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.
Highlights
As the repeating unit of chromatin, the nucleosome is the canvas upon which the epigenetic histone code is written
Hexasomes can be transformed into nucleosomes upon addition of H2A/H2B dimers, and we demonstrate that oriented hexasomes are an ideal substrate for generating uniform populations of asymmetric nucleosomes with uniquely modified H2A/H2B dimers
The hexasome is a stable sub-nucleosomal particle lacking one of the two H2A/H2B dimers (Arimura et al, 2012; Kireeva et al, 2002; Mazurkiewicz et al, 2006), and we confirmed by SDS-polyacrylamide gel electrophoresis (PAGE) analysis that the faster migrating species in our nucleosome preparations were hexasomes (Figure 1D)
Summary
As the repeating unit of chromatin, the nucleosome is the canvas upon which the epigenetic histone code is written. A fundamental characteristic of the histone code is the combinatorial diversity achieved from multiple marks, which may or may not reside on the same histone tail (Ruthenburg et al, 2007; Tee and Reinberg, 2014). Both through post-translational modifications (PTMs) and substitution of histone variants, additional chemical diversity arises from asymmetric modifications of nucleosomes. In addition to generating nucleosomes with asymmetric H3K4me3/H3K27me, PRC2 is activated by the mark it deposits, with substrate preference for asymmetric nucleosomes containing one H3K27me (Lechner et al, 2016; Margueron et al, 2009). While recognition of asymmetric H3K27me is believed to be important for maintenance and spreading of heterochromatin, and the bivalent H3K4me3/H3K27me signature has been well established for stem cell
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