Kinetics and Thermodynamics of Nucleosome Winding and Unwinding

Abstract

Nucleosome post-translational modifications (PTMs), histone variants, and cofactors can activate and repress transcription of the DNA they package. Modified and variant nucleosomes are thought to regulate transcription by two mechanisms acting independently or in concert: “Tuning” the DNA binding affinity, or recruiting additional cofactors. To truly understand the regulation of nucleosome packaging processes, it is essential to deconvolute the effects of cofactor recruitment from the effects of changes in binding affinity. To distinguish these two mechanisms, we use single molecule optical trap measurements of nucleosomes under force to measure the kinetics of transitions between nucleosome states. Using a system of known phenotypic response (the sin mutant nucleosomes) we find that nucleosome variations affect the transition rates between states of nucleosome unwinding. From these rates, we are able to determine the free energy difference induced by a nucleosome variation, thus correlating a phenotypic response with rates and energy and giving mechanistic meaning to chromatin “loosening”. Our measurement of the energy serves as a benchmark against which the kinetics and free energies of nucleosomes containing histones with PTMs, variants, or cofactors may be compared to in future studies.