Structural basis of nucleosome transcription mediated by Chd1 and FACT

Abstract

Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through nucleosomes. Efficient Pol II passage through nucleosomes depends on the chromatin remodelling factor Chd11 and the histone chaperone FACT2. How Chd1 and FACT mediate Pol II passage through nucleosomes remains unclear. Here we first show that Chd1 and FACT cooperate with the elongation factors Spt4/5 and TFIIS to facilitate Pol II transcription through a nucleosome in a defined biochemical system. We then determine cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT at 2.9 Å and 3.1 Å resolution, respectively. In the first structure, transcribing Pol II has partially unwrapped nucleosomal DNA and exposed the proximal histone H2A/H2B dimer, which is bound by the acidic N-terminal region of Spt5 (Spt5N). The inhibitory DNA-binding region of Chd1 is released3 and the Chd1 translocase adopts an activated state that is poised to pump DNA towards Pol II. In the second structure, transcribing Pol II has generated a partially unravelled nucleosome that binds FACT in a manner that excludes Chd1 and Spt5N. These results suggest a dynamic model of Pol II passage through a nucleosome. In the model, Pol II enters the nucleosome4, activates Chd1 by releasing its DNA-binding region, and thereby stimulates its own progression. Pol II progression then enables FACT binding, liberates Chd1 and Spt5N, and eventually displaces a complex of FACT with histones that is transferred to upstream DNA.

Spt4 Facilitates the Movement of RNA Polymerase II through the +2 Nucleosomal Barrier

“…Indeed, Spt5 interacts with free DNA in vitro suggesting such dynamics between the Spt4/5 and DNA is also possible (Crickard et al, 2016). Either through an interaction with histones or nucleosomal DNA (or both), our model supports a function for Spt4 facilitating RNAPII movement on the nucleosomal barriers and aligns well with an in vitro model suggesting that together with FACT or Chd1, Spt4/5 contributes to effective RNAPII transcription through a nucleosome (Farnung et al, 2020). This function of Spt4 would also explain why RNAPII accumulates at the upstream face of the nucleosomes.…”

“…This raises the possibility of distinct affinities by these different TEFs for specific conformations of histones with elongating RNAPII, leading to the oscillations. Like histone chaperones, Spt5 bears an acidic domain that is predicted to interact with H2A/H2B during transcription (Ehara et al, 2019;Farnung et al, 2020). Spt4 does not have charged domains, but the affinity of Spt4 for nucleosomes could change indirectly through Spt5.…”

“…Furthermore, the accumulation of RNAPII around 170 nt from the TSS in the absence of Spt4 brings about the possibility of a transcriptional barrier around this point. As in vitro studies have shown that the Spt4/5 complex does not help RNAPII progress over non-nucleosomal transcription barriers (Xu et al, 2020), but does aid RNAPII movement over nucleosomal barriers (Ehara et al, 2019;Farnung et al, 2020), we sought in vivo evidence for this by investigating the change in the distribution on RNAPII relative to nucleosome positions in the absence of Spt4.…”

Characterization of functional disordered regions within chromatin-associated proteins

“…Within the same IDR but C-terminal to the DNA binding region is a stretch that facilitates interaction with HDAC1/2, leading to gene repression. While cryo-EM studies have been carried out on CHD4 in complex with the nucleosome (Farnung et al, 2020), a structure of the MBD2/NURD complex has yet to be completed. This would provide substantial insight into the mechanism of MBD2 IDR function in the proper context.…”

Sophisticated Conversations between Chromatin and Chromatin Remodelers, and Dissonances in Cancer

“…In contrast to Chd1, in which the reorientation of the DBD contributes to unwrapping of the distal DNA, the binding of CHD4 to a nucleosome does not induce DNA unwrapping, an observation consistent with the role of CHD4 in gene repression and heterochromatin formation and maintenance [ 72 ] ( Figure 3 B). Furthermore, the engagement of the RNA Polymerases II (RNAPII) with a nucleosome activates Chd1 by releasing its DBD interaction with extranucleosomal DNA and its contact with the DNA second gyre, leading to the progression of RNAPII through the nucleosome [ 107 , 108 ]. Of note, some of the CHD-subfamily motor subunits harbor additional tandem plant homeodomain (PHD) fingers in their N-termini while others contain a CHD1 helical C-terminal (CHCT) domain in their C-termini [ 109 ] that might further contribute to DNA binding.…”