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| Status |
Public on May 29, 2020 |
| Title |
Protein O-GlcNAcylation Silences Methylated Promoters in Mammalian Genomes |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
Methylated 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 TIF1β) protein. An internally controlled interaction screen identified O-linked β-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.
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| |
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| Overall design |
RNA-seq analysis of Dnmt1-/- ES cells and WT ES cells untreated or treated with dCas9-OGA or dCas9-OGA-D242A with four sgRNAs targeting IAP elements.
O-GlcNAc ChIP-seq analysis of WT ES cells
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| |
|
| Contributor(s) |
Boulard M, Edwards JR, Bestor TH |
| Citation(s) |
32522876 |
| Submission date |
Jan 12, 2017 |
| Last update date |
Jul 07, 2020 |
| Contact name |
John R. Edwards |
| E-mail(s) |
jredwards@wustl.edu
|
| Organization name |
Washington University School of Medicine
|
| Department |
Center for Pharmacogenomics
|
| Street address |
660 S. Euclid Ave, Campus Box 8220
|
| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL21103 |
Illumina HiSeq 4000 (Mus musculus) |
|
| Samples (13)
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|
| Relations |
| BioProject |
PRJNA361065 |
| SRA |
SRP096630 |
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