GSE128015
DamC reveals principles of chromatin folding in vivo without crosslinking and ligation [HiC]
Mammalian chromosomes are folded into an intricate hierarchy of structural domains, within which topologically associating domains (TADs) and CTCF-associated loops partition the physical interactions between regulatory sequences. Current understanding of chromosome folding largely relies on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after crosslinking of chromatin. To measure chromosome structure in vivo, quantitatively and without relying on crosslinking and ligation, we have implemented a new method named damC. DamC combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and a biophysical model of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of TADs and CTCF loops, confirms 3C-based measurements of the scaling of contact probabilities within TADs, and provides evidence that mammalian chromatin in vivo is essentially rigid below 5 kilobases. Combining damC with transposon-mediated genomic engineering shows that new loops can be formed between ectopically introduced and endogenous CTCF sites, which alters the partitioning of physical interactions within TADs. This establishes damC as a crosslinking- and ligation-free framework to measure and modify chromosome interactions combined with a solid theoretical background for rigorous data interpretation. This orthogonal approach to 3C validates the existence of key structural features of mammalian chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated. Overall design: Mouse ESC lines #94.1_2.7 (carrying random insertions of 50x TetO arrays spanning approx. 2.7kb each) and #94.1_216_C3 (carrying different random insertions of the same 50x TetO cassette flanked by 3 CTCF sites faced outwards) were generated starting from an X0 clone of the PGKT2 line in Masui et al (Cell 145:447-458, 2011). The remaining X chromosome additionally carries the deletion of the Linx promoter within the X inactivation center (Nora et al., Nature 485:381-385, 2012). Random insertions were generated using the piggyBac transposon system. Both lines stably express rTetR-EGFP-Dam-ERT2 under the control of an ectopic Rosa26 promoter. Cells were cultured on gelatin-coated culture plates in Dulbecco Modified Eagle's medium (Sigma) in the presence of 15% foetal calf serum (Eurobio Abcys), 100 µM ß-mercaptoethanol, 20 U/ml leukemia inhibitory factor (Miltenyi Biotec, premium grade) and 250 µg/mL hygromycin in 8% CO2 at 37°C. 6x106 mESC were harvested and diluted in 1x PBS to final 1x106 cells/ml, then crosslinked with 1% formaldehyde and quenched with 0,125M glycine for 5 min at RT. After two 1x PBS washes, cells pellets were obtained by centrifugation, snap frozen and stored at -80°C. Pellets were thawed on ice and resuspended in 500 ul lysis buffer (10 nM Tris-HCl pH8.0, 10 nM NaCl, 0.2%NP40, 1x Roche protease inhibitors) and left 30 min on ice. Cells were then pelleted by centrifugation (954 x g, 5 min, 4°C ), washed once with 300 ul 1x NEB2 buffer and nuclei were extracted by 1 h incubation at 37°C in 190 ul 0.5%SDS 1xNEB2 buffer. SDS was neutralized by diluting the sample with 400 ul NEB2 buffer and adding 10% Triton X-100. After 15 min of incubation at 37°C, nuclei were pelleted, washed once in PBS and resuspended in 300 ul NEB2 buffer. 400U of MboI (NEB, 25 000 units/ml) were added and incubated at 37°C overnight. The next day, nuclei were pelleted again, resuspended in 200 ul fresh NEB2 buffer and additional 200U of MboI were added for two more hours before heat inactivation at 65°C for 15 min. 43 ul of end-repair mix (1.5 µL of 10 mM dCTP; 1.5 µL of 10mM dGTP; 1.5 µL of 10 mM dTTP; 37.5 µL of 0.4 mM Biotin-11-dATP (Invitrogen) and 1 µL of 50U/µL DNA Polymerase I Large Klenow fragment (NEB)) were added to the nuclear suspension, incubated at 37°C for 45 min and heat inactivated at 65°C for 15 min. The end repair mix was exchanged with 1.2 ml of ligation mix (120µL of 10X T4 DNA Ligase Buffer; 100 µL of 10% Triton X-100; 6 µL of 20 mg/mL BSA; 969µL of H2O) plus 5 ul of T4 ligase (NEB, 2000 units/ml) and ligation was performed at 16°C overnight. Nuclei were reconstituted in 200 ul fresh NEB2 buffer followed by RNA digestion in 0.5 mg/ml RNAse A for 10 min at 37°C. Samples were de-crosslinked with Proteinase K at 65°C overnight and DNA was purified using phenol/chloroform. 2 ug of DNA sample were sonicated using Diagenode Bioruptor Pico. MyOne Streptavidin T1 (Life Technologies # 65601) magnetic beads were used to capture biotinylated DNA followed by A-tailing. Adapter ligation was performed according to NEB Next Ultra DNA Library prep kit instructions. Two independent PCR reactions with multiplex oligos for Illumina sequencing were performed and pooled for the final PCR clean-up by magnetic AMPure bead (Beckman Coulter) purification. The final libraries were eluted in nuclease-free water, QCed by Bioanalyzer and Qubit. HiC libraries were sequenced on a Illumina Nextseq500 platform (2x42bp paired end). Please note that processed data was generated from both replicates and is linked to the corresponding 'replicate 1' sample records.