FACT weakens the nucleosomal barrier to transcription and preserves its integrity by forming a hexasome-like intermediate.

Transcription of yeast RNA polymerase II through nucleosomes requires the assistance of the histone chaperone FACT (facilitates chromatin transcription). Yet, how FACT modulates the nucleosomal mechanical barrier to affect the polymerase's elongation dynamics is poorly understood. Using high-resolution single-molecule optical tweezers, we show that FACT greatly decreases the magnitude of the barrier by favoring the unwrapping of DNA from the distal H2A-H2B dimer, which, in turn, weakens the contacts near the dyad, significantly reducing the enzyme's crossing time. We show that barrier crossing depends on the asymmetric flexibility of the nucleosome arms, an asymmetry we find across the genome. Mechanical unwrapping of Cy3-H2A nucleosomes reveals that FACT reduces their unwrapping force and stabilizes a hexasome-like intermediate that retains both labeled dimers during successive unwrapping cycles. This intermediate is also observed after transcription. In conclusion, FACT facilitates nucleosomal transcription by weakening the barrier and actively assisting the maintenance of nucleosomal integrity after enzyme passage.