Dual function of Swc5 in SWR remodeling ATPase activation and histone H2A eviction

Abstract

The chromatin remodeler SWR deposits histone H2A.Z at promoters and other regulatory sites via an ATP-driven histone exchange reaction that replaces nucleosomal H2A with H2A.Z. Simultaneous binding of SWR to both H2A nucleosome and free H2A.Z induces SWR ATPase activity and engages the histone exchange mechanism. Swc5 is a conserved subunit of the 14-polypeptide SWR complex that is required for the histone exchange reaction, but its molecular role is unknown. We found that Swc5, although not required for substrate binding, is required for SWR ATPase stimulation, suggesting that Swc5 is required to couple substrate recognition to ATPase activation. A biochemical complementation assay was developed to show that a unique, conserved domain at the C-terminus of Swc5, called Bucentaur (BCNT), is essential for the histone exchange activity of SWR, whereas an acidic region at the N-terminus is required for optimal SWR function. In vitro studies showed the acidic N-terminus of Swc5 preferentially binds to the H2A–H2B dimer and exhibits histone chaperone activity. We propose that an auxiliary function of Swc5 in SWR is to assist H2A ejection as H2A.Z is inserted into the nucleosome.

VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

“…ZxZ adducts were observed in the extracts from the SWC5 strain, but not swc5∆ ( Figure 4B ). Additional mutants of SWC5 that were previously shown to prevent H2A.Z deposition include a truncation of the highly conserved BCNT domain [ swc5(1-232) ] and substitution of the LDW motif within BCNT with alanines [ swc5(LDW→3A) ] ( Sun and Luk, 2017 ) ( Figure 4A ). These mutants also significantly reduced ZxZ crosslinking efficiency in VivosX ( Figure 4B–C ).…”

“… ( A ) The cartoon depicts the domain organization of yeast Swc5 ( Sun and Luk, 2017 ). ( B,C ) The yeast strain, HTZ1(T46C) FL swc5∆ , was transformed by a single-copy plasmid containing either the wild-type SWC5 or the indicated SWC5 mutants or by the control vector ( swc5∆ ).…”

“…Notwithstanding a minor contribution of the uncrosslinked monomers a result of inefficient crosslinking, the bulk of the uncrosslinked species should represent the non-nucleosomal pools of A-B and Z-B dimers, which constitute 39% and 26% of total H2A and H2A.Z, respectively ( Figure 3A lane 6, 3B lane 6, and 3C lanes 2 and 4). These unincorporated histones could be A-B and Z-B dimers that are associated with histone chaperones or chromatin remodelers, like SWR ( Luk et al, 2007 ; Sun and Luk, 2017 ; Wu et al, 2005 ). These dimers could also be associated with chromatin in the form of hexasomes (partial nucleosomal particles missing one A-B or Z-B dimer) or non-nucleosomal histone-DNA complexes ( Andrews et al, 2010 ; Arimura et al, 2012 ).…”

Overcoming off-targets: assessing Western blot signals for Bcnt/Cfdp1, a tentative component of the chromatin remodeling complex

“…Members of the Bcnt family generally consist of an acidic N-terminal region, a highly conserved C-terminal region with approximately 80 amino acids (BCNT-C domain), and a hydrophilic region between them ( Figure 1 and [1]). Recently, the BCNT-C domain of the budding yeast Swc5 was found to be essential for the histone exchange reaction [10]. On the other hand, a deletion mutant induced by one-base replacement of the Drosophila ortholog, Yeti, which entirely lacks the BCNT-C domain, shows substantial chromosomal abnormalities, resulting in lethality before pupation [11].…”

Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

“…Fig. S2.1), but essential 99 for SWR1 activity(Haruki et al, 2008;Sun and Luk, 2017;Tramantano et al, 2016). In 100 the AA system, rapamycin mediates heterodimerization of FRB and FKBP12 moieties 101 fused to Swc5 and the ribosomal protein RPL13A, respectively (i.e.…”