Author response: The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h (2018)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract ISWI family chromatin remodeling motors use sophisticated autoinhibition mechanisms to control nucleosome sliding. Yet how the different autoinhibitory domains are regulated is not well understood. Here we show that an acidic patch formed by histones H2A and H2B of the nucleosome relieves the autoinhibition imposed by the AutoN and the NegC regions of the human ISWI remodeler SNF2h. Further, by single molecule FRET we show that the acidic patch helps control the distance travelled per translocation event. We propose a model in which the acidic patch activates SNF2h by providing a landing pad for the NegC and AutoN auto-inhibitory domains. Interestingly, the INO80 complex is also strongly dependent on the acidic patch for nucleosome sliding, indicating that this substrate feature can regulate remodeling enzymes with substantially different mechanisms. We therefore hypothesize that regulating access to the acidic patch of the nucleosome plays a key role in coordinating the activities of different remodelers in the cell. https://doi.org/10.7554/eLife.35322.001 eLife digest Every human cell contains nearly two meters of DNA, which is carefully packaged to form a dense structure known as chromatin. The building block of chromatin is the nucleosome, a unit composed of a short section of DNA tightly wound up around a spool-like core of proteins called histones. The tight structure of the nucleosome prevents the cell from accessing and ‘reading’ the genes in the packaged DNA, effectively switching off these genes. So the exact placement of nucleosomes helps manage which genes are turned on. Changing the position of the nucleosomes can ‘free’ the DNA and make genes available to the cell. Enzymes called chromatin remodelers move nucleosomes around – for example, they can make the histone core slide on the DNA strand. However, it is still unclear how these enzymes recognize nucleosomes. Previous research indicates that many proteins bind to nucleosomes by using a surface on the histone proteins called the acidic patch. Could chromatin remodelers also work by interacting with this acidic patch? To address this further, Gamarra et al. investigate how a chromatin remodeler enzyme known as SNF2h interacts with a nucleosome. By default, SNF2h is inactive because two of its regions called AutoN and NegC act as brakes. The experiments show that the acidic patch helps to bypass this inactivation and switches on SNF2h. Gamarra et al. propose that, when SNF2h docks on to the nucleosome, the patch provides a landing pad for the AutoN and NegC modules; this interaction activates the enzyme, which can then start remodeling the nucleosome. However, another type of chromatin remodeler also uses the patch to interact with nucleosomes but it does not have the AutoN and NegC regions. This suggests that chromatin remodelers work with the acidic patch in different ways. Overall, the findings deepen our understanding of how DNA is packaged in cells, and how this process may go wrong and cause disease. https://doi.org/10.7554/eLife.35322.002 Introduction Eukaryotic genomes are packaged into chromatin, enabling large amounts of DNA to fit into the spatial constraints of the nucleus. This packaging has long been appreciated as a passive barrier to DNA access by nuclear factors. The discovery that chromatin regulators play critical roles in virtually all nuclear processes has informed a more nuanced view of chromatin as a dynamic regulatory platform that coordinates access to the genetic material. The smallest unit of chromatin is the nucleosome, a DNA-protein complex composed of ~150 bp of DNA wrapped around an octamer of histone proteins (Luger et al., 1997). Nucleosomes can further interact with each other and with other factors to form higher-order structures (Luger et al., 2012). Consequently, cells have evolved several sophisticated strategies to regulate chromatin structure at the nucleosome level. These include the covalent modification of histone proteins and DNA, as well as non-covalent changes to the position or composition of nucleosomes at specific genomic loci. Many of the non-covalent transformations, ranging from sliding nucleosomes to the complete disassembly of the histone