Proteomics of centromere repeat in distinct chromatin states reveals pathways to heterochromatin nucleation and transmission

Heritable heterochromatin domain formation is initiated by dynamic changes in the proteome on specialized loci, leading to chromatin modifier recruitment, heterochromatin assembly and propagation. Here we characterize the proteomes associated with a defined heterochromatin nucleation region in three distinct chromatin states: before de novo H3K9 methylation nucleation, following heterochromatin assembly and upon heterochromatin disassembly. Our analyses separate sequence-dependent from sequence-independent processes, providing a systematic examination of the template-driven nucleation and epigenetic wiring underlying heterochromatin transmission. We identify functional interactions that reveal an integral role for the conserved nucleolytic Grc3 complex, and its associated network, as pioneer factors in heterochromatin assembly. More broadly, we demonstrate the power of quantitative proteomics-based strategies for the unbiased scrutiny of the single regions assembled in distinct chromatin states. wt-phet & wt_pcontrol H3K9me2 ChIP-seq