ISWI-family nucleosome remodeling enzymes need the histone H4 N-terminal tail to mobilize nucleosomes. Here we mapped the H4-tail binding pocket of ISWI. Surprisingly the binding site was adjacent to but not overlapping with the docking site of an auto-regulatory motif, AutoN, in the N-terminal region (NTR) of ISWI, indicating that AutoN does not act as a simple pseudosubstrate as suggested previously. Rather, AutoN cooperated with a hitherto uncharacterized motif, termed AcidicN, to confer H4-tail sensitivity and discriminate between DNA and nucleosomes. A third motif in the NTR, ppHSA, was functionally required in vivo and provided structural stability by clamping the NTR to Lobe 2 of the ATPase domain. This configuration is reminiscent of Chd1 even though Chd1 contains an unrelated NTR. Our results shed light on the intricate structural and functional regulation of ISWI by the NTR and uncover surprising parallels with Chd1.DOI:http://dx.doi.org/10.7554/eLife.21477.001 In the cells of animals, plants and other eukaryotes, DNA wraps tightly around proteins called histones to form structures known as nucleosomes that resemble beads on a string. When nucleosomes are sufficiently close to each other they interact and clump together, which compacts the DNA and prevents the genes in that stretch of DNA being activated.But how do cells mobilize their nucleosomes? A nucleosome remodeling enzyme called ISWI can slide nucleosomes along DNA. ISWI becomes active when it interacts with a ‘tail’ region of a histone protein called H4. However, the H4 tail prefers to interact with neighboring nucleosomes instead of with ISWI. Therefore when ISWI slides a nucleosome close to another one, the H4 tail of the nucleosome binds instead to its new neighbor so that ISWI cannot continue to slide. By this mechanism, ISWI is proposed to pile up nucleosomes, which subsequently compact, leading to the inactivation of this part of the genome.To investigate how ISWI recognizes the H4 tail, Ludwigsen et al. mapped where the H4 tail binds to ISWI by combining the biochemical methods of cross-linking and mass spectrometry. In addition, mutagenesis experiments identified a new motif in the enzyme that is essential for recognizing the H4 tail. In the absence of the nucleosome, this motif – called AcidicN – works with a neighboring motif called AutoN to keep ISWI in an inactive state. The two motifs also work together to enable ISWI to distinguish between nucleosomes and DNA. Further evidence suggests that other remodeling enzymes have similar regulation mechanisms; therefore this method of controlling nucleosome remodeling may have been conserved throughout evolution.Further studies are now needed to detect the shape changes that occur in ISWI as it recognizes the histone tail and work out how this leads to nucleosome remodeling. Inside cells, ISWI is usually found within large complexes that consist of many proteins. It therefore also remains to be discovered whether the proteins in these complexes impose additional layers of regulation and complexity on the activity of ISWI.DOI:http://dx.doi.org/10.7554/eLife.21477.002 Eukaryotic cells package their DNA into chromatin. Chromatin organization allows cells to compact, protect and regulate their genomes. Nucleosomes are the primary building blocks of chromatin. These particles consist of ~150 bp of DNA that wrap almost twice around an octamer of histones. Nucleosomal DNA, however, is not accessible to most nuclear factors. Nature therefore evolved ATP-dependent nucleosome remodeling complexes that can alter the position or the structure of nucleosomes as necessary.Numerous remodeling complexes with distinct activities are active in any cell. Some move nucleosomes along DNA, eject histones or exchange them for histone variants, and some can even perform several of these activities . How theRemodelers of the ISWI family – like most other remodelers – can reposition nucleosomes along DNA in a process termed nucleosome sliding. ISWI’s activity is directly regulated by the histone H4 N-terminal tail and by DNA that flanks the nucleosome, so called linker DNA. The regulation imposed by these epitopes has direct consequences for the biological output of ISWI remodelers. By measuring the length of linker DNA, ISWI can generate arrays of evenly spaced nucleosomes , a charaHow ISWI senses the H4 tail is largely unknown. Evidence points to the ATPase domain of ISWI directly binding the H4 tail , consist17H18R19 within the H4 tail, which are part of a stretch of amino acids called basic patch  motif, so named because it follows the post-HSA motif in remodelers of the Snf2 family . Besides AutoN, the NTR contains additional motifs: an acidic region that we termed AcidicN, the ‘2 family , and a w2 family . Their fHere, we systematically interrogated the functions of all conserved motifs within the NTR by mutagenesis and a series of quantitative biochemical assays in vitro and in vivo. We paid particular attention to probe for possible crosstalk between these motifs and the H4 tail to understand its recognition process. Using protein crosslinking followed by mass spectrometry and protein structural modeling we obtained information about the general structural architecture of the NTR-ATPase module. With similar approaches, we mapped the H4-tail binding site. We interpret our results within a unified structural and functional framework for the combined inhibition of ISWI by the NTR and recognition of the histone H4 tail. Contrary to current models, we propose that AutoN does not occlude the binding pocket of the H4 tail and that inhibition by AutoN involves a more elaborate mechanism than simple mimicry of the H4 basic patch.Multiple sequence alignment of ISWI homologs revealed several sequence motifs in the NTR of ISWI . To asseSaccharomyces cerevisiae  at elevated temperatures , supporting the notion that mutations in the NTR destabilize ISWI structure.Toxicity at high expression levels could be caused by structural instability of the N-terminally truncated Isw1 variants. Indeed, analogous ISWI derivatives from Drosophila ISWI proteins.Toxicity of the Isw1 NTR deletions precluded a detailed analysis in vivo. Importantly, the in vivo results left open the possibility that NTR-deleted Isw1 was catalytically inactive. We therefore continued to study the function of the NTR motifs in vitro using purified 2, GB1, NusA, TrxA), fusion to or co-expression of chaperones  and inclusion of protease sites (3C) at three locations in the NTR to cleave off parts of the N-terminus after purification. The strategies that proved successful are summarized schematically in Although ISWI variants carrying mutations or deletions in the NTR generally expressed well, we failed to purify them using standard protocols. For each ISWI variant, we screened through a variety of expression and purification strategies to improve the yield of soluble protein. The strategies that we employed included fusion to solubility tags (ZΔppHSA) or both ppHSA and AT-hook  against the activity of wild-type ISWI (ISWIWT). We used saturating ATP and nucleic acid concentrations as indicated by control experiments with varying levels of ligands  and visualized the analogous surface on a homology model of ISWI  into this hypothetical binding interface in ISWI  on Lobe 2 of the ATPase module using the PISA algorithm , indicating that the efficiency of remodeling per hydrolyzed ATP was unchanged , further justifying the use of ISWIH483B for crosslinking experiments.We first tested whether mutagenesis of H483 impacted catalysis. The H483B mutation diminished the DNA- and chromatin-stimulated ATPase activity of full-length ISWI by fourfold each, a result that may not be surprising given that the mutation is located in the conserved ‘block D’ of Snf2 ATPases  Figure . Importanchanged . We concH483B was induced by UV irradiation, and the crosslinks were mapped by high accuracy mass spectrometry (MS) , which lacked the HSS domain and non-conserved N-terminal amino acids (data not shown).Crosslinking of full-length ISWItry (MS) . RemarkaSA motif ; Table 1In our previous work, we incorporated Bpa in a variety of places on Lobes 1 and 2 of the ATPase domain but never observed crosslinks to the NTR . We therab initio docking runs yielded a large cluster of models that identified the preferred binding site for AutoN and AcidicN . All three  AcidicN . Docking AcidicN . We valiStrikingly, AcidicN, which is predicted to be α-helical , contactDue to sequence similarity, the H4 tail and AutoN may compete for the same binding site . We thus26-648 in the presence of DNA , and bound these peptides to ISWIe of DNA . After ie of DNA . We mappe of DNA ; Table 2e of DNA .10.7554/Because the peptides may not exclusively bind ISWI in the physiological binding pocket, we pursued a second approach. We reconstituted entire nucleosomes bearing a photo-reactive benzophenone on the N-terminal tail of H4. Benzophenone labeling was achieved by chemical modification of single cysteine mutants of H4 (T1C and L10C). These nucleosomes bound to full-length ISWI and stimulated its ATPase activity like wild-type nucleosomes  suggestiTo identify the H4-tail binding pocket we turned to crosslink-guided in silico docking of the H4-tail peptide. We only used the five crosslinks for this analysis that passed stringent quality controls . Some ofInterestingly, the predicted docking interface of AcidicN was in close proximity to the H4-tail interface. This prompted us to investigate the function of AcidicN and – in the following section – its potential involvement in the H4-tail recognition process.