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| Status |
Public on Jan 31, 2021 |
| Title |
The chromatin remodeler Lsh modulates genome-wide cytosine hydroxymethylation |
| Organism |
Mus musculus |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. While Tet proteins have been associated to epigenetic repression or activation complexes, our understanding of the molecular mechanisms involved in Tet-mediated regulation of gene transcription remains limited. Here, we showed that Tet directly interact with lymphoid-specific helicase (Lsh), a chromatin remodelling factor belonging to the ISWI family. This specific interaction seems to regulate Tet enzymatic activity since Lsh knock-out leads to a substantial reduction of 5-hydroxymethylation global level in mouse embryonic fibroblasts (MEFs) and in embryonic stem cells (ES). Whole genome sequencing of 5hmC in wild-type versus Lsh knock-out MEFs and ESCs showed that in absence of Lsh some regions of the genome gain while others loose 5hmC marks, with a weak correlation to gene expression changes . We further demonstrated that 5hmC modifications upon Lsh loss are not a direct consequence of 5mC decrease as DhMR (differentially hydroxymethylated regions) did not overlap with DMR (differentially methylated regions) underlying that these modifications occurred at different genomic loci. Altogether, our results suggest that DNA 5-hydroxymethylation and nucleosome folding are linked phenomena, highlighting novel means by which Tet proteins may influence gene regulation.
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| Overall design |
5hmC level in wild-type mouse or LSH KO embryonic stem cells and MEF cells using the hydroxymethyl collector kit (Active Motif). Genome: mm9.
Briefly, 1µg of genomic DNA was diluted in ultra-pure water to 35 ng/μL and then sonicated in cold water with a Bioruptor sonicator (Diagenode) to obtain fragments averaging 300 bp in size. The fragmented DNA was used in combination with the hydroxymethyl collector (Active Motif) following the manufacturer’s protocol. Briefly, a glucose moiety that contains a reactive azide group was enzymatically linked to hydroxymethylcytosine in DNA, creating glucosyl-hydroxymethylcytosine. Next, a biotin conjugate was chemically attached to the modified glucose via a “click reaction”, and magnetic streptavidin beads were used to capture the biotinylated-hmC DNA fragments. After extensive washing steps and chemical elution, the hydroxymethylated DNA fragments released from the beads were used in sequencing experiments.
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| Contributor(s) |
de Dieuleveult M, Colin L, Defrance M, Bizet M, Calonne E, Deplus R, Fuks F |
| Citation(s) |
33960278 |
| Submission date |
Feb 05, 2018 |
| Last update date |
May 17, 2021 |
| Contact name |
Maud de Dieuleveult |
| E-mail(s) |
maud.dedieuleveult@gmail.com
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| Organization name |
Free University of Brussels
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| Lab |
Laboratory of Cancer Epigenetics
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| Street address |
Route de Lennik 808
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| City |
Brussels |
| ZIP/Postal code |
1070 |
| Country |
Belgium |
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| Platforms (1) |
| GPL16173 |
Illumina HiScanSQ (Mus musculus) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA432929 |
| SRA |
SRP132180 |
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