Abstract
BackgroundThe KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation.ResultsHere, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others.ConclusionsOur results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression.
Highlights
The KRABcontaining zinc finger proteins (KZFPs)/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation
KAP1 maintains tri-methylation of Lysine on histone H3 (H3K9me3) at ICRs and TEs and protects them from ten-eleven translocation (TET)‐dependent hydroxymethylation To decipher the role of the KZFP/KAP1 complex in the maintenance of heterochromatin and DNA methylation in ground-state murine embryonic stem cells (mESCs), we characterized the genomewide profiles of KAP1 binding and H3K9me3 enrichment by chromatin immunoprecipitation/deep sequencing (ChIP-seq) and compared them with recently published DNA methylation data [54]
We examined the presence of hydroxymethylated DNA, a product of TET activity, in wild-type and KAP1-depleted mESCs by hMeDIP-Seq, using antibodies against 5hmC
Summary
The KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. KAP1 is recruited to particular genomic loci by KRABcontaining zinc finger proteins (KZFPs), a large family of rapidly evolving sequence-specific DNA-binding factors [2,3,4,5,6]. It serves as a scaffold for a macromolecular complex that induces notably the di- and tri-methylation of histone 3 and DNA methylation on CpG dinucleotides, generating a highly heterochromatic environment characterized by the presence of the H3K9me2/3 repressive mark and the presence of 5mC (5-methylcytosine) [1]. Mutations in ZFP57 are responsible for transient neonatal diabetes [20]
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