octamer, are catalyzed by ATP-dependent chromatin remodeling enzymes (Zhou et al., 2016). Underscoring their central role in chromatin regulation, remodeling enzymes play essential roles in many processes including transcription, DNA replication, and DNA repair (Falbo and Shen, 2006; Hota and Bruneau, 2016; Price and D'Andrea, 2013). How a relatively small number of remodeler types carry out such diverse regulatory functions remains an area of active research, not least because much remains unknown regarding remodeler mechanisms for substrate recognition and the coupling of that recognition to activity. Chromatin remodelers are members of the SF2 superfamily of nucleic acid motors, which catalyze noncovalent changes to nucleic acid substrates (Zhou et al., 2016). Chromatin remodelers, however, are unique in that they specifically mobilize DNA in the context of the nucleosome, where DNA is tightly bound to histone proteins. Remodelers are further classified into families based on the domain architecture of their ATPase subunit. These families differ in their specific biochemical activities (Zhou et al., 2016). Substantial progress in our general understanding of remodeling mechanisms has been made by asking what elements of the nucleosome are important for remodeling in different families. For example, maximal remodeling by ISWI family remodelers, which primarily slide nucleosomes, requires DNA flanking the nucleosome and the N-terminal tail of histone H4 (Clapier et al., 2001; Yang et al., 2006). Conversely, maximal remodeling by SWI/SNF family remodelers, which carry out the most diverse set of changes to nucleosome structure, does not require these nucleosomal epitopes (Guyon et al., 1999). Less is known about how these substrate cues are recognized and mechanistically coupled to remodeling. Some important insights have come from biochemical analyses and structures of remodelers in the absence of nucleosomes, which suggest that in the ground state, chromatin remodelers are held in an inactive conformation by family-specific autoinhibitory motifs (Clapier and Cairns, 2012; Hauk et al., 2010; Xia et al., 2016; Yan et al., 2016). Binding to specific nucleosomal epitopes is thought to relieve this autoinhibition via conformational changes in the remodeler, but the details of this process remain unclear. At the same time, structures of several nucleosome-protein complexes are revealing that many of these proteins interact with a conserved acidic patch formed by histones H2A and H2B on the top surface of the nucleosome (McGinty and Tan, 2016). These proteins interact with the acidic patch using an ‘arginine anchor’ which nestles into a pocket formed by the α1–2 helices of histone H2A (McGinty and Tan, 2016). It is therefore plausible that chromatin remodelers also recognize the acidic patch. Indeed recent work has indicated that mutating the acidic patch reduces the activity of ISWI, SWI/SNF and some CHD family remodeling enzymes (Dann et al., 2017). To investigate the mechanistic role of the acidic patch in nucleosome remodeling by the ISWI family of enzymes we used a combination of ensemble and single molecule methods in the context of the human ISWI enzyme, SNF2h. We observe that interactions with the acidic patch activate SNF2h by relieving auto-inhibition mediated by two conserved domains of ISWI enzymes. Our results further suggest that contacts with the acidic patch helps control the distance the nucleosome is moved during translocation events. Finally we find that the acidic patch also stimulates the activity of the INO80 complex. Together, these results highlight the broad and essential role the acidic patch plays in chromatin regulation. Results The acidic patch of the nucleosome is important for nucleosome sliding by SNF2h The human ISWI ATPase, SNF2h, preferentially slides nucleosomes toward longer flanking DNA (Yang et al., 2006). As a consequence of this activity, SNF2h slides mononucleosomes towards the center of a DNA strand, an activity that can be detected by a native gel mobility assay (Yang et al., 2006). Recent work has shown that mutating residues in the nucleosome acidic patch reduces the ability of SNF2h to expose nucleosomal DNA to restriction enzymes (Dann et al., 2017). To investigate the mechanism by which such mutations affect the nucleosome sliding activity of