De novo methylation of an embryonic globin gene during normal development is strand specific and spreads from the proximal transcribed region

Abstract

The recently discovered de novo methyltransferases DNMT3a and DNMT3b have been shown to be critical to embryonic development. However, at a single gene level, little is known about how the methylation pattern is established during development. The avian embryonic -globin gene promoter is completely unmethylated in 4-day-old chicken embryonic erythroid cells, where it is expressed at a high level, and completely methylated in adult erythroid cells, where it is silent. The methylation pattern of the -globin gene promoter, proximal transcribed region, and distal transcribed region on both DNA strands was examined during development in chicken erythroid cells. It was found that de novo methylation targets the CpG-dense proximal transcribed region on the coding (top) strand initially, followed by spreading into the 3 region and into the promoter region. Methylation of the template (bottom) strand lags behind that of the coding strand, and complete methylation of both strands occurs only after the gene has been silenced. The results of the study indicate that establishment of the de novo methylation pattern involves strand-specificity and methylation spreading. IntroductionDNA methylation in eukaryotes involves addition of a methyl group to the carbon 5 position of the cytosine ring. This reaction is catalyzed by DNA methyltransferase in the context of the sequence 5Ј-CG-3Ј, which is also referred to as a CpG dinucleotide. 1 Eukaryotic genomes are not methylated uniformly but contain methylated regions interspersed with unmethylated domains. 2 Approximately 70% to 80% of the CpG residues in most vertebrates are methylated. 3 In contrast to the rest of the genome, smaller regions of DNA called CpG islands are unmethylated and possess the expected CpG frequency. 4,5 During early development a dramatic reduction in methylation levels occurs in the preimplantation embryo. 6 This is followed by a wave of de novo methylation involving most CpG residues. However, CpG island-associated promoter regions are protected from methylation by mechanisms which remain unclear. 7 The methylation profile of genes in the adult is stable over many cell generations. Genomic methylation patterns are conserved after DNA replication by the DNA methyltransferase Dnmt1, which is the major maintenance methyltransferase. 8 Dnmt1 is recruited to replicating DNA to reproduce the methylation pattern of the parental strands in the daughter strands. 9 Inactivation of the mouse Dnmt1 gene by gene targeting resulted in extensive demethylation of all sequences examined. 10,11 However, ES cells completely lacking Dnmt1 were still capable of methylating retroviral DNA de novo. 11 The search for the de novo methyltransferases led to the discovery of Dnmt3a and Dnmt3b. 12 These were found to be essential for de novo methylation and for mouse development. 13 However, it remains unclear how de novo methylation patterns are established during development, over what time intervals these changes occur, or if this process involves any strand-or sequence-specifici...

Preferential Epigenetic Suppression of the Autonomous MusD over the Nonautonomous ETn Mouse Retrotransposons

“…We propose that such repression, resulting in the observed lower transcription level of MusD compared with ETnII elements in early embryos (5) and ES cell lines (this study), stems from a higher probability of repressive chromatin and DNA methylation originating in the downstream CpG-rich retroviral region. A similar case was reported for the rho globin gene in chicken embryonic erythroid cells, in which, during normal developmental silencing, de novo methylation originates in the downstream CpG-dense region and then spreads into the promoter, silencing transcription and resulting in complete DNA methylation in adult erythroid cells (58). Notably, roughly half of the ETnII copies in the genome have methylation levels similar to MusD, but the other half are essentially unmethylated (Fig.…”

MBD2 is a critical component of a methyl cytosine-binding protein complex isolated from primary erythroid cells

“…MEL cells were transfected with the -globin mini-locus that had been methylated to recapitulate the pattern of methylation observed at the endogenous gene in adult chicken RBCs. 50 After selection and dilution cloning, DNA slot blotting confirmed that the -globin mini-locus was present in MEL cells, termed MEL-cells ( Figure 6D). …”

Methylation-Mediated Repression of GADD45α in Prostate Cancer and Its Role as a Potential Therapeutic Target

“…The sequencing gel was dried and exposed to a phosphorimager screen (BioRad Laboratories). Methylation analysis was carried out by quantitating the intensity of C and T bands using Quantity One software (Bio-Rad Laboratories) and calculating the percentage of C/(C + T) bands, as previously described (18).…”