Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin. These classifications are more accurate than the histone marks alone in predicting transcriptional silence and resistance of alternate fate genes to activation during direct cell conversion. Our proteomics of H3K9me3-marked srHC and functional screens revealed diverse proteins, including RBMX and RBMXL1, that impede gene induction during cellular reprogramming. Isolation of srHC with gradient-seq provides a genome-wide map of chromatin structure, elucidating subtypes of repressed domains that are uniquely predictive of diverse other chromatin properties.

异染色质通过阻止改变细胞命运的基因激活来维持细胞身份。异染色质区域与组蛋白 3 赖氨酸 9 三甲基化 (H3K9me3) 或 H3K27me3 相关，但这些修饰也存在于允许转录的常染色质区域中。我们发现，对超声处理的抗性是异染色质状态的可靠指标，并且我们开发了一种生物物理方法（梯度测序）来区分抗超声处理异染色质 (srHC) 和常染色质中的 H3K9me3 和 H3K27me3 结构域亚型。在预测直接细胞转化过程中的转录沉默和替代命运基因对激活的抵抗力方面，这些分类比单独的组蛋白标记更准确。我们对 H3K9me3 标记的 srHC 的蛋白质组学和功能筛选揭示了多种蛋白质，包括 RBMX 和 RBMXL1，它们在细胞重编程过程中阻碍基因诱导。使用梯度序列分离 srHC 提供了全基因组染色质结构图谱，阐明了抑制域的亚型，这些亚型可以独特地预测多种其他染色质特性。