+3 and ISWI+6 respectively  upregulated compared to ISWIWT , carrying amino acid substitutions just outside of the predicted AcidicN binding interface, however, retained its ability to discriminate chromatin over DNA in the ATP hydrolysis assay  also depended less on the H4 tail during remodeling than ISWIWT . The thi2RA mutation in AutoN  were found to be functionally important for ISWI enzymes  (leum (denoted ‘+’ in leum (‘++’), UASgal-A9-Pcyc (‘+++’), and UASgal-Pgal (‘++++’). Destination vectors were XbaI and ClaI digested and gel purified. Isw1 derivatives were PCR-amplified from yeast genomic DNA, gel purified and ligated into the destination vectors by Gibson assembly. All spotting assays employed untagged Isw1 variants. All constructs were sequence verified before transformation. As an empty vector control, the UAS promoter, coding and terminator sequences were removed from the Gal4pBS2-Pleum plasmid by AscI and MluI digest and subsequent self-ligation.in, USA) . The folYTT227 (TKO), YTT225 (DKO) and W1588-4c 5 linker and a TAP tag. YTT227 that expressed TAP-tagged Isw1 variants was induced with galactose as above, diluted to OD 0.1 and grown to OD 1.0 in 10 ml SC-Ura + Galactose media. YFMP047  YFMP047 , containDrosophila ISWIWT  served as the template for all ISWI variants. An overview over cloned ISWI variants is presented in 6-tag. To generate ISWIΔppHSA and ISWIΔppHSA; ΔAT-hook, a 3C cleavage site was introduced at the desired site by QuikChange mutagenesis or polymerase incomplete primer extension. The trigger factor gene was amplified from pTf16 (Takara Bio Inc.) and fused to the ISWIWT gene by Gibson assembly. ISWI+6 was subcloned into the pET-Z2 plasmid .A pPROEX-HTb–based expression plasmid with the gene encoding 6-tagged ISWIWT and its derivatives was performed essentially as described . Whereas ISWI+3 preparations were not directly compared, independent Z2-ISWI+6 preparations also yielded same results in remodeling assays.Expression and purification of Hisescribed  with theescribed . ISWIH48escribed . During E. coli cells. Protein expression was performed in 2x YT medium  supplemented with 34 mg/l chloramphenicol and 100 mg/l ampicillin. Expression of SNF2H was induced by addition of 0.4 mM IPTG at 18°C for approximately 18 hr. Bacteria cells were resuspended in 20 ml lysis buffer per 1 l culture  supplemented with protease inhibitors  per 1 l culture, and lysed by French Press (Thermo Spectronic) and ultrasonication (Branson). Per 1 l lysed bacteria culture, 1000 U Benzonase (Merck Millipore) were added. The lysate was clarified by centrifugation . The N-terminal His6-tagged SNF2H was purified by nickel affinity chromatography . An elution gradient was applied with 25 mM HEPES pH 7.0, 300 mM KCl and 400 mM Imidazole and enzyme-containing fractions were pooled. Contaminating DNA was removed by passing the sample over an anion exchange column  that was pre-equilibrated in SEC buffer . The flow-through of the column was collected. The protein sample was concentrated to 0.5–1 ml per 1 l of original E. coli culture in centrifugal filters . TEV protease (prepared in-house) was added to a final concentration of 0.075–0.15 mg/ml and the concentrated protein sample was dialyzed against 1 l SEC buffer overnight in dialysis tubing . The protein sample was loaded onto a size exclusion chromatography column  pre-equilibrated in SEC buffer. Elution fractions were pooled according to purity and, as necessary, concentrated and dialyzed into storage buffer  for at least 16 hr.A pBH4-based expression plasmid encoding full-length human SNF2H  was transformed into Rosetta competent Drosophila histones were purified as described  (escribed . The 187escribed . Mononuc arrays) . The con2, 0.1 mM EDTA, 10% glycerol, 0.2 g/l BSA and 1 mM DTT at 26°C in the presence of a ATP regenerating system as described  and of nucleic acids ligands were used . Saturation of DNA and chromatin was controlled by varying the concentration of the ligands at least 16-fold (ATP hydrolysis was monitored by an NADH-coupled ATP hydrolysis assay . Saturat 16-fold . Occasio2+ (1 mM) and KpnI (2 U/ml). Reactions were quenched with SDS (0.4%) and EDTA (20 mM) before the samples were deproteinized, ethanol precipitated and resolved by agarose gel electrophoresis benzophenone (Sigma) was dissolved to 100 mM in N,N-Dimethylformamide (DMF) and added to a final concentration of 3 mM to denatured single cysteine variants of H4 (1 mg/ml) in 20 mM Tris/HCl pH 7.1, 7 M Guanidine-HCl, 5 mM EDTA, 2 mM TCEP for 2 hr at room temperature. After a 3 hr incubation in the dark, the labeling reaction was stopped by adding 20 mM DTT for 20 min.26-648 (0.1 mg/ml) and a histone H4 peptide comprising the 24 N-terminal amino acids of H4 carrying a Bpa substitution at position 1 or 10 was carried out in the presence of 13 µM 59 bp DNA duplex in 25 mM HEPES-KOH, pH 7.6, 50 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 10% glycerol and 1 mM DTT for 3 hr as above. Samples were subsequently digested with benzonase before further processing. Crosslinking between Bpa variants of ISWI (H483B) was carried out as described (UV-Crosslinking was performed in uncoated 384-well plates or 96-well plates (Greiner) on ice using the 365 nm irradiation of a BioLink UV-Crosslinker (Peqlab) for the indicated durations. Crosslinking between benzophenone-labeled nucleosomes  and stoichiometric amounts of ISWI or SNF2H was performed in 20 mM Tris/HCl, pH 7.7, 100 mM KCl, 0.1 mM EDTA, 3 mM DTT. Crosslinking between ISWIescribed .UV-irradiated samples and unirradiated control samples were separated by SDS-PAGE and Coomassie stained. Protein bands were excised and trypsin digested for subsequent mass spectrometry as described .6). The ten most intense peptide ions with charge states between 3 and 5 were sequentially isolated to a target value of 1 × 105, and fragmented at 27% normalized collision energy. Typical mass spectrometric conditions were: spray voltage, 1.5 kV; no sheath and auxiliary gas flow; heated capillary temperature, 250°C; ion selection threshold, 33.000 counts.For LC-MS/MS, 5 µl were injected in either an Ultimate 3000 system (Thermo) and desalted on-line in a C18 micro column  or desalted offline using C18 Stagetip and injected in an Ultimate 3000 RSLCnano system (Thermo). Desalted sample was then separated in a 15 cm analytical C18 micro column  with a 40 to 60 min gradient from 5% to 60% acetonitrile in 0.1% formic acid. The effluent from the HPLC was directly electrosprayed into an LTQ-Orbitrap XL as described before  or a Q EEach Thermo binary raw file was converted to a dta file using Decon2LS  or to anCrosslink candidates were independently validated by the authors J.L., S.P., N.H. and F.M.-P. and rated as high, medium and low confidence. The validation comprised a general assessment of the spectrum quality, removal of wrong product ion assignments, and evaluation of the actual evidence for the presence of the two peptides within the crosslink. