Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes

Abstract

Chromatin-remodelling complexes (CRCs) mobilize nucleosomes to mediate the access of DNA-binding factors to their sites in vivo. These CRCs contain a catalytic subunit that bears an ATPase/DNA-translocase domain and flanking regions that bind nucleosomal epitopes. A central question is whether and how these flanking regions regulate ATP hydrolysis or the coupling of hydrolysis to DNA translocation, to affect nucleosome-sliding efficiency. ISWI-family CRCs contain the protein ISWI, which uses its ATPase/DNA-translocase domain to pump DNA around the histone octamer to enable sliding. ISWI is positively regulated by two 'activating' nucleosomal epitopes: the 'basic patch' on the histone H4 tail, and extranucleosomal (linker) DNA. Previous work defined the HAND-SANT-SLIDE (HSS) domain at the ISWI carboxy terminus that binds linker DNA, needed for ISWI activity. Here we define two new, conserved and separate regulatory regions on Drosophila ISWI, termed AutoN and NegC, which negatively regulate ATP hydrolysis (AutoN) or the coupling of ATP hydrolysis to productive DNA translocation (NegC). The two aforementioned nucleosomal epitopes promote remodelling indirectly by preventing the negative regulation of AutoN and NegC. Notably, mutation or removal of AutoN and NegC enables marked nucleosome sliding without the H4 basic patch or extranucleosomal DNA, or the HSS domain, conferring on ISWI the biochemical attributes normally associated with SWI/SNF-family ATPases. Thus, the ISWI ATPase catalytic core is an intrinsically active DNA translocase that conducts nucleosome sliding, onto which selective 'inhibition-of-inhibition' modules are placed, to help ensure that remodelling occurs only in the presence of proper nucleosomal epitopes. This supports a general concept for the specialization of chromatin-remodelling ATPases, in which specific regulatory modules adapt an ancient active DNA translocase to conduct particular tasks only on the appropriate chromatin landscape.

Figure 1. AutoN resembles the histone H4 tail basic patch and restricts ISWI ATPase activity

a, Alignment of ISWI (Drosophila) and the histone H4 N-terminus, with the basic patch depicted. b, ISWI protein and domains/regions, including ATPase, HAND-SANT-SLIDE (HSS), and new functional regions (AutoN and NegC), with zoom of AutoN alignments for ISWI orthologs: Drosophila (dm), Xenopus (xl), human, and Saccharomyces cerevisiae (sc). c, Mutation of AutoN (2RA) hyperactivates ISWI ATPase activity and bypasses H4 tail stimulation by peptides. Values, mean of ≥3 experiments, normalized to ISWI^wt with DNA. Error bars +/− s.e.m. d, AutoN (2RA) mutation stimulates ISWI ATPase activity with mononucleosomes: native (wt), H4 tailless/globular (g4), or basic patch mutations (A17A19). Color code (inset) reflects nucleosome sliding activity (in Figure 2c,d). Values, mean of ≥3 experiments, normalized to ISWI^wt. Error bars +/− s.e.m.

Figure 2. AutoN mutation (ISWI^2RA) increases DNA translocation and nucleosome sliding

a, Comparative DNA translocation activity (see Supplementary Fig. 2) of ISWI derivatives. Translocation generates supercoiled topoisomers (SC). b, Comparative sliding of extended nucleosomes by ISWI derivatives, as a time course, and c, their reliance on H4 tail epitopes, in titration series. d, Comparative sliding activity on core nucleosomes reveals a reliance on H4 tail epitopes. For b and d, enzyme:substrate molar ratio is 1:2.

Figure 3. NegC inhibits the coupling of ATP hydrolysis to DNA translocation, and nucleosome sliding

a, NegC conservation in ISWI orthologs. ISWI protein, with zoomed alignment of NegC from ISWI orthologs: Drosophila (dm), Xenopus (xl), human, and Saccharomyces cerevisiae (sc). b, Modelisation of ISWI^ΔC697 showing NegC (orange) traversing the cleft (red dash line) from the ATPase lobe #2 (blue) to ATPase lobe #1 (green). Model generated using Phyre; depicted with PyMol. c, The AutoN/2RA mutation elicits robust DNA-dependent ATPase activity independent of the HSS. Values, mean of three experiments, normalized to ISWI^wt, with 200bp DNA fragment. Error bars +/− s.e.m. d, Deletion of NegC via truncation restores DNA translocation. e, Impact of NegC region on sliding activities of core and extended nucleosomes (and H4 tailless versions). ATPase activities (values, colored by impact on sliding: high (green), moderate/low (orange), absent (red)). Enzyme:substrate molar ratio 1:2 . B (Buffer).

Figure 4. Expression of ISWI derivatives in vivo, and regulation model for ISWI

a, Isw1 lacking AutoN and NegC regulation greatly impairs growth of an S. cerevisiae strain sensitized for chromatin misregulation (rsc7Δ). Isw1 derivatives expressed in an isw1Δ rsc7Δ [p RSC7⁺.URA3] strain. 5-FOA enforces the loss of the URA3-marked RSC7⁺ plasmid, imposing a rsc7Δ genotype. b, Logic of ISWI ATPase regulation. The ISWI ATPase activity is positively regulated by DNA in the ATPase cleft. ISWI is also negatively regulated by two intrinsic domains: AutoN and NegC. AutoN inhibits the ATP hydrolysis rate, and is relieved by the basic patch (R17-R19) of the H4 tail, whereas NegC inhibits ATPase coupling to DNA translocation, and is relieved by the HSS domain binding to sufficient extranucleosomal DNA: defining the ‘inhibition of inhibition’ mode of ISWI regulation. (Note: an additional lysine (H4K12) also promotes coupling.)