SNF2h and to determine if these mutations affected SNF2h binding or catalysis, we first used a mutant H2A in which four key residues in the H2A acidic patch are replaced with alanines (E61A, E64A, D90A, and D92A) (Figure 1A). We call nucleosomes in which all four of these H2A residues have been mutated to alanines ‘APM’ (acidic patch mutant) nucleosomes. Using a native gel mobility assay, we measured remodeling rates of wild type (WT) and APM mononucleosomes containing 60 bp of DNA flanking one end of the nucleosome (0/60) (Figure 1B). APM nucleosomes are centered substantially slower than WT nucleosomes under conditions where SNF2h is in excess of nucleosomes (Figure 1B and C). Since the acidic patch has been shown to be critical for nucleosome binding by several chromatin proteins, we expected the apparent Km, Kmapp, to be increased with APM nucleosomes. Interestingly however, Kmapp is affected substantially less by mutation of the acidic patch than the maximal rate constant for remodeling, kmax (Figure 1D, 61 nM to 280 nM, corresponding to ~4.5 fold increased Kmapp, versus 2.5 min−1 to 0.012 min−1, corresponding to ~200 fold reduced kmax). These results indicate that the acidic patch plays a larger role in regulating maximal nucleosome sliding activity than nucleosome binding. We also measured ATP hydrolysis under conditions where nucleosomes are in excess of SNF2h. At saturating nucleosome concentrations, APM nucleosomes stimulate ATP hydrolysis 4-fold less than WT (Figure 1G, Figure 1—figure supplement 2). Together, these results imply that the acidic patch plays a critical role in coupling ATP hydrolysis to remodeling after the binding step. Figure 1 with 3 supplements see all Download asset Open asset The acidic patch is an important epitope for remodeling post-binding. (A) Charge profile of the nucleosome (left) and magnification of the acidic patch region (right) (PDBID: 1KX5, charge profile generated using ABPS and UCSF Chimera [Pettersen et al., 2004]). Residues of the acidic patch mutated in this study are shown in red. (B) Example of a Cy5-fluorescent scan of a gel-based remodeling assay with 0/60 WT and acidic patch mutant (APM) nucleosomes (1 µM SNF2h, 20 nM nucleosomes, saturating ATP). (C) Quantification of the gel in B and fits to a single exponential decay. Inset is zoomed to show faster time points with WT nucleosomes. (D) Gel remodeling rate as a function of enzyme concentration, plotted on a log(2) scale. APM nucleosomes are remodeled substantially slower even at saturating concentrations of enzyme. Data were fit to a cooperative binding model (WT nucleosomes: KMapp = 61 nM, kmax = 2.5 min−1, h = 1.5; APM nucleosomes: KMapp = 280 nM, kmax = 0.012 min−1, h = 1.2). (E) Remodeling is inhibited by competition for the acidic patch. WT nucleosomes were remodeled with sub-saturating SNF2h and in the presence of KSHV LANA peptide. Remodeling is inhibited by the peptide but not when the arginine anchor is mutated to alanine. (F) Inhibition curve with the LANA peptide (KI = 1.21 µM). Error bars represent standard errors on the mean for three replicates, except for the no-peptide condition in F, which had two replicates. (G) ATPase activity of WT SNF2h. Nucleosomes were in excess of SNF2h and at saturating concentrations. APM nucleosomes stimulated ATPase activity 4-fold weaker than WT. https://doi.org/10.7554/eLife.35322.003 Figure 1—source data 1 Values used to obtain plots. https://doi.org/10.7554/eLife.35322.009 Download elife-35322-fig1-data1-v3.xlsx To further investigate the importance of the acidic patch, we asked whether binding by another factor to the acidic patch could compete for nucleosome sliding by SNF2h. We used a peptide derived from the latency associated nuclear antigen (LANA) from Kaposi’s sarcoma-associated herpesvirus that has previously been shown to interact directly with the acidic patch via an arginine anchor (Barbera et al., 2006). We carried out these experiments using sub-saturating concentrations of SNF2h. Under these conditions most of the nucleosomes are unbound, allowing direct competition between the LANA peptide and SNF2h for the acidic patch. The presence of the LANA peptide dramatically slows remodeling by SNF2h in a dose-dependent manner (~200 fold reduction in rate constant at 50 µM LANA peptide), while a peptide with a mutant arginine anchor