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD005831.www.attract.ph.tum.de/peptide.html). The termini of the motifs (‘peptides’) were left uncharged, other parameters were set to the default values as described (Interactions between NTR motifs and the histone H4 tail with Lobe 2 were modeled using the fully blind peptide-protein docking protocol pepATTRACT  in the Aescribed . Brieflyescribed . The topescribed  and a shescribed . The pepThe structure of ISWI ATPase Lobe 2 (residues 352–637) was modeled by homology from the structure of Chd1 (PDB 3MWY) using MODELLER . We perf– provided three unique amino acid linkages. These linkages were used as upper harmonic distance restraints with a maximum distance of 20 Å to guide the modeling  (www.pymol.org).During docking of the histone H4 tail (residues 1–20) to Lobe 2, we used the five high confidence crosslinks , which –rotocol) . After m In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom, Jerry L Workman (Reviewer #1), is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor.Thank you for submitting your work entitled "Concerted regulation of ISWI by an autoinhibitory domain and the H4 N-terminal tail" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Reviewers 2 and 3 raise several technical issues with the study that could not be addressed in a timely manner. All three reviews are included below. We hope that you will find the comments in these reviews helpful in the continuation of this study.Reviewer #1:This is a nice biochemical study examining the auto regulation domains of the ISWI ATPase chromatin remodeling complex. The manuscript provides novel data that "correct" aspects of previous models of ISWI regulation while confirming other aspects. The most important finding in the manuscript is that the ppHSA domain of the N terminal region binds to lobe 2 of the ATPase domain to suppress the ATPase activity in the absence of the H4 tail similar to chromodomains of CHD1 binding the ATPase domain.1) While high profile papers have been published on examining auto regulation of ISWI including some which this study corrects, the impact of this work would be greatly enhanced if it were demonstrated that the ppHSA domain in other chromatin remodelers  were also found to bind and regulate their ATPases. Even one more example of this regulation would turn this detailed study of ISWI regulation into revealing novel mechanisms for a whole group of remodelers. I strongly recommend the authors try the cross linking experiment with another remodeler.2) ISWI is complexed with other proteins. The manuscript should discuss this in the Introduction and any implications other subunits might have for the activities shown here in the Discussion.3) The raw data for the restriction enzyme site accessibility assays should be shown somewhere in the manuscript.Reviewer #2:The ISWI chromatin remodeling enzymes are activated by interaction with the histone H4 N-terminal tail domain. In the current model for ISWI activation, the H4-tail binding site is blocked by the AutoN-motif in the N-terminal region (NTR) of ISWI, which contains a sequence very similar to the basic patch in the H4 tail. Consequently, it was proposed that H4 and AutoN compete for the same site on the ISWI ATPase domain. Using a combination of site-specific cross-linking, mass spectroscopy and structural modeling, the authors have mapped the approximate binding sites of the NTR and the H4 tail within purified ISWI enzymes in vitro. They deduce that the NTR and the H4 tail both bind to lobe 2 of the ATPase domain, but not at the same site. Their data are therefore inconsistent with the current model. They propose an allosteric model instead. This study also identifies two other important regions in the NTR: the ppHSA motif, which is shown to contribute to the structural stability of the NTR domain, and the AcidicN motif which, together with AutoN, is important for H4-dependent enzyme activation. Mutations in AcidicN or AutoN activate the ISWI ATPase in the absence of chromatin and allow remodeling of nucleosomal arrays even if the H4 tail is absent. These two motifs together confer H4-tail dependence on the enzyme. The authors also point out that ISWI is more similar to Chd1 than expected, given the lack of sequence homology between the NTRs of the two enzymes.Drosophila ISWI and human hSNF5) in the presence and absence of nucleosomes or H4 peptide, with consistent results. They also assessed the effects of NTR mutations in vivo, although this had to be done in yeast, with the ISW1 enzyme, which is unlike ISWI because it is inactive without Ioc subunits.Overall, this is a thorough and careful study with interesting and important conclusions concerning the mechanism of chromatin remodeling by ISWI enzymes. The authors used both UV and chemical cross-linking to map binding sites on two different ISWI enzymes , it is critical to determine if the modification interferes with the normal function of the protein. The authors have not shown if the modifications in ISWI or the H4 tail peptide interfered with their binding or the associated enzymatic activity. Since their results and conclusions are heavily dependent on these crosslinking data, it is imperative for them to do the necessary controls. They need to determine if the modifications interfered with the ATPase and nucleosome remodeling activity of ISWI and if modifications in the H4 tail interfere with it stimulating the ATPase activity of ISWI with DNA. The other approach uses chemical crosslinking of proximal lysines with a reagent called BS3 and identification of crosslinked pairs by mass spectrometry. The authors failed to present the full spectral dataset of these crosslinking experiments and how they were validated or confirmed. Instead they indicate only one of the potentially many crosslinks they obtained and in this case have picked the one that is consistent with the model. The data needs to be fully presented and properly vetted before it can be accepted.There are additional concerns about the photocrosslinking and the use of Bpa because of the type of photochemistry it entails. It is reasonably probable that the photocrosslinking captures a minor population of the protein and may not be a good representation of the bulk protein and the true state of the protein. Possibly only an inactive fraction of the protein is what is being productively crosslinked and thus additional tests need to be in place to confirm the protein interactions. The site-directed mutagenesis unfortunately do not accomplish this purpose, but only demonstrate that particular residues are important for the enzyme's activity. This is further compounded because unlike aryl azides, benzophenone has the ability to cycle between its excited state and ground state until it reaches the appropriate substrate and it has a high preference for Met. Therefore these reagents can search for quite a while until the right substrate presents itself and for these reason make it even more probable of trapping an inactive or inappropriate conformation that may occur infrequently.A major concern of this paper is that there is a lot of in silico work done that is more speculative in terms of determining the binding surface of NTR in the ATPase domain rather than direct experimental data. Based on only one crosslinked site and additional assumptions does not provide a high confidence in the sites proposed by the authors. This non-objective way of showing the data appears multiple times in this manuscript. And yet this is a major point of the manuscript. Also when they dock the H4 tail on the surface of the ATPase domain based on crosslinking at two sites on H4, they only show one model instead of the family of possible interactions obtained by their modeling method. The choice of only one site on the surface of the ATPase domain seems to be inadequate for probing the interactions of the NTR with the ATPase domain. The rationale for choosing the site based on the premise that it should be the same or similar to that of the chromodomains of Chd1 when there is no sequence homology between these two region doesn't have much supporting evidence and is very tenuous. There should at least by other sites within lobe 1 that are examined as controls in order to confirm the specificity of the observed contacts.Drosophila that contains most of the full length ISWI including the HSS tail, but then revert to one missing the HSS region. And then a third variation is using the human SNF2H when doing the chemical crosslinking with BS3. All of these variations are most likely going to matter. I think it is important for the authors to do all the experiments with the ISWI construct containing the HSS region, because the HSS region is very likely to matter in this regard.The choice of protein constructs was somewhat curious and varied. The authors start with ISWI from The other point of the paper is the ppHSA motif stabilizes the interactions of the NTR, however this does not seem to be well supported. The authors examine recombinant constructs of ISWI with serial truncations from the N-terminus. They examined the stabilities of these proteins using differential scanning