Nucleosomes, Transcription Factors and the Regulation of Gene Expression: Lessons from Single Molecule Experiments

Gene expression is a dynamic and tightly regulated process which requires continuous association and dissociation of proteins with DNA. In eukaryotic cells, additional complexity exists since the DNA is packaged into chromatin, which reduces the accessibility of regulatory proteins to DNA and interferes with the transcription process. Hence, the cellular machinery needs to constantly regulate the position of nucleosomes to modulate this accessibility, making the dynamics of chromatin an essential and well-studied part of the regulation of gene expression. However, the contribution of DNA sequence, histone variant usage and other factors to shaping the architecture of chromatin, and the mechanisms by which this architecture modulates expression of specific genes, are not yet completely understood. In our work, we use single-molecule optical tweezers experiments to study the roles that DNA sequence and histone variants play in shaping the structure and dynamics of the chromatin at the promoters of two model genes, Cga and Lhb, which encode, respectively, the alpha and beta subunits of luteinizing hormone (LH). We developed a single-molecule assay that allows measurement of the thermal diffusion of nucleosomes at bp precision and seconds to hours timescales. We discovered that although nucleosomes reposition spontaneously, their movement is constrained to ~10 bp. In certain genes, this restriction is overcome by the incorporation of the variant H2A.Z, leading to the production of larger DNA excursions. This distinct and enhanced diffusional properties result in an increase in the exposure of the transcription factor Egr-1 binding site, leading to its association with DNA. Our study highlights how the fundamental physical properties of promoter chromatin are modulated by the usage of local DNA sequence and alternative histone variants to synergistically modulate gene expression.