The nuclear matrix protein SAFB maintains heterochromatin architecture through RNA-dependent phase separation [RIP-seq]

Eukaryotic chromosomes fold into topologically associating domains (TADs), which further gather in active (A) or inactive (B) genomic compartments. Here we show that Scaffold Attachment Factor B (SAFB), a nuclear matrix-associated protein with RNA binding functions, modulates global chromatin condensation in a dosage-dependent manner. Upon the depletion of SAFB, the genome coverage of Lamina-associated-domains (LADs) decreases from 53.33% to 45.93%, and both inter- and intra-TAD chromatin-chromatin interactions in compartment B decrease significantly. In the nucleus, SAFB favorably co-localizes with H3K9me3, a marker of heterochromatin, and the disruption of SAFB leads to a collapse of H3K9me3 foci. Furthermore, we show that SAFB proteins can form liquid-liquid phase separation in the cell and in vitro. Intriguingly, SAFB interacts with RNAs from repetitive elements enriched in heterochromatin (e.g., Major Satellites and LINE1), which can promote SAFB-mediated phase separation, depending on the density of SAFB recognizing motifs in the RNAs. Overall design: Identification and characterization of RNAs associated with SAFB in AML12 cells

真核染色体折叠形成拓扑关联结构域（topologically associating domains, TADs），后者可进一步聚集为活性（A）或非活性（B）基因组区室。本研究证实，兼具RNA结合功能的核基质相关蛋白——支架附着因子B（Scaffold Attachment Factor B, SAFB），以剂量依赖方式调控全局染色质凝聚。在SAFB被敲低后，核纤层关联结构域（Lamina-associated-domains, LADs）的基因组覆盖度从53.33%降至45.93%，且B区室中TAD间与TAD内的染色质相互作用均显著下调。在细胞核内，SAFB倾向于与异染色质标记物组蛋白H3赖氨酸9三甲基化（H3K9me3）共定位；SAFB功能缺失会导致H3K9me3焦点结构崩解。此外，本研究证实SAFB蛋白可在细胞内及体外发生液液相分离。值得注意的是，SAFB可与异染色质富集的重复序列元件（如主要卫星序列与长散在核元件1, LINE1）所转录的RNA结合，且此类RNA可促进SAFB介导的液液相分离，这一过程依赖于RNA上SAFB识别基序的密度。整体实验设计：鉴定并表征AML12细胞中与SAFB结合的RNA。