fluorimetry and concluded the deletion of the extreme N-terminus containing the ppHSA motif is less stable than the full length ISWI. The data however shows that the truncated proteins are more complex that full length ISWI and did not give a simple one curve. Indeed there seems to be components within the sample that may actually be more stable than full length. The purity of these proteins as visualized by SDS-PAGE analysis are not very high and could lead to the complicated scan of these truncated proteins. The author average all this together to give them a lower thermal stability for the truncated constructs and is misleading. The also conclude that the ppHSA motif dies not contribute to the regulation of the activity of ISWI; however they see a 3.6 fold reduction when this motif is removed. I would have thought that was a substantial effect and should not be discounted. Reviewer #1:This is a nice biochemical study examining the auto regulation domains of the ISWI ATPase chromatin remodeling complex. The manuscript provides novel data that "correct" aspects of previous models of ISWI regulation while confirming other aspects. The most important finding in the manuscript is that the ppHSA domain of the N terminal region binds to lobe 2 of the ATPase domain to suppress the ATPase activity in the absence of the H4 tail similar to chromodomains of CHD1 binding the ATPase domain.1) While high profile papers have been published on examining auto regulation of ISWI including some which this study corrects, the impact of this work would be greatly enhanced if it were demonstrated that the ppHSA domain in other chromatin remodelers  were also found to bind and regulate their ATPases. Even one more example of this regulation would turn this detailed study of ISWI regulation into revealing novel mechanisms for a whole group of remodelers. I strongly recommend the authors try the cross linking experiment with another remodeler.We agree that additional crosslinking experiments with other remodeler families containing a ppHSA domain would be very interesting. We feel however that this is experimentally out of reach and may be beyond the scope of our study. Purifying megadalton complexes such as Ino80 and Snf2 is technically demanding, and incorporation of the photo-crosslinkable amino acids is not trivial either. A comparative analysis of the role of the ppHSA motif across multiple remodeler families -while certainly worthwhile in its own right- would possibly require a year or more for completion. In addition, it would not substantiate major conclusions of our study, which relate to the mechanism of H4 tail recognition by ISWI and the structure and function of its N-terminal region (NTR). For these reasons, we opted not to pursue crosslinking experiments with other remodelers.2) ISWI is complexed with other proteins. The manuscript should discuss this in the Introduction and any implications other subunits might have for the activities shown here in the Discussion.As suggested, we now include more references in the text to the presence of additional subunits in ISWI complexes. Their function is not well understood but they potentially provide a whole new layer of regulation:Introduction section: “Cells also adjust the subunit composition of remodeling complexes during development .”Introduction section, paragraph 4: “Another layer of regulation is imposed by the non-catalytic subunit termed ACF1, which associates with ISWI and sequesters the H4 tail under certain conditions .”Discussion section: “Our data do not rule out additional binding sites on other domains and on ISWI’s partner subunit ACF1 as proposed earlier .”Discussion section, last paragraph: “Sensitive biophysical assays will be instrumental in the future for resolving conformational changes that may occur during H4 tail recognition and for understanding their functional importance in ISWI complexes.”3) The raw data for the restriction enzyme site accessibility assays should be shown somewhere in the manuscript.As requested, we incorporated the raw data .Reviewer #2:The ISWI chromatin remodeling enzymes are activated by interaction with the histone H4 N-terminal tail domain. In the current model for ISWI activation, the H4-tail binding site is blocked by the AutoN-motif in the N-terminal region (NTR) of ISWI, which contains a sequence very similar to the basic patch in the H4 tail. Consequently, it was proposed that H4 and AutoN compete for the same site on the ISWI ATPase domain. Using a combination of site-specific cross-linking, mass spectroscopy and structural modeling, the authors have mapped the approximate binding sites of the NTR and the H4 tail within purified ISWI enzymes in vitro. They deduce that the NTR and the H4 tail both bind to lobe 2 of the ATPase domain, but not at the same site. Their data are therefore inconsistent with the current model. They propose an allosteric model instead. This study also identifies two other important regions in the NTR: the ppHSA motif, which is shown to contribute to the structural stability of the NTR domain, and the AcidicN motif which, together with AutoN, is important for H4-dependent enzyme activation. Mutations in AcidicN or AutoN activate the ISWI ATPase in the absence of chromatin and allow remodeling of nucleosomal arrays even if the H4 tail is absent. These two motifs together confer H4-tail dependence on the enzyme. The authors also point out that ISWI is more similar to Chd1 than expected, given the lack of sequence homology between the NTRs of the two enzymes.Overall, this is a thorough and careful study with interesting and important conclusions concerning the mechanism of chromatin remodeling by ISWI enzymes. The authors used both UV and chemical cross-linking to map binding sites on two different ISWI enzymes (Drosophila ISWI and human hSNF5) in the presence and absence of nucleosomes or H4 peptide, with consistent results. They also assessed the effects of NTR mutations in vivo, although this had to be done in yeast, with the ISW1 enzyme, which is unlike ISWI because it is inactive without Ioc subunits.We thank the reviewer for appreciating our efforts as “a thorough and careful study with interesting and important conclusions concerning the mechanism of chromatin remodeling by ISWI enzymes”.I have only one important concern. The weak point of the study is that the authors have to use structural modeling to interpret the cross-linking pattern. There is no good way around this problem in the absence of a crystal structure for a complete ISWI enzyme. The structural model in We realize that we did not sufficiently explain the modeling procedure and we would like to clarify this point. Contrary to the reviewer’s concern, the H4 tail was not modeled as an α-helix. Indeed, the modeling algorithm was not fed with any information on the tail structure. Accordingly, the family of 383 models that fit the distance restraints provided by the crosslinks, contained both helical und ‘unstructured’ H4 tails . The modeled interaction surface  was and still is contained in the colored interaction surface. See also our response to reviewer #3.Reviewer #3:The authors analyze the functional role of three conserved motifs in the N-terminus of ISWI, an ATP-dependent chromatin remodeler that spaces nucleosomes. Previous studies had focused on what is called the AutoN domain and its sequence similarity with the histone H4 tail. Earlier data had suggested the AutoN negatively regulates the ATPase and remodeling activities of ISWI which can be competed away and relieved by the H4 tail. They present a different explanation in which the AutoN and a flanking region called AcidicN bind to a region of the ATPase domain different than the H4 tail. Instead of competing the H4 tail, AutoN and AcidicN cooperate together to regulate ISWI in an H4 tail dependent manner. A third motif called ppHSA is also presented to bind to lobe 2 of the ATPase domain and stabilizes the interactions of AutoN and AcidicN with lobe 2.They map the interactions of the AutoN, AcidicN, ppHSA and H4 tail with the ATPase domain of ISWI using a combination of photoaffinity labeling and chemical crosslinking. They incorporate photoreactive amino acid analogs either into a site of the ATPase domain they propose to be the binding site of the NTR region or into the H4 tail. Photoaffinity labeling is a powerful technique, but there are certain concerns associated with this approach which need to be considered. Whenever a modification is made, especially one as bulky as benzophenone , it is critical to determine if the modification