Extended Data Fig. 1: SNAP experiments reveal differential responses of chromatin readers to nucleosomal modification signatures.

a, SILAC Nucleosome Affinity Purifications (SNAP). For SNAP experiments modified nucleosomes were immobilized on streptavidin beads and incubated with nuclear extracts from HeLa S3 cells grown in isotopically light (R₀K₀) or heavy (R₁₀K₈) SILAC media. In ‘forward’ experiments heavy extracts were incubated with modified and light extracts with unmodified nucleosomes, while in ‘reverse’ experiments the extracts were exchanged. Bound proteins were eluted using an on-bead digestion protocol and identified and quantified by mass spectrometry. For each SNAP experiment the SILAC ratios Heavy/Light (Ratio H/L) of the forward and reverse experiment of the identified proteins were measured and plotted in a logarithmic (log₂) graph (see Fig. 1c and panels b-d). The H/L ratios indicate binding preferences to the modified or the unmodified nucleosomes and allow the unbiased identification of proteins that are either recruited or excluded by the modifications, in addition to proteins that bind nucleosomes but do not show a strong response to the modifications. b, Exemplary SNAP experiment with H3K27me3-modified di-nucleosomes. The results show that the ORC subunit ORC2 and the PRC2 subunit EZH2 are recruited by the H3K27me3 modification as previously reported^25,74. c, SNAP experiment with H3K4me3- and H4K16ac-modified di-nucleosomes. This modification pattern recruits the H3K4me3 reader PHF8³² but excludes EZH2 through loss of PRC2 binding to the N-terminus of histone H3³³. d, SNAP experiment with di-nucleosomes combining di-methylation of lysine 20 and acetylation of lysines 5, 8, 12, and 16 on histone H4 (H4acK20me2). This nucleosome strongly recruits BRD4 through its interaction with H4ac via its bromodomains⁷⁵ as well as the ORC subunit ORC2 through recognition of H4K20me2 via ORC1⁷⁶. e. Results for SNAPs with the entire library of 55 modified di-nucleosomes. Tracking the signals of BRD4, EZH2, ORC2, and PHF8 as highlighted in b-d allows interrogation of their responses to the different modification signatures. The order of SNAP experiments corresponds to the order of di-nucleosomes shown in Fig. 1d.