Chromatin Structure and Gene Expression

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

1. Reprogramming of the chromatin landscape is a critical component to the transcriptional response in breast cancer. Effects of sex hormones such as estrogens and progesterone have been well described to have a critical impact on breast cancer proliferation. However, the complex network of the chromatin landscape, enhancer regions and mode of function of steroid receptors (SRs) and other transcription factors (TFs), is an intricate web of signaling and functional processes that is still largely misunderstood at the mechanistic level. We are exploring the dynamic interplay between TFs with chromatin and the reprogramming of enhancer elements. 2. We are adapting recent advances in live cell imaging technology to study the function of T factors in single cells in real time. Our approaches enable the coupling of population-based assays with real-time studies to address many unsolved questions about SRs and chromatin dynamics in normal mammalian cells, as well as cancer cells. 3. Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs-one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). We presented an analytical pipeline to evaluate which model best explains SMT data. We found that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we proposed a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template. 4. 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 remain unclear. We used single-molecule tracking and machine-learning based classification to directly measure the nuclear mobility of the glucocorticoid receptor (GR) in live cells. We discoverd two distinct and dynamic low-mobility populations. One accounts for specific binding to chromatin, while the other represents a confinement state that requires an intrinsically disordered region (IDR), implicated in liquid-liquid condensate subdomains. Further analysis showed that the dwell times of both subpopulations follow a power-law distribution, consistent with a broad distribution of affinities on the GR cistrome and interactome. Altogether, our findings link IDRs with a confinement state that is functionally distinct from specific chromatin binding and modulates the transcriptional output by increasing the local concentration of TFs at specific sites.