PhD student Penn State University State College, Pennsylvania, United States
Mai Huynh (Penn State University)| Satya Yadav (Penn State University)| Joseph Reese (Penn State University)| Tae-Hee Lee (Penn State University)
Aims: Nucleosomes impose physical barriers on enzymes translocating along nucleosomal DNA such as RNA Polymerase II (Pol II). As such, nucleosome dynamics is a molecular basis of gene regulation. We investigated nucleosome dynamics during transcription elongation by Pol II with three specific aims to 1) elucidate how lysine acetylation at histone H3K56 affects spontaneous DNA dynamics at the nucleosome termini during transcription elongation, 2) elucidate the (sub-)nucleosome structure after transcription elongation in both wild-type and H3K56 acetylated nucleosomes and 3) elucidate how histone chaperone Nap1 affects nucleosome dynamics during and after transcription elongation by Pol II.
Methods: We used two- and three-color single-molecule FRET (smFRET). A nucleosome-containing transcription template EC-42 were constructed by ligating DNA fragments followed by nucleosome assembly. DNA was labeled with a FRET pair Cy3 and Cy5 that reports DNA unwrapping and rewrapping during and after transcription elongation in wild-type and H3K56 acetylated nucleosomes with and without Nap1. Histone H2B was labeled with Cy5.5 to introduce a second FRET acceptor to report the histone H2A-H2B dynamics simultaneously with DNA dynamics.
Results: H3K56 acetylation (H3K56ac) significantly shortens the pause duration of Pol II near the entry region of the nucleosome, while Nap1 induces no noticeable difference. The elongation rate of Pol II through the nucleosome is unaffected by H3K56ac or Nap1. H3K56ac facilitates transcription elongation by inducing more frequent larger spontaneous DNA opening at the entry region, which in turn facilitates Pol II to resume from a pause, and that Nap1 does not affect Pol II dynamics. Following transcription, only 5 % of the nucleosomes remain intact, which is unaffected by H3K56 acetylation or Nap1.
Conclusions: These results suggest that 1) spontaneous nucleosome opening enables Pol II progression, that 2) Pol II mediates nucleosome re-assembly very inefficiently, and that 3) Nap1 in the absence of other factors does not promote nucleosome disassembly or reassembly during Pol II transcription. This study warrants further investigation into whether extended DNA at the exit side of the nucleosome would make Pol II stay longer with the nucleosome and facilitate nucleosome re-assembly.