DamC reveals principles of chromatin folding in vivo without crosslinking and ligation [capture-mapping]

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. 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 β-mercaptoethanol, 20 U/ml leukemia inhibitory factor (Miltenyi Biotec, premium grade) and 250 µg/mL hygromycin in 8% CO2 at 37°C. 2µg of genomic DNA were fragmented to an average of 500bp by sonication (Covaris S220, duty cycle: 5%, peak power: 175W, duration: 25sec). End-repair, A-tailing and ligation of full-length barcoded Illumina adapters were performed using the TruSeq DNA PCR-free kit (Illumina) according to the manufacturer guidelines with the exception that large DNA fragments were not removed. 750ng of libraries for each sample were pooled together, and fragments of interest were captured using biotinylated probes against the the piggyBac inverted terminal repeats (ITRs) sequence and the xGen Hybridisation Capture kit (IDT) according to the manufacturer protocol (probes concentration of 2.25pmol/µl). Following the capture, libraries were amplified for 12 cycles using the Kapa Hi-fi polymerase and the following primers: 5’-AATGATACGGCGACCACCGAGAT, 5’-CAAGCAGAAGACGGCATACGAGA. Final libraries were purified using AMPure XP beads (1:1 ratio), quality controlled and sequenced on the NextSeq500 platform (paired-end 300 cycles mid-output). Capture probe sequences are as follows: ITR3-1 [Btn]ATCTATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTT, ITR3-2 [Btn]TCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAA, ITR5-1 [Btn]TTAACCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAA, ITR5-2 [Btn]ATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAA ACTCAAAATTTCTTCTATAAAGTAACAAA.