Coordinated changes of DNA (de)methylation, nucleosome positioning and chromatin binding of the architectural protein CTCF play an important role for establishing cell type specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the Tet1 and Tet2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC) or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation and gene expression in DKO ESCs, and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, while unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild type cells but not from 5fC enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, e.g. at promoters, boundaries of chromatin loops and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to downregulation of neighbouring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions and DNA sequence that determines differential CTCF binding and regulates gene expression.
Overall design: Two WT and two Tet1/2-deficient (DKO) mouse ES cell lines were isolated from WT and Tet1/Tet2 double-mutant mice with a mixed 129 and C57BL/6 background as described (Daw+B1laty et al., 2013). ESCs were maintained in regular ESC medium (KnockOut DMEM supplemented with 15% fetal calf serum, 1x non-essential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine, 0.0008% β-mercaptoethanol and 1000 U/ml leukemia inhibiting factor) at 37 °C and 7% CO2 on inactivated MEF feeders seeded on gelatin (0.2%) coated dishes. For CTCF ChIP-seq experiments cells were trypsinized and pre-plated on gelatin coated dishes three times to remove feeders. ESCs were resuspended in medium, fixated for 10 min at room temperature and lysed as described (Wiehle and Breiling 2016). After nuclei lysis, samples were sonicated 4 × 5 min with 30 s on/off intervals (Bioruptor, Diagenode). SDS content was reduced by dilution as described (Wiehle and Breiling 2016). Chromatin from 6 x 10^6 cells was immunoprecipitated overnight at 4°C using 5 µg of antibody against CTCF (#61311, Active Motif). Further processing occurred according to instructions of the ChIP-IT High Sensitivity kit (Active Motif). Immunoprecipitated DNA was eluted from Protein A/G agarose beads and purified using the IPure kit (Diagenode). For ChIP-sequencing libraries were generated from 10 ng of DNA using the NEBNext ChIP-Seq Library Prep Master Mix Set (NEB) according to the manufacturer´s instructions. Final libraries were quality controlled and pooled to equimolar amounts and sequenced in 50 bp single-read mode on an Illumina HiSeq 2000 device. For MNase-assisted histone H3 ChIP-seq cells were cross-linked with 1% methanol-free formaldehyde for 10 min. After quenching with glycine, cells were washed three times with PBS. The cell pellet was treated with 40 U MNase for 5 min at 37°C, then stopped with 10x Covaris buffer (Covaris Ltd) and chromatin was sheared for 15 min with the Covaris S2 device (burst 200; cycle 20%; intensity 8). Immunoprecipitation was performed for ~5 x 10^6 cells with anti-H3 antibody (Abcam #ab1791, Lot: GR103864-1). Then chromatin was treated with RNaseA and proteinase K. Purified DNA was cloned into Illumina libraries with the NEBNext Ultra library preparation kit (NEB).
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