How DNA-Binding Proteins find their Target Sites in Human Cells

Abstract

Precise and timely expression of specific genes is fundamental for the viability of any organism. Specific DNA-binding proteins (transcription factors, TFs) transiently associate to promoter sequences on the genome to activate or repress transcription of specific genes. The way TFs explore the nuclear space, find, and bind to their target sequences is the key to decipher transcriptional control but is still subject of debate. We exploit a live-cell model system to identify what factors primarily orchestrate TFs target search. Our assay is based on human cells containing, at a single locus, specific DNA binding sites (‘target') for an exogenous DNA-binding protein (TetR, ‘searcher'). First, single-particle-tracking experiments revealed that individual TetR proteins move in a very composite way. They transiently interact with nonspecific DNA sequences, via their DNA-binding-domain, with residence times ranging from hundreds of millisecond up to few seconds. Furthermore, a subset of proteins shows confinement within micron-sized regions even when DNA-binding is impaired, which suggests that the organization of mammalian nuclei plays a role in search dynamics. Next, we directly quantified the association rate constant of proteins to their targets in living cells. Based on our binding assay, the measured rate constant resulted much slower than expected and local decondensation of chromatin, at the target site locus, did not affect the association rate. In other words, the effective size of a DNA binding site is much smaller than the size of the molecular interaction. The picture emerging from our experiments shows that, in human cells, specific DNA-binding proteins move in a complex environment where they may remain locally confined. They interact transiently also with nonspecific DNA sequences and their target search mechanism is consistent with a non-compact exploration of the nuclear space with very inefficient binding.