Single molecule tracking reveals distinct mobility states that contribute to the dynamics and function of nuclear receptors*

Transcription factors (TFs) regulate gene expression by binding to specific consensus motifs within the local chromatin context. The mechanisms by which TFs navigate the nuclear environment as they search for binding sites remains unclear. Single molecule tracking (SMT) has emerged as a powerful approach to explore mechanisms of TF movement and chromatin interactions in living cells. We used SMT in concert with a machine-learning based classification algorithm to directly measure the intranuclear dynamics of nuclear receptors. We apply this framework to study the dynamics of the peroxisome proliferator-activated receptor (PPAR)-γ2, which is an important regulator of glucose metabolism and insulin sensitivity in adipocytes. We show that PPARγ2 exhibits two distinct low mobility states, similar to other nuclear hormone receptors. Through mutagenesis and transient expression studies, we identify PPARγ2 domains that contribute to the dynamics of these two states. Our analyses constrain the physical mechanisms that underlie the origins of these two states and reveal their implications for transcriptional control. Our data provide the first dynamic characterization of PPARγ2 — a TF that is the focus of several anti-diabetes therapeutics.