interferes with the normal function of the protein. The authors have not shown if the modifications in ISWI or the H4 tail peptide interfered with their binding or the associated enzymatic activity. Since their results and conclusions are heavily dependent on these crosslinking data, it is imperative for them to do the necessary controls. They need to determine if the modifications interfered with the ATPase and nucleosome remodeling activity of ISWI and if modifications in the H4 tail interfere with it stimulating the ATPase activity of ISWI with DNA.We agree that photo-crosslinking is a powerful technique but that the functional consequences of incorporation of the benzophenone moiety into proteins need to be assessed. In the revised manuscript, we now show the requested quality controls that probe whether benzophenone incorporation affects function.We include now a figure that shows that benzophenone-containing H4 tail peptides retain their ability to stimulate ATP hydrolysis , that beH483B hydrolyzed ATP and remodeled nucleosomes 3- to 4-fold more slowly than ISWIWT (new H483B by its NTR was affected by the H483B mutation. Mutagenesis of the NTR (+3 and +6 mutation) had analogous effects on ISWIWT and ISWIH483B . Some of these modes may not be strongly populated or functionally active as crosslinking can in principle trap fleeting intermediates.”H483B, are fully supported by a crystal structure that was published during the revision of this manuscript . This structure therefore provided strong orthogonal validation of our crosslinking data, and we discuss our results in the light of the new structure briefly in the Discussion section now.We would like to point out that all our crosslinks, including the crosslinks formed by ISWIA major concern of this paper is that there is a lot of in silico work done that is more speculative in terms of determining the binding surface of NTR in the ATPase domain rather than direct experimental data. Based on only one crosslinked site and additional assumptions does not provide a high confidence in the sites proposed by the authors. This non-objective way of showing the data appears multiple times in this manuscript. And yet this is a major point of the manuscript.To clarify, we performed in silico docking only twice. We docked the H4 tail, and we docked AutoN- AcidicN. Docking of the H4 tail was well constrained by the experimentally observed crosslinks between H4 and the enzyme, for which we comprehensively disclosed all supporting spectra ; Table 3The reviewer is right, however, that we did not have distance restraints for the second docking that we performed, docking of AutoN-AcidicN. We therefore validated the proposed interface on Lobe 2 by mutagenesis during the revision new . RemarkaAlso when they dock the H4 tail on the surface of the ATPase domain based on crosslinking at two sites on H4, they only show one model instead of the family of possible interactions obtained by their modeling method.We apologize for being unclear about how the modeling results were being displayed. We do, actually, show the results of the whole family of models in To avoid giving the impression that we only show results of one model, we simply deleted the top ranking H4 tail model from the figure during the revision. Importantly, all information about the family of docked structures was and still is represented in the yellow to red interaction surface. See also our response to a comment from reviewer #2.The choice of only one site on the surface of the ATPase domain seems to be inadequate for probing the interactions of the NTR with the ATPase domain. The rationale for choosing the site based on the premise that it should be the same or similar to that of the chromodomains of Chd1 when there is no sequence homology between these two region doesn't have much supporting evidence and is very tenuous. There should at least by other sites within lobe 1 that are examined as controls in order to confirm the specificity of the observed contacts.This is a good point. We have done the requested specificity control already in a previous study where we inserted Bpa residues at different sites into the ISWI ATPase domain, including into Lobe 1. None of the positions formed crosslinks to the ISWI NTR. The NTR-Lobe2 interaction therefore appears to be specific to the region in the vicinity of H483. We have made the following changes to the text:Subsection “The NTR contacts Lobe 2 of the ATPase domain”: “In our previous work, we incorporated Bpa in a variety of places on Lobes 1 and 2 of the ATPase domain but never observed crosslinks to the NTR . We therefore suggest that the ppHSA motif specifically docked to a location in proximity of amino acid 483 in Lobe 2.”Of note, all our crosslinks between Lobe 2 and the NTR are fully consistent with a recent structure that came out during the revision of this manuscript . Thus, our crosslinking data and the crystal structure mutually validate each other.The choice of protein constructs was somewhat curious and varied. The authors start with ISWI from Drosophila that contains most of the full length ISWI including the HSS tail, but then revert to one missing the HSS region. And then a third variation is using the human SNF2H when doing the chemical crosslinking with BS3. All of these variations are most likely going to matter. I think it is important for the authors to do all the experiments with the ISWI construct containing the HSS region, because the HSS region is very likely to matter in this regard.Drosophila ISWI. Also the crosslinking experiments using benzophenone-labeled nucleosomes and ISWIH483Bincluded the HSS domain.The reviewer makes the point that we should use ISWI that includes the HSS domain for all experimental approaches. Maybe we were a bit unclear about this, but we did. The yeast Isw1 experiments included the HSS domain, as did the NTR mutants that we created in △HSS to a BPA-containing H4 tail peptide and crosslinking of a ISWIH483B, △HSS double mutant). Importantly, results from these experiments merely supported the evidence collected already with full-length ISWI so that none of our conclusions rest on results obtained with ISWI△HSS.In addition, we chose to support selected conclusions with independent data that were obtained with HSS-deleted ISWI , fusion to or co-expression of chaperones  and inclusion of protease sites (3C) at three locations in the NTR to cleave off parts of the N-terminus after purification. The strategies that proved successful are summarized schematically in The also conclude that the ppHSA motif dies not contribute to the regulation of the activity of ISWI; however they see a 3.6 fold reduction when this motif is removed. I would have thought that was a substantial effect and should not be discounted.We agree and revised the wording. Please note, however, the ppHSA motif relative to AcidicN seems to play at most a modest role for catalysis.Subsection “The ppHSA motif does not substantially contribute to catalysis”: “In conclusion, ATPase and remodeling data suggested that both ppHSA and AT-hook are not absolutely required for catalysis in vitro. The modest decreases in remodeling activities could be due to lower stability of these enzymes (see above).”ReferencesStructure and binding of the H4 histone tail and the effects of lysine 16 acetylation. Phys Chem Chem Phys, 2011. 13(7): p. 2911-21.1. Yang, D. and G. Arya, Identification of a non-basic domain in the histone H4 N-terminus required for repression of the yeast silent mating loci. EMBO J, 1992. 11(6): p. 2201-9.2. Johnson, L.M., G. Fisher-Adams, and M. Grunstein, Structure and function of the core histone N-termini: more than meets the eye. Biochemistry, 1998. 37(51): p. 17637-41.3. Hansen, J.C., C. Tse, and A.P. Wolffe,


