Loss of H3K9me3 heterochromatin at protein coding genes enables developmental lineage specification (RNA-Seq)

Domains of transcriptionally repressed heterochromatin, decorated by histone 3 lysine 9 trimethylation (H3K9me3), are reduced in embryonic stem cells compared to fully differentiated cells. However, the establishment and dynamics of closed regions of chromatin at protein coding genes, in natural embryologic development, has not been described. We developed a novel, antibody-independent method to isolate and map compacted heterochromatin from low cell number samples. Unexpectedly, we uncovered extensive high levels of H3K9me3-decorated, compacted heterochromatin at protein coding genes in early, uncommitted cells in the three germ layers, undergoing profound rearrangements and reduction upon differentiation, concomitant with cell type-specific gene expression. Perturbation of the three H3K9me3-related methyltransferases revealed that H3K9me3 heterochromatin is required to maintain cell lineage fidelity. We propose a key role for chromatin-based restriction of gene activity via H3K9me3 during embryologic development Study is an analysis by RNA-seq of pancreatic and hepatic development, with a trajectory including definitive endoderm (e8.25 ENDM1+), hepatic precursor (e10.5 Liv2+), and adult hepatocytes as well as pancreatic precursor (e10.5 PDX1+), immature beta cells (e18.5 INS-RFP+) and mature beta cells (8 week INS-RFP+); Ctrl and TKO 1 month old livers; each stage has two replicates; ENDM1+ is treated for some analyses as a baseline condition与完全分化的细胞相比，胚胎干细胞中被组蛋白3赖氨酸9三甲基化（H3K9me3）修饰的转录抑制型异染色质结构域丰度显著降低。然而，自然胚胎发育过程中，蛋白质编码基因位点处染色质闭合区域的建立与动态变化尚未被报道。我们开发了一种全新的无需抗体的方法，可从低细胞数样本中分离并绘制致密异染色质的基因组定位图谱。出乎意料的是，我们在三胚层早期未定型细胞的蛋白质编码基因位点处，发现了广泛分布的高水平H3K9me3修饰致密异染色质；该异染色质在细胞分化进程中发生显著重排并逐渐减少，同时伴随细胞类型特异性基因表达的调控。对三种H3K9me3相关甲基转移酶的功能扰动实验证实，H3K9me3异染色质对于维持细胞谱系保真度不可或缺。我们提出，在胚胎发育过程中，H3K9me3通过染色质介导的基因活性限制机制发挥关键调控作用。本研究通过RNA测序（RNA-seq）分析了胰腺与肝脏的发育进程，覆盖的发育轨迹包括：定型内胚层（e8.25 ENDM1+）、肝脏前体细胞（e10.5 Liv2+）与成年肝细胞，以及胰腺前体细胞（e10.5 PDX1+）、未成熟β细胞（e18.5 INS-RFP+）与成熟β细胞（8周龄INS-RFP+）；此外还包含对照组（Ctrl）与1月龄TKO小鼠肝脏样本；每个实验阶段均设置两个生物学重复；部分分析以ENDM1+作为基线对照条件。