SPACE exploration of RNA-binding proteins as major chromatin components

Many RNA-binding proteins (RBPs) play roles in regulating co-transcriptional RNA-processing, transcription and DNA damage response, but our knowledge of the repertoire of chromatin-associated RBPs (caRBPs) and their molecular functions remains limited. This is partly because the reliable and efficient identification of chromatin-associated proteins is technically challenging. Here, we present SPACE (Silica Particle Assisted Chromatin Enrichment), a rapid and highly reproducible method that works with as few as 20,000 formaldehyde-crosslinked cells. Applied to mouse embryonic stem cells, SPACE identified 1,825 chromatin-associated proteins, 40% of which are annotated as RBPs; thus, an unexpectedly large proportion of proteins hold dual roles in chromatin and RNA-binding. To detect the specific protein regions that interfaces with chromatin, we developed a highly stringent supplemental method, SPACEmap, by incorporating double tryptic digestion. We defined such regions in 596 caRBPs, which predominantly bind chromatin via intrinsically disordered regions (IDRs) that are missed in crystal structures, thus highlighting the multivalent interactions that exist between RBPs and chromatin components. Finally, we applied comparative SPACE to ground and meta-stable states of mouse embryonic stem (mES) cells, which identified striking changes of several RBPs in the chromatome, including Dazl. Collectively SPACE reveals an unprecedented repertoire of caRBPs and provides a useful tool for studying chromatin-protein networks in future.