Structure of complete Pol II–DSIF–PAF–SPT6 transcription complex reveals RTF1 allosteric activation

Vos, Seychelle M.; Farnung, Lucas; Linden, Andreas; Urlaub, Henning; Cramer, Patrick

doi:10.1038/s41594-020-0437-1

Cited by 125 publications

(126 citation statements)

References 77 publications

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“…di-nucleosomes) together with Chd1, RNAPII, and perhaps other elongation factors. Several cryo-EM structures have recently been solved that should help in that venture (Ehara et al, 2019;Ehara et al, 2017;Farnung et al, 2018;Farnung et al, 2017;Kujirai et al, 2018;Liu et al, 2020;Vos et al, 2018;Vos et al, 2020;Xu et al, 2017). Collectively, the work presented here ties previous in vitro information to a relevant in vivo context (transcription) and provides a ground state for the dissection of FACT chaperoning mechanisms, both in vivo and in vitro.…”

Section: Discussionmentioning

confidence: 81%

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

Jeronimo

Angel

Poitras

et al. 2020

Preprint

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The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models for how FACT finds its way to transcriptionally active chromatin include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails and binding partially disassembled nucleosomes. Here, we systematically tested these and other scenarios in Saccharomyces cerevisiae and found that FACT binds transcribed chromatin, not RNAPII. Through a combination of experimental and mathematical modeling evidence, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.

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Section: Discussionmentioning

confidence: 81%

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

Jeronimo

Angel

Poitras

et al. 2020

Preprint

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“…Consistent with previous observations (data not shown), TFIIS is lost from the elongation complex during size exlusion chromatography. Fractions containing the complex were individually cross-linked and dialyzed against dialysis buffer (100 mM NaCl, 20 mM Na•HEPES pH 7.4, 3 mM MgCl2, 1 mM TCEP) for three hours as described previously 45 . The dialyzed complexes with an approximate concentration of 50-100 nM were applied to R2/2 gold grids, Au 200 mesh (Quantifoil).…”

Section: Reconstitution Of Transcribing Pol Ii-nucleosome Com-mentioning

confidence: 99%

Structural basis of nucleosome transcription mediated by Chd1 and FACT

Farnung

Ochmann

Engeholm

et al. 2020

Preprint

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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.

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“…In the absence of Paf1, RNAPII accumulates at the downstream face of the +2 nucleosome ( Figure S7C, D). Paf1C is a TEF complex generally associated with productive elongation as it takes part in co-transcriptional histone PTMs (Van Oss et al, 2017) and increases the processivity of RNAPII in vitro (Vos et al, 2020). Therefore, the movement of RNAPII through the upstream face of the +2 nucleosome might rely more on the function of Spt4.…”

Section: Discussionmentioning

confidence: 99%

“…A wide range of factors are implicated in assisting this route of RNAPII through nucleosomes but how they function in the cell is not yet clear (Clapier et al, 2017;Ehara et al, 2019;Farnung et al, 2018;Gurova et al, 2018;Venkatesh and Workman, 2015). One such factor is the DRB-sensitivity inducing factor (DSIF) complex in metazoans, also known as the Spt4/5 complex in yeasts, required for efficient transcription on chromatin (Crickard et al, 2017;Ehara et al, 2019;Kujirai and Kurumizaka, 2020;Vos et al, 2020). Spt4 is one of the most highly conserved transcription elongation factors (TEFs) in archaea and eukaryotes and its partner Spt5 is conserved in all three kingdoms, known as NusG in prokaryotes (Hartzog and Fu, 2013;Ponting, 2002).…”

Section: Introductionmentioning

confidence: 99%

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

Uzun

Brown

Fischl

et al. 2021

Preprint

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Spt4 is a transcription elongation factor, with homologues in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, yet the in vivo function of Spt4 is unclear. Here we assessed the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

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Structure of complete Pol II–DSIF–PAF–SPT6 transcription complex reveals RTF1 allosteric activation

Cited by 125 publications

References 77 publications

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

Structural basis of nucleosome transcription mediated by Chd1 and FACT

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

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