Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Methylation profiling by high throughput sequencing
Summary
TET family enzymes successively oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine, leading to eventual demethylation. 5hmC and TET enzymes occupy distinct chromatin regions, suggesting unknown mechanisms controlling the fate of 5hmC within diverse chromatin environments. Here, we report that SALL4A preferentially associates with 5hmC in vitro and occupies enhancers in mouse embryonic stem cells in a largely TET1-dependent manner. While most 5hmC at SALL4A peaks undergoes further oxidation, this process is abrogated upon deletion of Sall4 gene, with a concomitant reduction of TET2 at these regions. Thus, SALL4A facilitates further oxidation of 5hmC at its binding sites, which requires its 5hmC binding activity and TET2, supporting a collaborative action between SALL4A and TET proteins in regulating stepwise oxidation of 5mC at enhancers. Our study identifies SALL4A as a 5hmC binder, which facilitates 5hmC oxidation by stabilizing TET2 association, thereby fine-tuning expression profiles of developmental genes in mouse embryonic stem cells.
Overall design
In several cell lines, including TT2, Sall4 het, Sall4 KO, Sall1/Sall4 DKO, Tet1 KD, Tet1/2/3 TKO, Sall4a, Sall1 and Tet1 is ChIPed by antibody. ChIPed fragments were sequenced by Illumina GA II or Illumina HiSeq 2000 platform. 5mC and hmC were also immunoprecipitated by its antibody as well.
Less...
More...