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Learn MoreMethylated mammalian promoters are transcriptionally silenced even in the presence of all the factors required for their expression. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 (also known as KAP1 and TIF1) protein. An internally controlled interaction screen identified O-linked -N-acetylglucosamine transferase (O-GlcNAc transferase or OGT) as a protein that was complexed with TRIM28 in wild type em-bryonic stem cells but not in Dnmt1-/- cells that had severely demethylated genomes. In the ab-sence of DNA methylation, multiple proteins associated with TRIM28 failed to undergo modifica-tion by N-Acetylglucosamine (GlcNAc). Mass spectrometry identified several of these proteins as known mediators of transcriptional silencing. The most active transposon in the mouse ge-nome is the IAP LTR retrotransposon, which have been previously shown to be repressed by DNA methylation. A Bacteroides O-GlcNAc hydrolase was fused to a catalytically inactive Cas9 and targeted to methylated IAP retrotransposon promoter sequences via IAP-specific guide RNAs; fulminating reactivation of IAP transcription was induced. These data revealed that Glc-NAcylation is directly involved in the transcriptional repression of methylated promoters. SOURCE: John,R.,Edwards (jredwards@wustl.edu) - Washington University School of Medicine
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