Extended Data Fig. 3: The Nucleosome Destabilization model predicts rTetR-VP48 binding in chromatin.

A) Relationship between average TF occupancy and ATAC-seq signal (RPM) for a selection of single enhancer amplicons from multiple backgrounds. Each dot represents a single enhancer. Error bars represent the 95% CI from four (SMF) or two (ATAC-seq) biological replicates. B) Schematic describing how rTetR-VP48 is installed with PiggyBac transposase and expressed in K562 cells, selected with hygromycin, and assayed for activity with mCherry under a dox-responsive promoter. C) Flow cytometry distributions for mCherry fluorescence with and without dox for two technical replicates. D) Histogram of the predictions of number of TFs bound per molecule for the enhancer with 7 TetO sites from the Simple Competition model (gray) fit to the binding data of rTetR-VP48 and these predictions with Gaussian noise (SNR = 1) added to TF energy (black), effectively varying the concentration of the TF per cell/molecule (see Methods). E) Average rTetR-VP48 occupancy with variable TetO number at 24 and 48 h with dox. F) Probability of each TetO site being occupied on nucleosome-free molecules for the enhancer with six TetO sites (two biological replicates). Dashed line indicates the average occupancy. G) Fraction of molecules with >0 TFs bound for enhancers with increasing TetO number (four biological replicates) fit to either the Simple Competition (r² = 0.44) or Nucleosome Destabilization (r² = 0.99) models. H) Occupancy distributions across all enhancers (left) with matching simulations from the Nucleosome Destabilization model (right). Each row represents a single enhancer and each column represents the number of TFs bound. The pixel intensity at (row i, column j) indicates the fraction of molecules with i TetO sites that have j TFs bound. Rows sum to 1. I) Full molecular state representations for 10,000 measured (left) or simulated (according to the Nucleosome Destabilization, right) molecules with six TetO sites, where each column represents a TetO site and each row represents a molecule. Sites are colored by their occupancy status. J) Best-fit parameters for binding of rTetR-VP48 using the Nucleosome Destabilization model (four biological replicates). K) Three alternate TF cooperativity models (left) and their fits to average occupancy and occupancy distributions (right) for rTetR-VP48.