Survival represents the single most important indicator of successful HIV treatment. Routine monitoring fails to capture most deaths. As a result, both regional assessments of the impact of HIV services and identification of hotspots for improvement efforts are limited. We sought to assess true mortality on treatment, characterize the extent under-reporting of mortality in routine health information systems in Zambia, and identify drivers of mortality across sites and over time using a multistage, regionally representative sampling approach.3 (IQR 111–312), the 2-year cumulative incidence of mortality increased from 1.9% (95% CI 1.7%–2.0%) to a corrected rate of 7.0% (95% CI 5.7%–8.4%)  and from 2.1% (95% CI 1.8%–2.4%) to 8.3% (95% CI 6.1%–10.7%) (new ART users). Revised provincial mortality rates ranged from 3–9 times higher than naïve rates for new ART users and were lowest in Lusaka Province (4.6 per 100 person-years) and highest in Western Province (8.7 per 100 person-years) after correction. Corrected mortality rates varied markedly by clinic, with an IQR of 3.5 to 7.5 deaths per 100 person-years and a high of 13.4 deaths per 100 person-years among new ART users, even after adjustment for clinical  and contextual  factors. Mortality rates  were highest year 1 after treatment at 4.6/100 person-years (95% CI 3.9–5.5), 2.9/100 person-years (95% CI 2.1–3.9) in year 2, and approximately 1.6% per year through 8 years on treatment. In multivariate analysis, patient-level factors including male sex and pretherapy CD4 levels and WHO stage were associated with higher mortality among new ART users, while male sex and HIV disclosure were associated with mortality among all ART users. In both cases, being late (>14 days late for appointment) or lost (>90 days late for an appointment) was associated with deaths. We were unable to ascertain the vital status of about one-quarter of those lost and selected for tracing and did not adjudicate causes of death.We enumerated all HIV infected adults on antiretroviral therapy (ART) who visited any one of 64 facilities across 4 provinces in Zambia during the 24-month period from 1 August 2013 to 31 July 2015. We identified a probability sample of patients who were lost to follow-up through selecting facilities probability proportional to size and then a simple random sample of lost patients. Outcomes among patients lost to follow-up were incorporated into survival analysis and multivariate regression through probability weights. Of 165,464 individuals , median CD4 201 cells/mmHIV treatment in Zambia is not optimally effective. The high and sustained mortality rates and marked under-reporting of mortality at the provincial-level and unexplained heterogeneity between regions and sites suggest opportunities for the use of corrected mortality rates for quality improvement. A regionally representative sampling-based approach can bring gaps and opportunities for programs into clear epidemiological focus for local and global decision makers. To improve estimates of mortality for patients who initiate HIV treatment, Charles Holmes and colleagues use a multistage sampling approach to find and trace health outcomes for patients lost to follow-up after starting antiretroviral therapy in Zambia. Previous studies from cohorts in South Africa and parts of East Africa have suggested that site-level reporting of mortality is incomplete.We wanted to understand the degree to which this phenomenon was impacting HIV outcomes at a broader scale, in this case at the provincial level in Zambia, a country with one of the highest burdens of HIV.We also wanted to gain an in-depth understanding of differences between the outcomes of clinical care sites in order to assess the role of mortality as a potential quality improvement target.From a source population of patients in 4 provinces  who visited government-operated HIV treatment sites in these provinces, we conducted a multistage sampling approach of a stratified selection of sites and a random sample of patients lost to follow-up.Lost patients were traced and their vital status was ascertained, which was used to enable a corrected regionally representative estimate of survival after starting antiretroviral therapy (ART) as well as corrected site-specific mortality estimates.Of 165,464 individuals, the 2-year cumulative incidence of mortality increased from 1.9% to 7.0% for all ART users and from 2.1% to 8.3% for new ART users, and provincial-level mortality rates rose 3- to 8-fold once corrected for true outcomes.Being late (>14 days late for appointment) or lost (>90 days late for an appointment) was associated with death.Deaths are under-reported within the Zambian HIV program, and mortality rates are highly variable across sites and provinces.Our findings enable national- and global-level policy makers to correct existing underestimates of mortality, link these data to quality improvement efforts, and reprioritize interventions to target regional and site-level reductions in mortality as a goal of HIV programs.We have also established that this methodology is feasible for use as a representative surveillance tool for accurate monitoring of provincial and potentially national levels of mortality, even as vital status registries and data systems are further developed and strengthened. As the global response to HIV moves towards universal treatment, with the intent of ending AIDS as a public health crisis by 2030, a rigorous understanding of contemporary program effectiveness demonstrates progress as well as identifies important opportunities for improvement . AlthougDespite the importance of mortality, relatively little data exist about long-term survival after initiation of HIV treatment in lower- and middle-income countries . In SoutIn order to rigorously assess mortality, we apply for the first time, to our knowledge, a multistage sampling design to obtain representative estimates of mortality in 4 provinces in Zambia as well as site-level estimates with enough precision to identify site-to-site variation. This approach offers a rigorous estimate of (1) the magnitude of deaths after starting antiretroviral therapy (ART) among those already on treatment at the start of the study and among new treatment initiates, (2) when deaths occur, (3) which groups are at highest risk of death, and (4) whether these factors differ by region, facility, or other important factors such as prior healthcare utilization patterns. Both the approach and findings have implications for assessment of program effectiveness and program improvement efforts.The protocol and study were approved by the University of Zambia Biomedical Research Ethics Committee (UNZABREC) (004-06-14) and the IRB of the University of Alabama, Birmingham, School of Medicine (F160122006), and the submitted protocol is available in the supplemental material. In this paper, we used a number of analytic innovations not specified in the analysis protocol, including the use of a modified Lorenz curve to depict the distribution of deaths across facilities and survival analyses of mortality in which deaths are classified by retention histories. In addition, analyses of pretreatment experience as specified in the protocol are also planned but not included in this paper.The target population for this analysis is the contemporary population of HIV-infected adults on ART in Zambia. We considered “contemporary” to be patients who had a clinical encounter while on ART (including the ART initiation visit) in the 24 months before the evaluation, and they are referred to as “all ART users” and provide the most complete picture of treatment outcomes. We also designate a subpopulation of this group as “new ART users,” i.e., those who initiated ART during the 24-month period prior to the evaluation—this group is more readily comparable to outcomes from other analyses of those starting ART. Our source population consisted of patients in 4 provinces  who visited government-operated HIV treatment sites in these provinces, each of which receives technical assistance support from Centre for Infectious Disease Research in Zambia (CIDRZ), a local Zambian organization.The analysis population was enumerated using a multistage sampling approach to yield estimates representative of the entire population of patients on treatment as well as to enable comparison, through stratification, across 4 provinces and 3 facility types . We selected using probabilities proportional to size a minimum of 2 to 10 facilities from each of 12 strata defined by facility type and province for a total of 32 facilities. In each selected facility, we enumerated all adults on ART who made a visit in the previous 24 months and identified loss to follow-up from this cohort (defined as >90 days late at the time of sampling). From patients lost to follow-up, we selected a simple random sample for tracing inverse proportional to size. This approach seeks to enable both a regionally representative estimate of survival after starting ART and site-specific estimates with approximately 95% confidence estimates from 5% to 15% in a setting where mortality is estimated to be 10% [As described in previous work, we used data from an electronic medical records system to enumerate the cohort and to obtain sociodemographic and clinical data. Peer health workers traced lost patients intensively in the community to ascertain vital status . TracingWe estimated