“Stripe” transcription factors provide accessibility to regulatory DNA in mammalian genomes_1
Single molecule raw data is visualized with ImageJ software. Further analysis is required for the trajectory generation, residence time and mean square displacement (MSD) of single molecules. To generate the trajectories in Figure 7A and S7A, single molecules are localized and tracked using SLIMfast and evalSPT, implementing the Multiple-Target-Tracing algorithm for localizing and tracking single molecules. Single molecules are first localized with 2D Gaussian fitting subject to a log-likelihood ratio test with a localization error. A maximal expected diffusion constant was set to connect localizations between consecutive frames. To perform the residence time analysis for Figure 7B,C,D and Figure S7C,D, the dwell time was obtained by calculating the ensemble distribution of bound times for Smad3 and Smad7 in different cells from each biological replicate and corrected by dividing the exponential component estimated in the H2B dwell time distribution analysis (S(t)=e^γt S_E (t), where S(t) corresponds to the survival distribution after photobleaching correction. To perform the MSD analysis in Figure S7B, Perturbation Expectation Maximation (pEM) together with Bayesian Inference Criterion (BIC) was used to classify the trajectories of the protein into the least number of diffusive modes (sub-diffusion, diffusion and super-diffusion). The number of reinitializations were set up to 10, number of perturbations to 50, maximum number of iterations to 10000, convergence criteria for change in log-likelihood to 1e-7 and the number of features of the covariance matrix to 3. The posterior probability weighted MSD for each diffusive state was computed. To calculate the diffusion coefficient (D) for a diffusive state (Brownian motion), the variance of the instantaneous velocity vector v was related to the diffusion coefficient as 〈v^2 〉=4D/∆t , ∆t is the acquisition interval and D is the diffusion coefficient of the particle.