Imprinted DNA methylation reconstituted at a non-imprinted locus.

David H Taylor,Alex B Wang,Chelsea M Mclean,Paul D Soloway,Warren L Wu

doi:10.1186/s13072-016-0094-0

Abstract

Background In mammals, tight regulation of cytosine methylation is required for embryonic development and cellular differentiation. The trans-acting DNA methyltransferases that catalyze this modification have been identified and characterized; however, these proteins lack sequence specificity, leaving the mechanism of targeting unknown. A cis-acting regulator within the Rasgrf1 imprinting control region (ICR) is necessary for establishment and maintenance of local imprinted methylation. Here, we investigate whether 3-kb of sequence from the Rasgrf1 ICR is sufficient to direct appropriate imprinted methylation and target gene expression patterns when ectopically inserted at the Wnt1 locus.ResultsThe Rasgrf1 ICR at Wnt1 lacked somatic methylation when maternally transmitted and was fully methylated upon paternal transmission, consistent with its behavior at the Rasgrf1 locus. It was unmethylated in the female germline and was enriched for methylation in the male germline, though not to the levels seen at the endogenous Rasgrf1 allele. Wnt1 expression was not imprinted by the ectopic ICR, likely due to additional sequences being required for this function.ConclusionsWe have identified sequences that are sufficient for partial establishment and full maintenance of the imprinted DNA methylation patterns. Because full somatic methylation can occur without full gametic methylation, we infer that somatic methylation of the Rasgrf1 ICR is not simply a consequence of maintained gametic methylation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0094-0) contains supplementary material, which is available to authorized users.

Highlights

In mammals, tight regulation of cytosine methylation is required for embryonic development and cellular differentiation
The repetitive element, which is required for proper methylation establishment [15] and maintenance after fertilization [18], acts as a promoter for a piRNAtargeted noncoding RNA that is transcribed across the differentially methylated domain (DMD) in e16.5 testes [16]. piRNAs normally silence transposable elements in the male germline; a subset of these primary piRNAs interact with two loci within the pitRNA, called sites 1 and 2
Generation of targeted mice The Wnt1tm1pds vector was assembled using genomic clones from Rasgrf1, and PCR products from Wnt1. It included a loxP site at the Wnt1 DMD that enabled us to distinguish it from the endogenous Rasgrf1 DMD, and which we previously showed did not interfere with DNA methylation at Rasgrf1 [18]

Summary

Introduction

Tight regulation of cytosine methylation is required for embryonic development and cellular differentiation. 5mC is usually associated with transcriptional repression, but is known to be a complex and dynamically regulated modification Research into this regulation has included descriptive studies such as methylome mapping [1, 2] or functional studies of the proteins that regulate methylation in trans, such as DNMT1 [3], and isoforms of DNMT3 [4,5,6]. Few studies have yielded much information about the cis-acting DNA sequences that target these trans-acting proteins to the DNA in a locus-, tissue-, or time-specific manner. The pitRNA is subsequently processed to secondary piRNAs, and de Taylor et al Epigenetics & Chromatin (2016) 9:41

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