Abstract 2477 The biogenesis and dynamic regulation of chromatin accessibility for transcription

Generation of DNA accessibility in chromatin constitutes the first stage of eukaryotic transcriptional control. The establishment and maintenance of DNase hypersensitive, Nucleosome Depleted Regions (NDRs) at promoter and enhancer DNA elements require collaboration between ATP-dependent chromatin remodeling complexes that mobilize nucleosomes, and so-called 'pioneer' transcription factors (TFs) that are unimpeded by nucleosome architecture. We use single-molecule fluorescence microscopy to directly observe and quantify protein kinetics in vitro and in living cells, in real time. The results show that chromatin remodelers bind to chromatin transiently and frequently with dwell times of several seconds in live cells. Integration of factor kinetics with changes in chromatin accessibility upon remodeler depletion suggests that NDRs undergo dynamic length expansion and contraction, providing a highly transient window of DNA accessibility at promoters and enhancers on a seconds-timescale for recruitment of the eukaryotic transcription machinery. 'Pioneer' TFs such as yeast General Regulatory Factors Reb1 and Abf1, and Drosophila GAGA factor (Trithorax-like) exhibit dwell times of ∼100 seconds on genomic targets in live cells, an order of magnitude longer than the binding of other TFs. Furthermore, despite microscale on-off kinetics, GAGA factor persists on chromatin at very high factor occupancies for extended time periods. This high occupancy is dependent on its expressed abundance, the DNA binding, multimerization and intrinsically disordered Q-rich domains, the local clustering of cognate binding sites, and intrinsic nucleosome affinity. Importantly, GAGA factor physically recruits ISWI and SWI/SNF remodeling enzymes to increase chromatin accessibility for TF neighbors. The corresponding NDRs prevail throughout Drosophila development, enabling timely recruitment of other TFs in response to physiological and environmental signaling. This work was supported by grants from the National Institutes of Health R01GM132290, R01GM145844, and R35GM149291.