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 TIF1β), a scaffolding protein without intrinsic repressive 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 β-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.
Highlights
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
The only such factor that was strongly enriched in this screen was O-linked β-N-acetylglucosamine transferase (OGT), the sole protein glycosyltransferase that is active in the nucleus and cytoplasm
While many glycosyltransferases modify secreted proteins and the extracellular domains of membrane proteins, OGT is the only glycosyltransferase that modifies nuclear and cytosolic proteins, and O-GlcNAcylation is the only form of glycosylation that is known to be highly dynamic and reversible [14]
Summary
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. Local chromatin de-GlcNAcylation 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. The repression of methylated retrotransposon promoters requires the TRIM28 protein ( known as KAP1 and TIF1β) [10], as does the methylation-dependent monoallelic expression of imprinted genes [11], but TRIM28 is a structural factor that does not bind to DNA and lacks repressor activity [12, 13]. We developed a combined genetic and biochemical screen to identify factors that interact with TRIM28 in a methylationdependent manner The only such factor that was strongly enriched in this screen was O-linked β-N-acetylglucosamine transferase (OGT), the sole protein glycosyltransferase that is active in the nucleus and cytoplasm. These data show that O-glycosylation is an essential component of the system that represses methylated promoters
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