Epigenetic priming of embryonic enhancer elements coordinates developmental gene networks [CUT&TAG]

Embryonic development requires the accurate spatiotemporal execution of cell lineage-specific gene expression programs, which are controlled by transcriptional enhancers. Developmental enhancers adopt a primed chromatin state prior to their activation; however how this primed enhancer state is established, maintained, and how it affects the regulation of developmental gene networks remains poorly understood. Here, we use comparative multi-omic analyses of human and mouse early embryonic development to identify subsets of post-gastrulation lineage-specific enhancers which are epigenetically primed ahead of their activation, marked by the histone modification H3K4me1 within the epiblast. We show that epigenetic priming occurs at lineage-specific enhancers for all three germ layers, and that epigenetic priming of enhancers confers lineage-specific regulation of key developmental gene networks. Surprisingly in some cases, lineage-specific enhancers are epigenetically marked already in the zygote, weeks before their activation during lineage specification. Moreover, we outline a generalisable strategy to use naturally occurring human genetic variation to delineate important sequence determinants of primed enhancer function. Our findings identify an evolutionarily conserved program of enhancer priming and begin to dissect the temporal dynamics and mechanisms of its establishment and maintenance during early mammalian development. Overall design: CUT&Tag libraries were generated according to the EpiCypher protocol (Kaya-Okur et al., 2019) with minor modifications. As described for ATAC-seq, hiPSCs were harvested with accutase (StemCell Technologies; 07922). After resuspension in 1X DPBS (Life Technologies; 14190144), 2 x 105 cells per CUT&Tag reaction underwent centrifugation at 300 x g for 3 min at RT. Cells were then lysed in 100µl of cold Nuclear Extraction (NE) Buffer (20mM HEPES–KOH, pH 7.9, 10mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5mM Spermidine (Sigma-Aldrich; 05292), 1x cOmpleteTM, Mini, EDTA-free Protease Inhibitor (Roche; 11836170001)) per CUT&Tag reaction for 10 min on ice. The nuclei were then centrifuged for 3 min at 600 x g at RT before resuspension in cold NE Buffer to achieve a final concentration of 1.2x106 nuclei/ml. For each CUT&Tag reaction, 11µl of concanavalin A (ConA) beads (EpiCypher; 21-1401) were washed twice on a magnetic stand with 100 µl of Bead Activation (BA) Buffer (20mM HEPES, pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2). The beads were then resuspended in 11µl of BA Buffer, and 10µl beads were aliquoted into a 0.2ml tube. Each tube of activated ConA beads was then incubated with 100µl of nuclei for 10 min at RT. The supernatant was then removed with a magnet and the beads were resuspended in 50µl of cold Antibody150 Buffer (20mM HEPES, pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x cOmpleteTM, Mini, EDTA-free Protease Inhibitor, 0.01% digitonin (Sigma-Aldrich, D141-100MG), 2mM EDTA) containing a 1:50 dilution of rabbit primary antibody (anti-H3K4me1 (Active Motif; 39298) or anti-H3K27ac (Abcam; ab4729)) and incubated overnight at 4°C on a rocker. The following day, the supernatant was discarded with a magnet and the beads were incubated in 50µl of Digitonin150 Buffer (20mM HEPES, pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x cOmpleteTM, Mini, EDTA-free Protease Inhibitor, 0.01% digitonin) containing 0.5µg of anti-rabbit secondary antibody (Epicypher; 13-0047) for 1 hour at RT. The beads were then washed twice on a magnet with Digitonin150 Buffer, then incubated in 50µl of Digitonin300 Buffer (20mM HEPES, pH 7.5, 300mM NaCl, 0.5mM Spermidine, 1x cOmpleteTM, Mini, EDTA-free Protease Inhibitor, 0.01% digitonin), and 1.25µl of CUTANA pAG-TN5 (EpiCypher; 15-1017) for 1 hour at RT. The beads were then washed twice with a magnet by resuspension in Digitonin300 Buffer, then incubated in 50µl of chilled Tagmentation Buffer (Digitonin 300 Buffer, 10mM MgCl2) for 1 hour at 37°C. The supernatant was then discarded with a magnet and the beads were washed once with 50µl RT TAPS Buffer (10mM TAPS, pH 8.5, 0.2mM EDTA). The supernatant was again removed with the magnet and the beads were resuspended in 5µl of RT SDS Release Buffer (10mM TAPS, pH 8.5, 0.1% SDS) and vortexed on maximum speed for 10 sec followed by a brief centrifugation. The beads were then incubated for 1 hour at 58°C before the addition of 15µl of RT SDS Quench Buffer (0.67% Triton-X 100 in Molecular grade H2O), vortexing at maximum speed for 10 sec, and a brief centrifugation. For library amplification, samples were combined with 2µl of 5µM i5 primer, 2µl of 5µM i7 primer and 25µl of CUTANA High Fidelity PCR mix (EpiCypher; 15-1018). The following PCR programme was then used for all libraries: 58°C for 5 min, 72°C for 5 min, and 98°C for 45 sec. This was followed by 11 cycles for anti-H3K27ac and 13 cycles for anti-H3K4me1 of [ 98°C for 15 sec and 60°C for 10 sec], before a final extension at 72°C for 1 min. Libraries then underwent two 1X AMPure XP bead (Beckman Coulter; A63881) cleanups. Following QC on a Bioanalyzer, libraries were multiplexed and sequenced (paired-end 150 bp) using a NovaSeq 6000 instrument (Illumina).