Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading.

Heterochromatin formation in Schizosaccharomyces pombe requires the spreading of histone 3 (H3) Lysine 9 (K9) methylation (me) from nucleation centers by the H3K9 methylase, Suv39/Clr4, and the reader protein, HP1/Swi6. To accomplish this, Suv39/Clr4 and HP1/Swi6 have to associate with nucleosomes both nonspecifically, binding DNA and octamer surfaces and specifically, via recognition of methylated H3K9 by their respective chromodomains. However, how both proteins avoid competition for the same nucleosomes in this process is unclear. Here, we show that phosphorylation tunes the nucleosome affinity of HP1/Swi6 such that it preferentially partitions onto Suv39/Clr4’s trimethyl product rather than its unmethylated substrates. Preferential partitioning enables efficient conversion from di-to trimethylation on nucleosomes in vitro and H3K9me3 spreading in vivo. Together, our data suggests that phosphorylation of HP1/Swi6 creates a regime that relieves competition with the “read-write” mechanism of Suv39/Clr4 for productive heterochromatin spreading. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) probing H3K9me2 and H3K9me3 levels in the context of delta swi6, wildtype swi6, or S18A/S24A swi6. Strain background contained our heterochromatin spreading sensor at the MAT locus.

粟酒裂殖酵母（Schizosaccharomyces pombe）的异染色质形成，需要通过H3K9甲基转移酶Suv39/Clr4以及读取蛋白（reader protein）异染色质蛋白1（HP1/Swi6），将组蛋白3（H3）赖氨酸9（K9）甲基化（me）信号从成核中心扩散开来。为完成这一过程，Suv39/Clr4与HP1/Swi6必须既能通过结合DNA与核小体八聚体表面实现非特异性结合，又能通过各自的染色质域（chromodomain）识别甲基化H3K9实现特异性结合。然而，目前尚不清楚这两种蛋白如何避免在该过程中竞争结合同一核小体。本研究证实，磷酸化可调节HP1/Swi6的核小体亲和力，使其优先结合Suv39/Clr4的三甲基化产物，而非非甲基化底物。这种优先分配效应可在体外实现核小体上二甲基化向三甲基化的高效转化，并在体内促进H3K9me3的扩散。综上，我们的数据表明，HP1/Swi6的磷酸化可形成一种调控机制，缓解其与Suv39/Clr4的“读写”机制之间的竞争，从而保障异染色质的高效扩散。本研究通过染色质免疫共沉淀测序（ChIP-seq），检测了Δswi6缺失菌株、野生型swi6菌株以及S18A/S24A突变型swi6菌株中H3K9me2与H3K9me3的水平。所有受试菌株的遗传背景均在MAT基因座处整合了异染色质扩散传感器。