Electron microscopy analysis of ATP-independent nucleosome unfolding by FACT

Abstract

FACT is a histone chaperone that participates in nucleosome removal and reassembly during transcription and replication. We used electron microscopy to study FACT, FACT:Nhp6 and FACT:Nhp6:nucleosome complexes, and found that all complexes adopt broad ranges of configurations, indicating high flexibility. We found unexpectedly that the DNA binding protein Nhp6 also binds to the C-terminal tails of FACT subunits, inducing more open geometries of FACT even in the absence of nucleosomes. Nhp6 therefore supports nucleosome unfolding by altering both the structure of FACT and the properties of nucleosomes. Complexes formed with FACT, Nhp6, and nucleosomes also produced a broad range of structures, revealing a large number of potential intermediates along a proposed unfolding pathway. The data suggest that Nhp6 has multiple roles before and during nucleosome unfolding by FACT, and that the process proceeds through a series of energetically similar intermediate structures, ultimately leading to an extensively unfolded form.

FACT modulates the conformations of histone H2A and H2B N-terminal tails within nucleosomes

“…This structure highlights that pAID of hFACT retains the nucleosome core structure instead of DNA. Similar interactions between FACT and unwrapped nucleosomes have been recently observed [53][54][55] ; however, the histone tails are not visualized in those cryo-EM structures. For the 112-bp DNA/pAID nucleosome, our previous NMR study clarified that the H3 N-tails, which are invisible in the cryo-EM structure, adopt two distinct conformations reflecting their asymmetric locations; a conformation of contact to DNA, as in the canonical nucleosome where the H3 N-tail is buried in two DNA gyres (DNA side); and a conformation of reduced contact to DNA and pAID (pAID side) 24 .…”

“…In a recent report, mono-ubiquitination at Lys119 in the H2A C-tail was shown to impede hFACT binding on nucleosome even together with the DNA stretching 58 . Taken together, direct and indirect interactions between hFACT and each histone tail are important for hFACT binding on nucleosome, although the histone tails are disordered in the structures of nucleosome-FACT complexes 10,[53][54][55] .…”

Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

“…We hypothesize that Nhp6 promotes partial destabilization of nucleosomes and that partially evicted basic histones could activate Spt6 binding, consistent with the fact that Spt6 can bind histones (both H2A–H2B dimers and H3–H4 tetramers) as observed here and previously ( 24 , 25 ). Alternatively, the Spt6 autoinhibition could be regulated by direct interaction of the acidic N-terminal region of Spt6 with basic Nhp6, similar to what has been shown for FACT ( 61 ). Furthermore, it is possible that Spt6 is activated as it undergoes dynamic conformational changes by association with Pol II.…”

Comparison of nucleosome unfolding by yeast and human FACT: electron microscopy analysis

“…Linear dimensions of the 2D-classes were measured with ImageJ. Results: Here using TEM we studied human FACT (hFACT) and yeast FACT (yFACT) flexibility alone and in complexes with nucleosomes [3]. All studied complexes are highly flexible and adopt broad ranges of configurations.…”