\title{Methylation-directed glycosylation of chromatin factors represses retrotransposon promoters}

\author{Mathieu Boulard, Sofia Ruci\'a, John R. Edwards, and Timothy H. Bestor}

\begin{abstract}
The mechanisms by which methylated mammalian promoters are transcriptionally silenced even in the presence of all of the factors required for their expression have long been a major unresolved issue in the field of epigenetics. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 protein (also known as KAP1 and TIF1$\beta$), a scaffolding protein without intrinsic repressor or DNA-binding properties. The identity of the key effector within this complex that represses transcription is unknown. We developed a methylation-sensitized interaction screen which revealed that TRIM28 was complexed with O-linked $\beta$-N-acetylglucosamine transferase (OGT) only in cells that had normal genomic methylation patterns. OGT is the only glycosyltransferase that modifies cytoplasmic and nuclear protein by transfer of N-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed that O-glycosylated proteins and TRIM28 were specifically bound to promoters of active retrotransposons and to imprinting control regions, the two major regulatory sequences controlled by DNA methylation. Furthermore, genome-wide loss of DNA methylation caused a loss of O-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal of O-GlcNAc was directed against retrotransposon promoters. Local chromatin de-GlcNAcylation specifically reactivated the expression of the targeted retrotransposon family without loss of DNA methylation. These data revealed that O-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons.
\end{abstract}

\section{Introduction}
It has been known for many years that the methylation of mammalian promoters induces heritable transcriptional repression \cite{1,2,3}. Genome-wide demethylation reactivates expression of silenced retrotransposons \cite{4} and causes the biallelic expression of imprinted genes \cite{5}, which are normally expressed from only the allele of maternal or paternal origin. After introduction into cells, artificially methylated Pol II-dependent promoters are actively transcribed for a brief period prior to heritable silencing \cite{6,7}. This indicates that recruitment of methylation-dependent repressive factors rather than a direct effect of cytosine methylation on the transcriptional machinery is responsible for silencing.

Biochemical studies identified proteins that bind to methylated DNA in vitro and had the properties expected of methylation-dependent transcriptional repressors. However, ablation of the genes that encode MeCP2 and other methylation-dependent DNA-binding proteins singly or in combination did not reactivate methylated promoters in vivo \cite{8}. Ablation of methylated DNA-binding proteins produces phenotypes that are much less severe than the phenotypes caused by deletions of DNA methyltransferase genes \cite{9}.

The components of the methylation-dependent repressive complex and the actual mechanisms that repress transcription are not known. The repression of methylated retrotransposon promoters requires the TRIM28 protein (also known as KAP1 and TIF1$\beta$) \cite{10}, as does the methylation-dependent monoallelic expression of imprinted genes \cite{11}, but TRIM28 is a structural factor that does not bind to DNA and lacks repressor activity \cite{12,13}. We developed a combined genetic and biochemical screen to identify factors that interact with TRIM28 in a methylation-dependent manner. The only such factor that was strongly enriched in this screen was O-linked $\beta$-N-acetylglucosamine transferase (OGT), the sole protein glycosyltransferase that is active in the nucleus and cytoplasm. OGT has important regulatory functions in multiple pathways \cite{14}, but had not previously been directly related to DNA methylation. Whole-genome analysis showed that TRIM28 and proteins modified by OGT colocalize at transposon promoters and at imprinting control regions. In the absence of DNA methylation, multiple proteins with key roles in gene silencing failed to undergo modification by OGT. Targeted protein deglycosylation by a novel editing method reactivated the transcription of methylated retrotransposon promoters. These data show that O-glycosylation is an essential component of the system that represses methylated promoters.

\section{Results}
\subsection{Ablation of TRIM28 Phenocopies Mutations that Cause Genome-Wide Demethylation}
Homozygosity for a strongly hypomorphic allele of Trim28 in mouse embryos does not cause appreciable demethylation