DamC reveals principles of chromatin folding in vivo without crosslinking and ligation [damC]

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.To induce nuclear translocation of the rTetR-Dam fusion protein to the nuclei, mESC were trypsinized and directly seeded in culture medium containing 4-hydroxy-tamoxifen (4-OHT) at various concentration as indicated in the sample titles. Binding of the Dam fusion protein to the TetO arrays was induced by simultaneously adding 2.5 μg/ml doxycycline (Dox). After 18 hours, 3x106 cells were harvested using trypsin. Genomic DNA was extracted using the Qiagen blood and tissue kit adding RNaseA in step 1. Genomic DNA was eluted in 80ul ddH2O. DNA concentration was measured using the Qbit DNA Broad Range kit. Genomic DNA (100ng input) was treated with Shrimp Alkaline Phosphatase treatment (NEB, 1U), followed by DpnI digestion (ThermoFisher Scientific, 10U), A-tailing (0.6mM final dATP, 5U Klenow exo-, ThermoFisher Scientific), and UMI adapters ligation (30U T4 DNA ligase, PEG4000, ThermoFisher Scientific) performed within the same tube and buffer (Tango 1X, ThermoFisher Scientific) by heat inactivating each enzymatic step followed by adjustment with the reagents required for the next step. UMI adapters were made by annealing the following oligos: 5’-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T and 5’-pGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT. Ligation reactions were treated with Exonuclease I (20U, ThermoFisher Scientific ) then purified using AMPureXP beads (1:0.8 ratio, Agencourt) and the second sequencing adapter (5’ TGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNN*N 3’, IDT) was tagged using heat denaturation and second strand synthesis (5U T4 DNA Polymerase, ThermoFisher Scientific). The tagging reaction was purified using AMPure XP beads (1:1 ratio) followed by a final library amplification (10 cycles) using 1U of Phusion polymerase, 2μl 10 μM DAM_UMIindex_PCR (5’ AATGATACGGCGACCACCGAGATCTACA*C 3’), and 2μl 10μM NEBnext indexed primer (NEB). Final libraries were purified AMPure XP beads (1:1 ratio) and QCed using Bioanalyser and Qbit. DamC libraries were sequenced on a NextSeq500 (75 cycles single-end) with a custom sequencing protocol (dark cycles at the start of read1 to "skip" the remaining DpnI site TC sequence). Samples index were determined using index1 read, and UMI sequence using index2 read. UMIs are contained in the readname of each read in each fastq file Please note that processed data was generated from multiple samples (as indicated in the corresponding sample description field and in the README.txt) and is linked as Series supplementary file.