H3K9me3 heterochromatin, established by lysine methyltransferases (KMTs) and compacted by heterochromatin protein 1 (HP1) isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3 heterochromatin stability is presently limited to individual domains and DNA repeats. Here we engineered Suv39h2-knockout mouse embryonic stem cells to degrade remaining two H3K9me3 KMTs within 1 hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12–24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A ‘binary switch’ governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMT depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening and exit from pluripotency within 12 h. Unexpectedly, receding H3K9me3 domains unearth residual HP1β peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3 heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency.

H3K9me3 异染色质由赖氨酸甲基转移酶 （KMT） 建立并被异染色质蛋白 1 （HP1） 亚型压缩，可抑制替代谱系基因和 DNA 重复序列。我们对 H3K9me3 异染色质稳定性的理解目前仅限于单个结构域和 DNA 重复序列。在这里，我们改造了 Suv39h2 敲除小鼠胚胎干细胞，使其在 1 小时内降解剩余的两个 H3K9me3 KMT，并发现被动稀释和主动去除都会导致 H3K9me3 在 12-24 小时内腐烂。我们发现了基因组中的四种不同的 H3K9me3 衰变速率以及预测稳定性类别的染色质特征和转录因子结合模式。“二元开关”控制异染色质压缩，HP1 在 KMT 耗竭后迅速与异染色质解离，并且 HP1 的特定阈值水平限制了先锋因子结合、染色质在 12 小时内打开和退出多能性。出乎意料的是，后退的 H3K9me3 结构域发现了富含异染色质诱导蛋白的残留 HP1β 峰。我们的研究结果揭示了控制基因网络的不同 H3K9me3 异染色质维持动力学，并重复它们共同保护多能性。