Extended Data Fig. 8: TF–histone contacts are relevant in vitro and in vivo.

a, Scheme of the experiment: MYC-MAX (WT or mutant), labelled with JF549, is injected into flow cells containing immobilized Alexa647-labelled NCPs. Dynamic MYC-MAX binding events are detected by colocalization single-molecule (sm) TIRF imaging. b, Detection of DNA or nucleosome (NCP) localizations using smTIRFM in 640/694 nm channel and single MYC-MAX binding events are detected at DNA positions by smTIRFM in 532/582 nm channel, through a colocalization algorithm. Scale bars: 2 μm. The images are representative of 3 independent experiments. The statistical details for each experiment are listed with the quantification of the signal. c, Extracted fluorescence time trace for 2 nM MYC-MAX WT, showing stochastic binding events to NCPs. d, Fluorescence time trace for MYC-MAX^Y73A,R76A binding to NCPs. e, Dwell-time histogram for MYC-MAX WT binding to NCPs. For fit results, yielding two dwell times (τoff,1; τoff,2) see j, k. f, Dwell-time histogram for MYC-MAX^Y73A,R76A binding NCPs. For fit results, yielding two dwell times (τ_off,1; τ_off,2) see j,k. g, Scheme of the experiment: MYC-MAX (WT or mutant) with Alexa647-labelled DNA. h, Dwell-time histogram for MYC-MAX WT binding to DNA. For fit results, yielding two dwell times (τ_off,1; τ_off,2) see l,m. i, Dwell-time histogram for MYC-MAX^Y76A,R73A binding to DNA. For fit results, yielding two dwell times (τ_off,1; τ_off,2) see l,m. j,k, Dwell times (τ_off,1; τ_off,2) for MYC-MAX WT, MYC-MAX^Y73A,R76A and MYC^S405Y,A408R-MAX binding to NCPs. The indicated numbers are P values (two-tailed Student’s t-test, with n = 4 (MYC-MAX^Y73A,R76A), 7 (MYC-MAX WT) and 4 (MYC^S405Y,A408R-MAX) ([independent experiments]). l,m, Dwell times (τ_off,1; τ_off,2) for MYC-MAX WT, MYC-MAX^Y73A,R76A and MYC^S405Y,A408R-MAX binding to DNA. The indicated numbers are P values (two-tailed Student’s t-test, with n = 3 (MYC-MAX^Y73A,R76A), 6 (MYC-MAX WT) and 3 (MYC^S405Y,A408R-MAX) ([independent experiments]). In j–m the bottom of the boxes defines the first quartile (Q1 or 25th percentile), the middle indicates the median (Q2 or 50th percentile), and the top the third quartile of the data (Q3 or 75th percentile). Whiskers are extended up to the most extreme data point that is no more than 1.5 × IQR. All data points are shown for each box with a mean shown in white. n, Dwell times for MYC-MAX proteins binding to the different substrates. o, The movies were pre-processed with cryoFLARE and the resulting movies were imported in RELION for particle picking. Multiple rounds of 2D and 3D classification (RELION) resulted in a homogenous subset of particles used for the final 3D reconstruction. The boxes defined by dashed line indicate the good models and set of particles used for the following step in the data processing workflow. p, Overlay of the cryo-EM map of the MAX-MAX- (at SHL+5.1 and SHL−6.9) bound nucleosome and the model showing MAX-MAX bound at SHL+5.1. q, Gold-standard FSC curve for the 7 Å resolution map highlighted by the red dashed box shown in o. r, Angular distribution for the particles leading to the 7 Å resolution map. s, Local-resolution filtered map (MonoRes) highlighted by red dashed box shown in o. t, DNA protection analysis at a CLOCK-BMAL1 enhancer by SMF. SMF was performed in mouse liver at a distal enhancer of the gene Por (chr. 5:135674788–135675224). Heat maps displaying protection from GpC methylation on each single DNA molecules at that enhancer, with unprotected/methylated cytosines coloured in yellow, and protected/unmethylated cytosines coloured in green (WT mouse at zeitgeber time (ZT) 6 or blue (Bmal1^−/− at ZT6). Shades of green and blue distinguish three biological replicates for each group. Reads from all 6 animals (n = 1,052 reads per sample) were clustered by the Binary Matrix Decomposition clustering algorithm in a total of 13 clusters. Each column illustrates protection at a single GpC, spanning 327 bp. The arrows at the bottom of the heat maps point to a GpC in a CLOCK-BMAL1 DNA-binding motif (E-box sequence shaded in green). The dashed boxes in clusters C6 and C7 indicate an enhanced protection region immediately upstream of a CLOCK-BMAL1 binding motif, suggesting protection by a nucleosome. For sequencing reads see Supplementary Table 3. Quantification of the percentage of reads ± s.e.m. in clusters C6 and C7 for both wild-type and Bmal1^−/− mice. u, The graph displays the percentage of protection at each GpC for cluster C7, with the lines and shaded area representing the average ± s.e.m. of three biological replicates for wild-type (green) and Bmal1^−/− (blue) mice. v, Genome browser view of BMAL1 ChIP–seq signal at Por gene locus in mouse liver. Sequencing data were retrieved from GSE39860²¹. The arrow and yellow-shaded area point to the distal enhancer analysed by SMF. Zoom in the whole amplicon analysed by SMF (chr5:135674788–135675224), with the blue area indicating the location of CLOCK-BMAL1 DNA-binding motif. w, Schematic representation of predicted DNA-bound proteins corresponding to the observed footprints.