the cumulative incidence of mortality using the Kaplan Meier (KM) approach as well as simple mortality rates. In KM estimates, we treated time zero as the date of ART initiation. Patients who started ART before the observation period were left-truncated, which yields an estimate of survival after ART initiation during the 2-year period of observation of this study that is contingent on surviving into this period. This approach is analogous to life expectancy estimates. Mortality estimates were carried out for all ART users as well as stratified by new ART initiators during the 2-year observation period and those already on ART at the start of the current period. These analyses are intended to convey the totality of the contemporary experience of the patient population, including both that of those on treatment for extended periods of time and that of important subpopulations. We carried out mortality estimates both using only deaths known to the program before tracing and after incorporating findings from tracing lost patients to “correct” estimates through use of probability weights. These individual weights were combined with facility-level weights for estimates in the entire patient population.y-axis displays the cumulative proportion of excess deaths and the x-axis the cumulative population proportion after adjustment for patient  as well as facility factors  [In additional survival analyses, we used the Aalen Johansen method to estimate the cumulative incidence of death events classified by their current and previous engagement history using the reweighted data. We defined patients as having “died in care” if they had died within 30 days of their last visit; the remaining patients were classified as having “died out of care.” In addition, patients were categorized as “previously lost” if they had had at least 1 episode of being out of care for more than 90 days after an appointment in the past and returned to care prior to our study sampling and “previously late” if they had at least 1 episode of being late for an appointment by more than 30 days but less than 90 days prior to our study sampling. These categories yielded 6 engagement states for the analysis ,15. We e volume) . Excess  volume) . The LorWe used 3 approaches to characterize deaths over time. First, we graphically displayed the retention history with all patients who died represented by a single horizontal bar. The length of the bar represents time since ART initiation, and colors represent time spent in care, late for care (>30 days and <90 days late for a visit), or lost (>90 days late for last appointment). Second, we examined mortality rates in each year after ART initiation among all patients as well as only among those patients who had made a visit in the last 2 years (and therefore survived into the current era) and tested for significance in the change over time. Third, we added time-varying covariates—(1) engaged, never late or lost; (2) engaged, but with a history of being late or lost; (3) late without a history of previous episodes of being late or lost; (4) late with a history of being lost; and (5) being lost (defined as being >90 days since last appointment)—in multivariable regression to capture the association between lapses in retention states and mortality. Based on the revised estimates, we quantified the fraction of deaths that fell into each category of visit histories and in each of the segments of time after ART initiation. We calculated the “population attributable fraction” of all deaths that occurred in each of these states—an estimate of how much mortality could be avoided if all measured characteristics were set at the lowest level of risk.In a network of 64 HIV delivery facilities operated by the Zambian Ministry of Health and supported by CIDRZ across 4 provinces, we identified a total of 165,464 individuals on ART and who had made a visit in the 2 years before the project period , which included 49,129 individuals newly initiating ART during that period and 116,335 individuals already on treatment at the opening of the observation period .3 (IQR 111–312), and 58% were WHO stage 1 or 2 at ART initiation. New ART users were similar demographically but had a higher median CD4 level at ART initiation of 262 cells/mm3 (IQR 138–288), and 65% were WHO stage 1 or 2 at enrollment. The median time on ART was 1,142 days  for all ART users in the cohort and 255 days (IQR 63–407) in new ART starters. The median date of ART initiation for all users was 14 February 2012 (IQR 03 June 2009–23 January 2014) and for new ART users was 24 July 2014 (IQR 3 February 2014–23 December 2014).In the entire cohort , 64% werRoutine program data before the tracing exercise suggested that 1% of all patients had died. A total of 17% of all patients were lost to follow-up, and this fraction lost was highly variable across clinic sites (IQR 10%–31%). Among the 28,111 lost patients from the entire cohort, we made an attempt to trace 2,892 (10%). Tracing efforts led to updated vital status in 2,163/2,892 (75%) of patients, but ascertainment of vital status varied from 54% to 93% across all sites . Among pThe deaths during the first year on treatment occur mostly in persons who have not missed appointments. There is an increase over time in the fraction of deaths that occur in patients who are either late for a visit or who have a history of being late or lost from care . The cumMortality varied markedly across geographical areas and facilities. At the provincial level, revised estimates of mortality were higher than naïve estimates, ranging in all ART users from 3-fold higher in Eastern and Southern Province to 8-times higher in Lusaka Province and in new ART users from 3-fold higher in Eastern Province to 9-fold higher in Lusaka Province . The higMultivariable regression models using patient and facility characteristics at ART initiation found that male sex, CD4 level (≥200 cells/μL) at ART initiation, and higher WHO stage were associated with higher mortality in new ART users, whereas facility size and type  were protective. For those already on ART at the start of observation, male sex; province; facility size, type, and setting; and disclosure of HIV status at treatment initiation were associated with mortality .A modified Lorenz curve to capture how “concentrated” mortality was across facilities showed that approximately half (57%) of excess deaths occurred in 15% of the population . The Ginp-value = 0.46). When examined alone, patients who started ART during the observation time for this study (new ART users) displayed the highest mortality in the first 90 days of treatment, at 7.9 per 100 person-years (95% CI 6.3–10.0); this fell to 4.6 per 100 person-years from 91–180 days and then fell to 3.3 per 100 person-years (95% CI 2.0–5.8). Regression models with time-varying factors capturing changing retention status found that being late and being lost were both associated with mortality, but a history of lateness or loss among those in care did not predict mortality after adjustment for other sociodemographic and clinical factors. These associations were more pronounced among all ART users than among new ART users , fell to 2.9 per 100 person-years (95% CI 2.1–3.9) in the second year, and then remained steady at approximately 1.6% per year for up to 8 years on treatment. The overall mortality rate in this group was 1.7 per 100 person-years (95% CI 1.5–2.0), and a test of difference in mortality rates by year on treatment for up to 10 years was not statistically significant (RT users .We estimated that 38.1% (95% CI 33.1%–43.4%) of mortality occurred among individuals in their first year on treatment, while the remaining 61.8% (95% CI 56.6%–66. 