Abstract: DNA is stored inside the cell nucleus at densities approaching crystal packing. Densely packaged DNA—and the genes for which they code—cannot be accessed by the cellular machinery. This requires that DNA can be packaged and unpackaged (turned on and off) during the cell life cycle. The fundamental structure of DNA packaging is the nucleosome, a 10 nm protein “spool” wrapped 1.7 times by DNA. Modified and variant nucleosomes are thought to regulate packaging by “tuning” the strength of DNA binding and/or recruiting cellular machines. To truly understand the regulation of nucleosome packaging, it is essential to deconvolute the effects of other molecular machines from the effects of changes in binding strength. To study this system, we apply force to single molecules of DNA packaged by nucleosomes and measure the kinetics of transitions between nucleosome states of unwinding. Using nucleosomes with modifications leading to known differences in gene expression, 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 gene expression with rates and binding free energy, giving mechanistic meaning to chromatin “loosening” as a change in DNA binding free energy.