Extended Data Fig. 3: CLOCK-BMAL1 competes with chromatin binders that bind both acidic patches.

a, Bar graph showing the number of cross-links obtained for the cryo-EM structures as a function of the obtained cross-link distances. b, Cross-link between histone H3 and CLOCK bHLH lysines (spheres). The cross-linker was DSSO and indicated distances (dashes) are between lysine Cα atoms. c–e, Map density around CLOCK-BMAL1 bHLH (c), interface between CLOCK PAS-B HI loop and H3α1 L1 (d) and PAS domains (e) at position SHL+5.8. The contour levels are 5.98 (c), 5.92 (d) and 5.86 (e). Maps were postprocessed by low-pass filtering or model-based local amplitude scaling (LocScale)⁸⁸. f, Alignment of the CLOCK-BMAL1 bHLH-PAS-AB crystal structure (apo) onto the CLOCK-BMAL1 bHLH-PAS-AB-nucleosome-bound structure at SHL+5.8. The alignment was performed by Needleman-Wunsch using the bHLH residues 29–89 of CLOCK in ChimeraX. The interaction of the PAS domains with the histone octamer is accommodated by flexible linkers (22 residues in BMAL1, 17 residues in CLOCK) connecting the PAS-AB domains and the bHLH domains. g,h, Sequence alignment of CLOCK (g) and BMAL1 (h) proteins across species using a multiple sequence alignment¹¹¹. Amino acid conservation is coloured according to Clustal using JalView¹¹². i, Overlay of CLOCK-BMAL1 at SHL±5.8 with the map of a BAF-bound nucleosome (EMD-0974). j, SDS–PAGE of BAF after size-exclusion chromatography. k, EMSA competition assays between CLOCK-BMAL1 (CB) and BAF. The NCP (20 nM) was incubated with either, BAF only (100 nM), BAF (100 nM) with increasing amounts of CLOCK-BMAL1 (125 nM, 250 nM and 500 nM) or with CLOCK-BMAL1 only (250 nM, 500 nM). Three independent replicates were performed and two representative EMSAs are shown. Asterisk (*) indicates the lane where competition is most evident with the appearance of a CLOCK-BMAL1-NCP complex. l, Model of CLOCK-BMAL1 (at SHL+5.8) and cGAS (PDB: 6y5e) co-binding a nucleosome. m, EMSA competition assays between CLOCK-BMAL1 and the immune signalling sensor cGAs. The NCP was incubated with either CLOCK-BMAL1 (250 nM), CLOCK-BMAL1 with increasing amounts of cGAS (18.75 nM, 37.5 nM, 75 nM and 150 nM) or cGAS (75 nM). 3 independent biological replicates were performed, and one representative replicate is shown. A higher-running band that is likely to correspond to a higher-order CLOCK-BMAL1-cGAS-NCP complex is observed when titrating cGAS to the CLOCK-BMAL1-NCP complex.