Endogenous Halo-tagging of RARα RXRα to characterize their diffusive behaviour.

(A) Schematic showing Type II nuclear receptors (T2NRs) like RXR-RAR bind direct response elements (DREs) as heterodimers to activate or repress transcription by recruiting coactivators (in presence of ligand) or coreporessors (in absence of ligand). A competitive interaction network between the obligate heterodimeric partner RXR with other T2NRs acts as a complex regulatory node for gene expression. Mechanastic features of protein-protein interaction within this regulatory node and its affect on chromatin binding in live cells is yet to be explored. (B) Cartoon showing Halo-tagging scheme of RARα and RXRα alongwith western blots of U2OS wild-type (WT) and knock-in (K.I) RARα (left) and RXRα (right) homozygous clones. (C) Fast single molecule tracking (fSMT). Likelihood of diffusion coefficients based on model of Brownian diffusion with normally distributed localization error for H2B-Halo (black), Halo-NLS (grey), RARα clones (blue) and RXRα clones (red) with black lines on top of the figure illustrating bound and moving polulations. Each line represents a nucleus. (D) Diffusive spectra, probability density function (top) and cumulative distribution function (CDF) (bottom) -with drawing illustrating bound states as heterodimers of RARα and RXRα bound to chromatin.

Chromatin binding of RARα and RXRα can be saturated and is limited by RARα.

(A) Schematic and western blot of stably integrated EF1α promoter driven Halo-tagged (HT) RARα (left) and RXRα (right) overexpression in WT U2OS cells. (B) Bar plot; y-axis shows number of Halo-tagged (HT) knock-in (K.I) and overexpressed (O.E) RARα (blue) and RXRα (red) molecules quantified using flow cytometry. (C) Bar plot; y-axis depicts chromatin bound fraction (fbound%) of K.I and O.E RARα, RXRα compared to Halo-NLS (control). (D) Assay condition schematics to determine which of the partners in the RARα/RXRα hetero-dimer complex is limiting for chromatin binding; parental K.I HT RARα or RXRα clones with O.E SNAP (orange), SNAP-RXRα (brown), RARα-SNAP (light blue), and RARRRα-SNAP (pink) using stably integrated EF1α promoter driven transgene. (E) Bar chart; y-axis denotes number of K.I HT RARα and RXRα molecules (depicted as blue and red cartoon respectively with ‘H’ labelled star attached) in presence or absence of trans-gene products. Error bars denote stdev of the mean from three biological replicates. (F) Bar plot showing fbound% of K.I HT RARa and RXRa in presence or absence of exogenously expresssed SNAP proteins. Error bars for (B), (E) denote stdev of the mean from three biological replicates. Error bars for (C), (F) represent stdev of bootstr-apping mean. P value ≤ 0.001(***), ≤ 0.01(**) & ≤ 0.05 (*).

PAPA-SMT shows direct interaction between Halo-tagged K.I and SNAP-tagged overexpressed RARα and RXRα in live cells.

(A) Schematic illustrates how PAPA signal is achieved. Firstly, SNAP-tagged(ST) protein is labelled with ‘receiver’ fluorophore like JFX650 (star with letter ‘S’) and Halo-tagged (HT) protein is labelled with ‘sender’ fluorophore like JFX549 (star with letter ‘H’). When activated by intense red light the receiver fluorophore goes into a dark state (grey star with S). Upon illumination by green light, the receiver and sender molecules distal to one another do not get photoactivated (red X) but receiver SNAP molecules proximal to the sender gets photoactivated (green √). Pulses of violet light can induce direct reactivation (DR) of receiver independent of interaction with the sender. PAPA experiments, (B) Plots showing PAPA versus DR reactivation, (C) Diffusion spectra of PAPA and DR trajectories obtained for ST proteins for the represented conditions; parental HT RARα knock-in (K.I) cell, stably expressing RXRα-SNAP (brown, left panel), as well as parental HT RXRα K.I cell stably expressing RARα-SNAP (light blue, middle panel) and RARαRR-SNAP (light pink, right panel). A linear increase in PAPA versus DR reactivation is seen for non-interacting SNAP controls and a sublinear increase is seen for interacting SNAP proteins. Respective colored lines show linear fits of the data (see residuals in Figure S8B). SNAP control data are replotted in middle and right panels. Cartoon inside diffusion spectra depicts if the expressed HT and ST proteins are expected to interact or not. fbound errors represent stdev of bootstrapping mean.

A model for RARα limited chromatin binding of RARα-RXRα heterodimers.

(A) Pool of RXRα (red) and RXR partners (RARα – blue, other T2NRs-yellow) along with some number of chromatin bound RARα-RXRα heterodimers exist under normal conditions. (B) When the pool of free RXRα is increased, the number of chromatin bound RARα-RXRα heterodimers does not change. (C) When the pool of RARα is increased, chromatin binding RARα-RXRα heterodimers increases, until it reaches saturation. Note: For simplicity we have omitted to show heterodimerization of other T2NRs (yellow) with RXRα (red).