does not have a detectable inhibitory effect (Figure 1E and F). We measured a KI of 1.2 µM for inhibition by the LANA peptide, within 5-fold of its published affinity for the nucleosome (Figure 1F) (Fang et al., 2016). These results indicate that remodeling by SNF2h requires an interaction with the acidic patch that is mutually exclusive with the binding of acidic patch interacting factors such as the LANA peptide. The results are also consistent with recent work showing that the LANA peptide can inhibit the restriction enzyme accessibility activity of the SNF2h containing complex, ACF (Dann et al., 2017). We next investigated if the acidic patch residues act independently or cooperatively by measuring the effects of individual mutations. Interestingly, except for residue E64, all the single alanine mutants have comparable defects as the four point mutants combined (Figure 1—figure supplement 3). This observation suggests that three of the four acidic patch residues tested here act cooperatively to promote sliding by SNF2h. The AutoN and NegC regions of SNF2h cooperate with the acidic patch to enable maximal remodeling We next investigated which regions of SNF2h might functionally interact with the acidic patch. In principle, these would include (i) regions that directly contact the acidic patch and (ii) regions that do not directly contact the acidic patch, but whose function is energetically coupled to the presence of the acidic patch. To investigate regions that may directly contact the acidic patch we carried out cross-linking mass spectrometry using the zero-length, carbodiimide based reagent EDC. This method catalyzes the formation of new amide bonds between protein carboxylates, such as the side chains of aspartate and glutamate residues, and amino groups and is therefore well suited to probing electrostatic interactors of the acidic patch. Hundreds of high confidence cross-linked residue pairs were identified using this approach (Supplementary file 1). To focus on mechanistically meaningful domain-domain interactions, we employed a semi-quantitative mass spectrometry method to compare the extent of SNF2h-nucleosome cross-linking in the presence of different nucleotides (Figure 2B). In previous work we have shown that ADP•BeFx mimics an activated state of the SNF2h-nucleosome complex (Leonard and Narlikar, 2015; Racki et al., 2014; Sinha et al., 2017). We therefore, focused on domain level interactions that were enriched at least two-fold in the presence of ADP•BeFx relative to ADP. Figure 2 with 5 supplements see all Download asset Open asset Dependence on the nucleosome acidic patch is linked to relief of autoinhibition. (A) Domain architecture of SNF2h. The two critical arginines (R142, R144) of AutoN are highlighted in blue, while the NegC region replaced with a flexible GGS linker (Leonard and Narlikar, 2015) is highlighted in brown. Intervening sequences with no known domain annotations are numbered as in B. (B) Direct domain-domain interactions of SNF2h nucleosomes probed by crosslinking mass spectrometry. SNF2h mononucleosomes were crosslinked with the zero-length reagent EDC in the presence of either the ATP transition state analog ADP•BeFx or ADP. Residue-residue crosslink data were aggregated into domain-domain level spectral counts. The fold change between ADP•BeFx and ADP conditions is displayed as the log2 of the spectral count ratio. The data were centered to the median value and the color scale depicts the interactions most enriched in ADP•BeFx in magenta, and ADP in blue. Light grey tiles indicate no crosslinks observed in either condition. Domains are listed from N- to C- term within each protein. The distribution of fold changes in domain-domain spectral counts is plotted as a histogram below. It should also be noted that due to the structure of the histone octamer and the 2:1 stoichiometry of the SNF2h-Nucleosome complex, we cannot distinguish between intramolecular and intermolecular SNF2h-SNF2h or Histone-Histone crosslinks. (C and D). Mutation of AutoN or NegC increases remodeling rate with WT and APM nucleosomes. Cy5 fluorescent scans of native gel remodeling assays. (E) Saturating remodeling rates (kmax) for WT and APM nucleosomes with WT, 2RA, and mNegC SNF2h. Mutation of the acidic patch has a substantially lower effect on the kmax for 2RA and mNegC SNF2h