imprinted in mouse, displaying paternal allele specific expression in neonatal brain. Paternal expression is accompanied by paternal-specific DNA methylation at a differentially methylated domain (DMD) within the locus. The cis-acting elements necessary for Rasgrf1 imprinting are known. A series of tandem DNA repeats control methylation of the adjacent DMD, which is a methylation sensitive enhancer-blocking element. These two sequences constitute a binary switch that controls imprinting and represents the Imprinting Control Region (ICR). One paternally transmitted mutation, which helped define the ICR, induced paramutation, in trans, on the maternal allele. Like many imprinted genes, Rasgrf1 lies within an imprinted cluster. One of four noncoding transcripts in the cluster, AK015891, is known to be imprinted.Here we demonstrate that an additional noncoding RNA, AK029869, is imprinted and paternally expressed in brain throughout development. Intriguingly, any of several maternally inherited ICR mutations affected expression of the paternal AK029869 transcript in trans. Furthermore, we found that the ICR mutations exert different trans effects on AK029869 at different developmental times.Few trans effects have been defined in mammals and, those that exist, do not show the great variation seen at the Rasgrf1 imprinted domain, either in terms of the large number of mutations that produce the effects or the range of phenotypes that emerge when they are seen. These results suggest that trans regulation of gene expression may be more common than originally appreciated and that where trans regulation occurs it can change dynamically during development.
Project description:Genomic imprinting is an epigenetic feature characterized by parent-specific monoallelic gene expression. The aim of this study was to compare the DNA methylation status of imprinted genes and imprinting control regions (ICRs), harboring differentially methylated regions (DMRs) in a comprehensive panel of 18 somatic tissues. The germline DMRs analyzed were divided into ubiquitously imprinted and placenta-specific DMRs, which show identical and different methylation imprints in adult somatic and placental tissues, respectively. We showed that imprinted genes and ICR DMRs maintain methylation patterns characterized by intermediate methylation levels in somatic tissues, which are pronounced in a specific region of the promoter area, located 200-1500 bp from the transcription start site. This intermediate methylation is concordant with gene expression from a single unmethylated allele and silencing of a reciprocal parental allele through DNA methylation. The only exceptions were seen for ICR DMRs of placenta-specific imprinted genes, which showed low levels of methylation, suggesting that these genes escape parent-specific epigenetic regulation in somatic tissues.
Project description:Genomic imprinting is an epigenetic phenomenon restricting gene expression in a manner dependent on parent of origin. Imprinted gene products are critical regulators of growth and development, and imprinting disorders are associated with both genetic and epigenetic mutations, including disruption of DNA methylation within the imprinting control regions (ICRs) of these genes. It was recently reported that some patients with imprinting disorders have a more generalised imprinting defect, with hypomethylation at a range of maternally methylated ICRs. We report a cohort of 149 patients with a clinical diagnosis of Beckwith-Wiedemann syndrome (BWS), including 81 with maternal hypomethylation of the KCNQ1OT1 ICR. Methylation analysis of 11 ICRs in these patients showed that hypomethylation affecting multiple imprinted loci was restricted to 17 patients with hypomethylation of the KCNQ1OT1 ICR, and involved only maternally methylated loci. Both partial and complete hypomethylation was demonstrated in these cases, suggesting a possible postzygotic origin of a mosaic imprinting error. Some ICRs, including the PLAGL1 and GNAS/NESPAS ICRs implicated in the aetiology of transient neonatal diabetes and pseudohypoparathyroidism type 1b, respectively, were more frequently affected than others. Although we did not find any evidence for mutation of the candidate gene DNMT3L, these results support the hypotheses that trans-acting factors affect the somatic maintenance of imprinting at multiple maternally methylated loci and that the clinical presentation of these complex cases may reflect the loci and tissues affected with the epigenetic abnormalities.
