Epigenetic priming of embryonic enhancer elements coordinates developmental gene networks [ATAC-seq]
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: ATAC-seq libraries were generated as described in (Corces et al. 2017), with minor modifications. After washing once with 1X DPBS (Life Technologies; 14190144), hiPSCs were harvested with accutase (StemCell Technologies; 07922) by incubation for 5 min at 37°C. After accutase neutralisation with TeSR-E8 medium cells were centrifuged at 300 x g for 3 min at RT. After resuspension in 1X DPBS, 50,000 cells underwent centrifugation at 500 x g for 5 min at 4°C in a swing arm rotor centrifuge, then lysis in 50µl of cold ATAC Resuspension Buffer (10mM TrisHCl pH 7.5, 10mM NaCl, 3mM MgCl2) containing 0.1% IGEPAL-630, 0.1% Tween-20, and 0.01% digitonin for 3 min on ice. The lysate was then topped up with 1ml cold ATAC Resuspension Buffer containing 0.1% Tween-20 and centrifuged at 500 x g for 10 min at 4°C in a swing arm rotor centrifuge. The pellet was then resuspended in 50µl of transposition mixture (1X Illumina Tagment DNA Buffer and 100nM Illumina TDE1 Tagment DNA Enzyme transposase (Illumina; 20034197), 0.33X DPBS, 0.1% Tween-20, 0.01% digitonin) and incubated at 37°C for 30 min with 1000 RPM mixing. After undergoing purification with the Zymo DNA Clean & Concentrator-5 kit (Zymo Research; D4003), samples were eluted in 21µl of elution buffer, and combined with 2.5µl of 25µM i5 primer, 2.5µl of 25µM i7 primer, and 25µl 2X NEBNext High-Fidelity 2X PCR Master Mix (NEB; M0541S). A PCR was then performed with the following conditions: 72°C for 5 min, 98°C for 30 sec, 8 cycles of [98°C for 10 sec, 63°C for 30 sec, 72°C for 1 min]. The samples then underwent a second Zymo DNA Clean & Concentrator-5 purification and were eluted in 30µl of elution buffer prior to a 1.2X AMPure XP bead (Beckman Coulter; A63881) cleanup. Following QC on a Bioanalyzer, libraries were multiplexed and sequenced (paired-end 50bp) using a HiSeq 2000 instrument (Illumina).