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. 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).

胚胎发育依赖细胞谱系特异性基因表达程序的精准时空执行，而此类程序由转录增强子（transcriptional enhancers）调控。发育增强子在激活前会处于预激活染色质状态，但目前学界对该预激活增强子状态的建立、维持机制，以及其对发育基因网络调控的影响仍知之甚少。
本研究通过对人类和小鼠早期胚胎发育开展比较多组学分析，鉴定了原肠胚形成后谱系特异性增强子的亚群：此类增强子在激活前即已发生表观遗传预激活，以上皮细胞中的组蛋白修饰H3K4me1作为标记。研究发现，三个胚层的谱系特异性增强子均存在表观遗传预激活现象，且增强子的表观遗传预激活可实现关键发育基因网络的谱系特异性调控。令人意外的是，部分案例中谱系特异性增强子早在受精卵阶段就已被表观遗传标记，较其在谱系特化阶段的激活时间早达数周。此外，本研究提出了一种可推广的策略，可利用自然存在的人类遗传变异来解析预激活增强子功能的关键序列决定因子。本研究的发现揭示了一套进化保守的增强子预激活程序，并开始解析哺乳动物早期发育过程中增强子预激活的时间动态及其建立与维持的机制。
ATAC-seq（转座酶可及性测序测定，Assay for Transposase-Accessible Chromatin using sequencing）文库的构建参照Corces等人2017年的研究方法，并进行了小幅优化。先用1×杜氏磷酸缓冲盐溶液（Dulbecco's Phosphate-Buffered Saline, DPBS；Life Technologies；14190144）洗涤细胞一次，随后采用accutase细胞解离液（StemCell Technologies；07922）在37℃孵育5分钟以收集人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）。用TeSR-E8培养基中和accutase后，将细胞于室温以300×g离心3分钟。重悬于1×DPBS后，取50,000个细胞于4℃下以500×g在转头式离心机中离心5分钟，随后置于冰上用50μl冰冷的ATAC重悬缓冲液裂解3分钟，该缓冲液配方为：10mM TrisHCl pH 7.5、10mM NaCl、3mM MgCl₂，且含有0.1% IGEPAL-630、0.1% Tween-20及0.01%洋地黄皂苷。随后向裂解液中补加1ml含0.1% Tween-20的冰冷ATAC重悬缓冲液，于4℃下以500×g在转头式离心机中离心10分钟。将沉淀重悬于50μl转座反应混合液，该混合液包含：1×Illumina Tagment DNA缓冲液、100nM Illumina TDE1 Tagment DNA转座酶（Illumina；20034197）、0.33×DPBS、0.1% Tween-20及0.01%洋地黄皂苷。随后将体系置于37℃、1000 RPM振荡条件下孵育30分钟。之后使用Zymo DNA Clean & Concentrator-5试剂盒（Zymo Research；D4003）纯化样品，用21μl洗脱缓冲液洗脱，再加入2.5μl 25μM i5引物、2.5μl 25μM i7引物及25μl 2×NEBNext高保真PCR预混液（NEB；M0541S）。随后按照以下程序进行PCR扩增：72℃孵育5分钟，98℃变性30秒，随后进行8个循环[98℃变性10秒、63℃退火30秒、72℃延伸1分钟]。样品再次经Zymo DNA Clean & Concentrator-5试剂盒纯化，用30μl洗脱缓冲液洗脱后，再进行1.2×AMPure XP磁珠（Beckman Coulter；A63881）纯化。在Bioanalyzer上完成质控后，将文库进行多重化处理，采用HiSeq 2000测序仪（Illumina）进行双端50bp测序。