Project description:Genomic imprinting is governed by allele-specific DNA methylation at imprinting control regions (ICRs), and the mechanism controlling its differential methylation establishment during gametogenesis has been a subject of intensive research interest. However, recent studies have reported that gamete methylation is not restricted at the ICRs, thus highlighting the significance of ICR methylation maintenance during the preimplantation period where genome-wide epigenetic reprogramming takes place. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. Furthermore, this activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. In addition, we showed that a specific 5' fragment of the H19 ICR is required for its paternal methylation after fertilization, while CTCF and Sox-Oct motifs are essential for its maternal protection from undesirable methylation after implantation.To ask whether specific cis elements are sufficient to reconstitute imprinted methylation status, we employed a TgM co-placement strategy for facilitating detection of postfertilization methylation activity and precise comparison of test sequences. Bacteriophage lambda DNA becomes highly methylated regardless of its parental origin and thus can be used as a neutral sequence bearing no inclination for differential DNA methylation. We previously showed that insertion of only CTCF and Sox-Oct binding motifs from the H19 ICR into a lambda DNA (LCb) decreased its methylation level after both paternal and maternal transmission. We therefore appended a 478-bp 5' sequence from the H19 ICR into the LCb fragment and found that it acquired paternal-allele-specific methylation, the dynamics of which was identical to that of the H19 ICR, in TgM. Crucially, transgene expression also became imprinted. Although there are potential binding sites for ZFP57 (a candidate protein thought to control the methylation imprint) in the larger H19 ICR, they are not found in the 478-bp fragment, rendering the role of ZFP57 in postfertilization H19 ICR methylation a still open question.Our results demonstrate that a differentially methylated region can be reconstituted by combining the activities of specific imprinting elements and that these elements together determine the activity of a genomically imprinted region in vivo.
Project description:Biallelic expression of Igf2 is frequently seen in cancers because Igf2 functions as a survival factor. In many tumors the activation of Igf2 expression has been correlated with de novo methylation of the imprinted region. We have compared the intrinsic susceptibilities of the imprinted region of Igf2 and H19, other imprinted genes, bulk genomic DNA, and repetitive retroviral sequences to Dnmt1 overexpression. At low Dnmt1 methyltransferase levels repetitive retroviral elements were methylated and silenced. The nonmethylated imprinted region of Igf2 and H19 was resistant to methylation at low Dnmt1 levels but became fully methylated when Dnmt1 was overexpressed from a bacterial artificial chromosome transgene. Methylation caused the activation of the silent Igf2 allele in wild-type and Dnmt1 knockout cells, leading to biallelic Igf2 expression. In contrast, the imprinted genes Igf2r, Peg3, Snrpn, and Grf1 were completely resistant to de novo methylation, even when Dnmt1 was overexpressed. Therefore, the intrinsic difference between the imprinted region of Igf2 and H19 and of other imprinted genes to postzygotic de novo methylation may be the molecular basis for the frequently observed de novo methylation and upregulation of Igf2 in neoplastic cells and tumors. Injection of Dnmt1-overexpressing embryonic stem cells in diploid or tetraploid blastocysts resulted in lethality of the embryo, which resembled embryonic lethality caused by Dnmt1 deficiency.
Project description:Genomic imprinting in mammals marks the two parental alleles resulting in differential gene expression. Imprinted loci are characterized by distinct epigenetic modifications such as differential DNA methylation and asynchronous replication timing. To determine the role of DNA methylation in replication timing of imprinted loci, we analyzed replication timing in Dnmt1- and Dnmt3L-deficient embryonic stem (ES) cells, which lack differential DNA methylation and imprinted gene expression. Asynchronous replication is maintained in these ES cells, indicating that asynchronous replication is parent-specific without the requirement for differential DNA methylation. Imprinting centers are required for regional control of imprinted gene expression. Analysis of replication fork movement and three-dimensional RNA and DNA fluorescent in situ hybridization (FISH) analysis of the Igf2-H19 locus in various cell types indicate that the Igf2-H19 imprinting center differentially regulates replication timing of nearby replicons and subnuclear localization. Based on these observations, we suggest a model in which cis elements containing nonmethylation imprints are responsible for the movement of parental imprinted loci to distinct nuclear compartments with different replication characteristics resulting in asynchronous replication timing.