FACT promotes efficient Pol II transcription by modulating the topographic features of the nucleosomal barrier

In eukaryotic cells, efficient transcription through nucleosomes by Pol II requires the help of several transcriptional regulators including the histone chaperon FACT (FAcilitates Chromatin Transcription). However, mechanistic details of how FACT modulates the magnitude of the mechanical barrier of a nucleosome and how this factor affects the elongation dynamics of the enzyme are poorly understood. Here, using a high-resolution single-molecule optical tweezers (SM-OT) instrument, we characterize the dynamics of Pol II during FACT-mediated nucleosome transcription. Our results show that FACT greatly increases the probability that Pol II crosses the nucleosome and greatly reduces its crossing time. FACT also significantly decreased the duration of both major Pol II pause sites at SHL-5 and -1 by weakening the strong DNA/H2A-H2B dimer contacts and favoring the unwrapping of the distal DNA. Interestingly, the mechanical unwrapping of H2A-Cy3 nucleosomes using OT combined with SM fluorescence detection shows that FACT allows the retention of both H2A-H2B dimers during nucleosome disassembly. Altogether our data suggest that FACT facilitates nucleosome transcription by modifying the strength and the transcriptional map features of the nucleosome barrier, while it promotes nucleosome reassembly in the wake of Pol II passage by its ability to keep the H2A-H2B dimers associated to the nucleosome.

Speaker: Carlos Bustamante, UC Berkeley