than for WT SNF2h (8-fold and 2-fold respectively vs. 200-fold). Error bars represent standard errors on the mean for N = 3 replicates. https://doi.org/10.7554/eLife.35322.010 Figure 2—source data 1 Values used to obtain plots. https://doi.org/10.7554/eLife.35322.019 Download elife-35322-fig2-data1-v3.xlsx Cross-links between the H4 tail and RecA lobe 2 of SNF2h are strongly enhanced in the ADP•BeFx state (Figure 2B). This result is consistent with previous work showing that the H4 tail activates ISWI remodelers and promotes a restricted active site conformation in the presence of ADP•BeFx (Clapier et al., 2001; Hamiche et al., 2001; Racki et al., 2014). We also found that the ADP•BeFx state promotes specific cross-links between the H2A/H2B acidic patch and lysines in the extended AutoN region, RecA lobe 1, the NegC region, and the DNA binding HAND-SANT-SLIDE (HSS) region of SNF2h (Figure 2—figure supplement 4). While cross-links between the acidic patch and the AutoN, HSS and NegC regions are compatible with structural constraints from previous studies, the cross-links with the RecA lobe one are not easily explained. Multiple studies of ISWI remodelers suggest that their RecA lobes bind at a location two DNA helical turns from the nucleosome dyad (SHL ±2), quite far from the H2A/H2B acidic patch (Dang and Bartholomew, 2007; Kagalwala et al., 2004; Schwanbeck et al., 2004; Zofall et al., 2006). We hypothesize that the RecA-acidic patch cross-links arise from a population of higher-order SNF2h-nucleosome aggregates in our mass-spec samples and therefore focus below on cross-links to the remaining three regions. To test the functional significance of cross-links to AutoN, we mutated the lysines that crosslink to the acidic patch in the ADP•BeFx state. However, none of the mutants remodeling by SNF2h (Figure 2—figure supplement 1). These results suggest that the lysines in the AutoN region are in to the acidic patch but do not make mechanistically We however, that other residues in AutoN might make functional interactions with the acidic patch. Previous work has indicated that many nucleosome binding proteins recognize the acidic patch via arginine the AutoN lysines that crosslink to the acidic patch a key autoinhibitory specific to ISWI family remodelers that contains two arginine residues (Figure Figure 2—figure supplement and by these arginine residues is by a patch on the H4 a nucleosomal epitope essential for maximal remodeling by remodelers (Clapier and Cairns, 2012; et al., We tested whether and functionally cooperate with the acidic patch by a mutated of SNF2h with the two critical arginines of AutoN mutated to alanine and measured the remodeling activity of this with previous 2RA SNF2h WT nucleosomes 2 fold faster than WT SNF2h (Clapier and Cairns, 2012; Yan et al., 2016). However, 2RA SNF2h APM nucleosomes 50 fold faster than WT SNF2h (Figure This to a fold reduced on the acidic patch for remodeling with 2RA SNF2h (Figure The same also by an ensemble FRET remodeling assay (Figure 2—figure supplement 2). Together, these data suggest that the acidic patch to relief of autoinhibition by and in Since arginine residues are known to this interaction in other binding of either of the two arginines in AutoN to the acidic patch could a for acidic patch However, that carbodiimide other used cross-linking arginine residues, we cannot determine whether and make direct contacts with the acidic patch based on our We were to observe detectable binding between an AutoN peptide containing the residues and the nucleosomal acidic patch not that either these residues do not interact with the acidic patch or the regions of the SNF2h protein are for binding. To investigate the functional significance of cross-links to the NegC region of SNF2h, we the effect of a of residues in NegC with a flexible linker NegC is another autoinhibitory region of SNF2h that flanking DNA on SNF2h by specifically remodeling of nucleosomes flanking DNA (Leonard and Narlikar, with previous mNegC SNF2h slides WT 0/60 nucleosomes 1.2 fold faster than WT SNF2h (Figure and (Leonard and Narlikar, However, mNegC SNF2h slides APM nucleosomes fold faster than WT SNF2h (Figure As a sliding of APM nucleosomes by mNegC SNF2h is fold slower than WT nucleosomes. the mNegC mutation the of the acidic patch These results suggest that residues