Asynchronous replication timing of imprinted loci is independent of DNA methylation, but consistent with differential subnuclear localization

Abstract

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 fluoroscent 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.[Keywords: Asynchronous replication; genomic imprinting; nuclear localization; DNA methylation; Igf2-H19; replication fork] Genomic imprinting is an epigenetic mechanism that marks the two parental alleles during gametogenesis, resulting in differential gene expression in the developing embryo and adult. The epigenetic modifications responsible for establishing and maintaining imprinted gene expression are thought to involve DNA methylation, chromatin modifications, and asynchronous replication timing. For several imprinted loci specific cis control elements, imprinting centers (ICs), have been identified which are required for regional control of imprinting, imprinted gene expression, and asynchronous replication timing. All identified ICs harbor regions that contain differentially methylated DNA sequences. These methylation imprints are established in the germline and are resistant to global demethylation during the early cleavages of the preimplantation embryo. To date, DNA methylation is the only epigenetic mark shown to be required for the maintenance of imprinted gene expression. Embryos deficient for the DNA maintenance methyl transferase gene, Dnmt1, lack imprints and die early during development (Li et al. 1992). Moreover, embryos derived from females deficient for Dnmt3L, a newly identified de novo DNA methyl transferase gene that lacks the catalytic domain shared by the other DNA methyl transferases, were shown to lack maternal imprints and fail to develop past 9.5 days postcoitum (dpc; Bourc'his et al. 2001;Hata et al. 2002). A similar phenotype was observed in embryos generated by fertilization of oocytes deficient for the de novo DNA methyl transferase Dnmt3a, suggesting that these prot...

Allelic silencing at the tumor-suppressor locus 13q14.3 suggests an epigenetic tumor-suppressor mechanism

“…In imprinting, discrimination of the two gene copies is achieved through epigenetic chromatin modifications, such as differential DNA methylation (13), histone modifications (14), and replication timing (15). We visualized asynchronous replication timing by fluorescence in situ hybridization under experimental conditions that separate replicated chromatids (16,17). Imprinted regions have been shown to replicate asynchronously (17,18), with the fraction of asynchronously replicating cells ranging from 21% to 41%, depending on the region analyzed and cell line used.…”

“…29). At the Ig loci, asynchronous replication timing is independent of the DNA methylation status (17) and transcriptional activity (16). Even engineered rearrangement of Ig loci, which induces changes in chromatin packaging, does not change their asynchronous replication timing (16).…”

“…We visualized asynchronous replication timing by fluorescence in situ hybridization under experimental conditions that separate replicated chromatids (16,17). Imprinted regions have been shown to replicate asynchronously (17,18), with the fraction of asynchronously replicating cells ranging from 21% to 41%, depending on the region analyzed and cell line used. In contrast, nonimprinted regions have shown asynchronous replication in only 9-19% of cells.…”

Incomplete penetrance and variable expressivity: is there a microRNA connection?

“…Differential transcription at this locus is correlated with variation in DNA methylation at the differential methylation region (DMR) upstream of the H19 locus, differential DNase I hypersensitivity and replication asynchrony between imprinted alleles. (16)(17)(18)(19) The consequence of these molecular correlates is that the enhancer present downstream of H19 gene is not accessible to Igf2 promoter on the maternal chromosome. (16) Modification of histones is an epigenetic marking reported in several cases, including Prader Willi and Angelman syndrome (PWS and AS) region comprising of SNRPN, IGF2R, and U2AF1-RS genes.…”

Heterochromatin?many flavours, common themes

“…(96,(98)(99)(100) Silenced imprinted alleles are also located towards the exterior of a nucleus. (101) Interestingly, PcG proteins are found on the surface of condensed chromatin domains, (102) suggesting that they need only a limited access to heterochromatin factors, most likely because they recruit many PcG-specific factors and only rarely HP1. It is also important to note that not all components of silent heterochromatin compartments should not be considered as static, as HP1 has a dynamic association with heterochromatin compartments.…”