The Chd1 chromatin remodeler shifts hexasomes unidirectionally

Abstract

Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.DOI: http://dx.doi.org/10.7554/eLife.21356.001

The Chd1 Chromatin Remodeler Shifts Nucleosomal DNA Bidirectionally as a Monomer

“…The major peak appears at high FRET of 0.9 as expected from the proximity between the Cy3 and Cy5 dyes (Figure 1B, top). A minor mid-FRET peak at 0.6 is likely due to the Cy5-labeled H2A at a distal position as seen in previous studies (Deindl et al, 2013; Levendosky et al, 2016). FRET peaks did not change upon addition of Chd1 protein alone without ATP (Figure 1B, middle).…”

“…Compared to nucleosomes, hexasomes lack one histone H2A/H2B dimer, and we recently demonstrated that Chd1 was unable to robustly shift hexasomes toward the side lacking H2A/H2B (Levendosky et al, 2016). To monitor Chd1 activity on poor hexasome substrates, we produced end-positioned hexasomes with the side lacking the H2A/H2B dimer adjacent to the long flanking DNA, such that the Cy3-labeled DNA end was on the side with the remaining H2A/H2B dimer (Figure 6A).…”

The Sequence of Nucleosomal DNA Modulates Sliding by the Chd1 Chromatin Remodeler

“…To determine if the slower rate for A 16 [SHL2.5/3.5-right] was due to a catalytic or a binding effect, we monitored the kinetics of sliding for 0-N-80 nucleosomes with saturating Chd1 using a fluorescence-quenching assay 31 . In this assay, Dabcyl was attached to the short DNA end of an 0-N-80 nucleosome, which placed it close enough to quench a Cy3B fluorophore attached to histone H2A at T120C.…”

“…The 601 is notably asymmetric: one side has a higher affinity for histones than the other, and the strength of histone-DNA are unevenly distributed, with strongest contacts near the dyad 31, 43, 44 . The central portion of 601 is also asymmetric with respect to periodic TA steps, with one side of the dyad having a TA step at four consecutive turns of DNA where the minor groove faces the histone core, whereas the other side possesses just a single TA step (Fig.…”

“…Chd1 is known for preferentially shifting mononucleosomes toward the center of short DNA fragments, which is believed to underlie its ability to evenly distribute nucleosomes in arrays 27–29 . To date, directional sliding by Chd1 has been shown to arise from responding to three distinct nucleosome characteristics: the length and availability of DNA flanking the nucleosome 28, 30 , the presence of a transcription factor bound at the nucleosome edge 4 , and the absence of the histone H2A/H2B dimer on the entry side of hexasomes 31 . Our work here reveals that Chd1 activity is also impacted by the DNA sequence adjacent to SHL2.…”