You might think our portrait of the marks that make up the epigenetic landscape is nearing completion, but a new feature is now filling in some of the blank space behind a key regulatory mechanism. It has been long known that DNA methylation represses transcription via the recruitment of methylation-dependent repressive factors. The components of the methylation-dependent repressive complex and the actual mechanisms that repress transcription remain unclear. TRIM28 is known to be required for the repression of methylated retrotransposon promoters. However, TRIM28 does not have any DNA binding or repressor activity.
The lab of Timothy Bestor at Columbia University wanted to identify the proteins TRIM28 interacts with in a methylation-dependent manner. They developed a screen to compare TRIM28 interactors in DNMT1-/- with DNMT1+/+ mouse embryonic stem cells; the former lacking the de novo DNA methylransferase and thus being globally depleted of DNA methylation. They used a TRIM28 antibody to pull down the protein complexes, then resolved them with SDS/PAGE and mass spectrometry. Here’s what they found:
- Only the glycosylation enzyme O-linked β-N-acetylglucosaminetransferase (OGT) shows a strong methylation-dependent association with TRIM28
- This was unexpected because there was previously no known connection between DNA methylation and protein glycosylation
- ChIP-seq against O-GlcNAc (the carbohydrate OGT adds to proteins) uncovered that Long terminal repeats (LTRs) of IAP transposons (the most active in mouse) are highly enriched for O-GlcNAc modified proteins as well as TRIM28
- Transposons not capable of transcription are not enriched for
- Isolation of TRIM28 interactors and characterization of their O-GlcNAc modification in both DNMT1-/- and DNMT1+/+ ES cells uncovered that several proteins with known roles in transcriptional repression were found to undergo methylation-dependent O-GlcNAcylation
- Finally, the authors developed a novel epigenetic editing method for targeted protein deGlcNAcylation: a chimeric dCas9-prokaryotic O-GlcNAc hydrolase
- Using sgRNAs against IAP retrotransposons, they found deGlcNAcylation reactivates the expression of these elements without loss of DNA methylation
This work demonstrates, for the first time, a functional relationship between protein glycosylation and DNA methylation. The experiments performed suggest that DNA methylation at retrotransposons directs chromatin factor O-GlcNAcylation, which is then required for repression of transcription.
Fill in the blanks of DNA methylation’s repressive mechanism over at PNAS, June 2020.