Abstract
In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation.
Highlights
In mammals, methylation at CG dinucleotides is required for the monoallelic expression of genes subject to genomic imprinting, transcriptional silencing of retrotransposons and X chromosome silencing in females [1]
Given that the hypomethylation observed in Droshadeficient embryonic stem (ES) cells occurs without any reduction in the expression of proteins known to impact DNA methylation and that genetically Drosha is necessary to stimulate DNA methyltransferase 1 (DNMT1) activity, we considered alternative mechanisms to account for our observations
A recent study revealed that RNAs capable of adopting stem–loop structures are capable of associating with and inhibiting DNMT1 methyltransferase activity [14] and a recent description of the interactome of the long non-coding RNA (lncRNA) Xist identified DNMT1 as a major interacting factor [38]
Summary
Methylation at CG dinucleotides is required for the monoallelic expression of genes subject to genomic imprinting, transcriptional silencing of retrotransposons and X chromosome silencing in females [1]. Genomic methylation patterns are initially established by the de novo methyltransferases Dnmt3A and Dnmt3B [2] following two stages of methylation loss that occurs prior to implantation of the embryo [3] as well as during the migration of primordial germ cells (PGCs) [4]. Genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase DNA methyltransferase 1 (DNMT1). Given the importance of faithful inheritance of cytosine methylation, understanding how DNMT1 is correctly regulated is crucial. Normal DNMT1 function is achieved by a combination of its correct targeting [5,6,7], control of protein stability [8,9] and regulation of its methyltransferase activity [10]. Our understanding of the mechanisms involved and how they interact remains incomplete
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