in NegC also of nucleosome sliding to acidic patch The of cross-links residues in the HSS regions. in these lysines reduced remodeling in to the 2RA and mNegC SNF2h not the defects by the acidic patch mutations not It is that the defects by these mutations direct contacts between the HSS residues and the acidic patch. However, because the HSS also contacts flanking DNA, the reduction in sliding rate could also arise from defects in binding To which crosslinks are dependent on H2A acidic patch we also SNF2h-nucleosome cross-linking with ADP•BeFx in the presence of the LANA peptide which for acidic patch binding (Figure of the LANA peptide substantially reduced crosslinks between the H2A acidic patch and AutoN and NegC (Figure 2—figure supplement In crosslinks between the HSS and the acidic patch were less strongly affected by LANA (Figure 2—figure supplement This suggests that HSS the acidic patch in the ADP•BeFx state is not dependent on direct binding to the region of the H2A acidic patch by the LANA peptide. As a we cannot patch crosslinks as mechanistically Together, these data suggest that acidic patch recognition is strongly linked to relief of autoinhibition by NegC and AutoN for nucleosome sliding by SNF2h. of SNF2h conformational changes that of these autoinhibitory domains and the HSS in to the acidic patch. The acidic patch is to promote the translocation of the SNF2h To into which in the remodeling interaction with the acidic patch, we turned to a FRET assay Figure This assay is to the ensemble FRET remodeling assay in Figure 2—figure supplement in that the activity of SNF2h in sliding nucleosomes from DNA increases the distance between a and and the measured FRET However, with we can the remodeling of nucleosomes, and insights into the activity of SNF2h that are in ensemble assays. has previously been used to study remodeling by several ISWI family as well as by remodeling complexes from the SWI/SNF and INO80 families et al., et al., et al., 2016; et al., 2014; et al., key by these studies is that ISWI family remodelers do not slide nucleosomes in a such that FRET time, but in a of a in which FRET therefore nucleosome remain and a in which the nucleosome is moved relative to the DNA These which far to be specific to ISWI, are essential to our understanding of the mechanism of of ISWI remodelers, because the remodeling rates observed in ensemble are by the of the not the translocation rate substrate cues such as the H4 tail and flanking DNA have been shown to be in the not the translocation for ISWI family remodelers et al., 2014). flanking DNA or mutation of the H4 tail increases the of the the remodeling while no effect on the sliding rate of the nucleosome. These results suggest a of and activity in ISWI translocation are by that for the of substrate This also the ability of ISWI remodelers such as ACF to nucleosomes if the nucleosome is the of this translocation by a new can translocation in the the nucleosome to a centered Figure 3 with 3 supplements see all Download asset Open asset The acidic patch interacts with AutoN to promote and (A) of the Nucleosomes on histone with a and on one end of the DNA with a Cy5 are on the surface of a slide and with a The nucleosomes used here have an high FRET also Figure supplement 2). As remodeling and the nucleosome is moved from the DNA the FRET is (B) Example of remodeling of a single WT nucleosome in the presence of saturating SNF2h and ATP nM and 1 indicates of enzyme and and are the of an fit used to and the and of the remodeling in with the ISWI we call the first the the the and FRET data here and in have been with a median for (C) Example of remodeling from top to WT nucleosomes by SNF2h, nucleosomes by SNF2h, WT nucleosomes by 2RA SNF2h, and nucleosomes by 2RA SNF2h. Saturating enzyme nM enzyme with WT 2 enzyme with and ATP (1 were used in all are shown in Figure supplement (D) Quantification of the first three a lower as shown in Figure supplement remodeling of nucleosomes by SNF2h is to by due to the competition between of the and remodeling by SNF2h. were Materials and (E) distribution functions of the change in nucleosome position during the first translocation and the translocation for different of nucleosome and enzymes. of the translocation by SNF2h on WT nucleosomes move the nucleosome bp or in nearly of translocation by