Stepwise Histone Replacement by SWR1 Requires Dual Activation with Histone H2A.Z and Canonical Nucleosome

Abstract

Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.

Tripartite organization of centromeric chromatin in budding yeast

“…It is possible that sonication caused loss of poorly cross-linked nucleosomes, and differences in the degree of cross-linking of different histones (8) might have resulted in discrepant ChIP efficiencies between them. The excellent concordance between our study using native chromatin and that of Luk et al (35) using cross-linked chromatin confirms that H2A is as abundant over centromeres as it is over the entire genome, as expected for a centromeric particle containing all four histones. Our findings are consistent with a report that the Mif2 kinetochore-specific protein coimmunopurifies with Cse4, H4, H2A, and H2B, but not detectably to H3, when purified without cross-linking or enzymatic DNA fragmentation (36).…”

“…Our native ChIP-seq analysis also showed that H2A is present at all 16 yeast centromeres at the same level as over the rest of the genome. We confirmed this result by analyzing X-ChIP data from a published study (35). Because that study was performed on formaldehyde cross-linked chromatin by the same laboratory that previously reported deficiency of H2A over centromeres (28), we might attribute the different outcomes to the use of sonication in the first study to fragment and solubilize DNA, versus the use of MNase in the second study (35).…”

“…We confirmed this result by analyzing X-ChIP data from a published study (35). Because that study was performed on formaldehyde cross-linked chromatin by the same laboratory that previously reported deficiency of H2A over centromeres (28), we might attribute the different outcomes to the use of sonication in the first study to fragment and solubilize DNA, versus the use of MNase in the second study (35). It is possible that sonication caused loss of poorly cross-linked nucleosomes, and differences in the degree of cross-linking of different histones (8) might have resulted in discrepant ChIP efficiencies between them.…”

Two distinct modes for propagation of histone PTMs across the cell cycle

“…Notably, H2A.Z levels increased within the next 10 h (Fig. 2E), consistent with replication-independent assembly (Luk et al 2010;Nekrasov et al 2012). Altogether, our data demonstrate that histone variants are not recycled with similar efficiency at replication forks and that H2A.Z marked sites may not be maintained after DNA replication.…”

Histone Variant H2A.Z and RNA Polymerase II Transcription Elongation

“…Histone H2A.Z is itself subject to posttranslational modifications, including acetylation, ubiquitylation, and sumoylation, and these may alter its chromatin association, exchange dynamics, structure, and function (8,32,39,41,43,66,81,84,95). H2A.Z is broadly but nonuniformly distributed throughout the chromosomes and is deposited by specific remodeling complexes, such as the yeast SWR1 complex (SWR1) (3,45,51,60,67).…”