9%) was distributed among patients who had been on treatment for longer periods of time . For patDespite the success of implementing HIV treatment in high-burden settings in Africa, we have identified high rates of mortality among those on treatment in Zambia, substantial under-reporting of deaths, and marked heterogeneity among provinces and sites. We applied for the first time, to our knowledge, a multistage sampling approach that included a sample of both sites and patients lost to follow-up in order to develop both provincially representative and site-level revised mortality estimates for an HIV program. In a sample representing over 160,000 patients across 4 provinces in Zambia, we found that 7% of all patients and 8% of new ART starters died within 2 years—from 10- to 20-fold higher than treated patients in Europe . ProvincAlthough mortality from childbirth, road accidents, and other factors are indeed higher in Africa, over 95% of the deaths ascertained in this cohort were due to “illness” rather than death from these competing causes. We believe the potential drivers of deaths include unmet needs for more advanced medical care, especially given the protective effect in our multivariable model of being treated in a hospital versus a health center, which may indicate a better capacity for advanced diagnostics and care in hospital-based clinics. Indeed, approximately half of deaths occur relatively shortly after a clinic visit. In a previous analysis, we found that the majority of patients who died after a recent visit had some opportunity for medical intervention that was missed—a more detailed assessment of the clinical encounters before death in these patients should be urgently undertaken . At a tiThe provincially representative nature of the study enabled a new and more nuanced understanding of regional mortality among those newly starting ART and among the cross-section of all ART users in a province. First, the ratio of the revised mortality rate versus the naïve rate was highest in Lusaka Province—8-fold higher for all ART users and 9-fold higher for new ART users—compared with the other 3 provinces (ratios between 3–5). One interpretation of this is that populations in dense urban population centers such as Lusaka may be more likely to have their mortality under-reported. Lusaka Province is dominated by the capital city and transport hub of Lusaka, which has an urban population of 2.4 million people. It is conceivable that high mobility and/or fewer community linkages to health facilities result in less passive reporting of mortality than in the less urban provinces. The highest absolute revised mortality rates were in Western Province, which is a largely rural floodplain of the Zambezi River with the lowest population density of the 4 provinces, an HIV prevalence equal to that in Lusaka Province (16%), and limited economic activity other than cattle farming. Although our overall multivariable analysis suggested an association of nonurban facility setting with reduced mortality, health services in much of Western Province region are notoriously difficult to deliver and up-referrals for advanced care can be particularly challenging in the rainy season because of flooding and long distances to higher-level facilities. We believe these and potentially other factors may account for the high revised mortality rates in Western Province, although further investigation is needed at the provincial and site level. Indeed, the differences between the revised provincial mortality rates and between the ratios of the naïve and corrected morality rates in the provinces serve as starting points for both quality improvement work by provincial health offices and for further study. For example, future investigation could examine the system- and human-level causes of low levels of passive mortality reporting that we found across all 4 provinces, but particularly in Lusaka Province, and could investigate differential utilization of up-referral for sick patients by province and facility type and the association of provincial mortality rates with the quality and availability of advanced care services in the region.3; women’s rates approached those of HIV-uninfected individuals by 48 months [Our results, drawn from a sample of patients visiting HIV treatment clinics between 2013 and 2015, indicate that even in an era of expanded eligibility for therapy and rising CD4 cell count thresholds, CD4 cell counts remain low at initiation and are a risk factor for mortality ,23. With8 months . These o8 months . Previou8 months , and the8 months .The marked heterogeneity of mortality across facilities is a crucial and novel observation made possible by our sampling scheme. The fact that mortality differs across facilities, even when higher-level systems support  is relatively uniform, implies that the next generation of improvement efforts should be focused at the facility level and with access to improved data for better decision-making. In South Africa, where facilities are now linked to the national vital status registry, this is already possible, but in most settings in the region, it is not. Another implication of the site-to-site variability is that attention should be targeted to the high-volume, high-mortality-rate facilities because the absolute reductions in mortality are likely to be the biggest through improvements at these sites. Third, the presence of high-performing facilities suggests that strategies such as exemplars by positive deviants—in which high-performing facilities are brought in to assess and guide lower-performing facilities—should be explored . UnexplaOverall, our findings also imply that in Zambia and other similar settings, even if the regular use of site and subnational region-level mortality data for improvement were attempted, inaccuracy in the underlying data would undermine the utility of the approach. As we have shown, under-recording of mortality is the norm, and variability is masked under typical approaches to monitoring and evaluation, as evidenced by the differences in the ordering of mortality burden by province with use of naïve versus revised mortality rates. Although Demographic and Health Surveys (DHSs) and other door-to-door surveys provide representative data that can be used to evaluate trends in mortality, they are often not linked to sites and are not conducted with the frequency required to monitor for program performance and to measure the proximal effects of health systems interventions . We propThe methodology is predicated on tracing a random sample of individuals lost to care. We were unable to ascertain the vital status of about one-quarter of those lost and traced, which could have biased our results. However, we did not find differences in the sociodemographic or clinical characteristics among those who were traced and found compared with those we were unable to find. We were also not able to adjudicate causes of death and therefore are unable to distinguish clearly all-cause mortality versus HIV-related mortality. Further studies are needed to better understand clinical causes of death among HIV-infected individuals on ART in Zambia. Lastly, although the study included a comprehensive sample of Ministry of Health (MOH) ART sites in the 4 provinces, faith-based and other sites were not included in the estimates.To our knowledge, this study is the first to apply a multistage sampling-based approach to generating provincially representative mortality rates for HIV programs in low- and middle-income countries (LMICs). In addition to demonstrating that provincial mortality is substantially under-reported and variable among HIV-infected individuals on ART, we found unexpectedly high site-to-site variability in mortality after starting HIV treatment under routine health service delivery conditions. This heterogeneity among sites suggests that service delivery has much room to improve and that greater attention to systematically generating and using corrected mortality rates is a logical next step for ongoing quality improvement for national programs and major donors. In addition, given the associations of late/missed visits with mortality, greater attention to patient-centered and differentiated care approaches that are also sensitive to the need for care intensification may have a role in not only improving retention but addressing the markedly high death rates, despite the widespread use and availability of ART.S1 Fig(TIF)Click here for additional data file.S2 FigTiming of deaths by care status and prior healthcare utilization patterns among (A) new antiretroviral therapy (ART) initiators and (B) all ART users.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableNaïve and revised provincial mortality rate estimates among (A) new antiretroviral (ART) initiators and (B) all ART users.(DOCX)Click here for additional data file.


Connochaetes taurinus) migration of approximately 1.3 million individuals, but it also has a growing population of hippopotami (Hippopotamus amphibius), which reside within the river and may contribute to the flux of suspended sediments. We used in situ pressure transducers and turbidity sensors to quantify the sediment flux at two sites for the Mara River and investigate the origin of riverine suspended sediment. We found that the combined Middle Mara—Talek catchment, a relatively flat but semi-arid region with large populations of wildlife and domestic cattle, is responsible for 2/3 of the sediment flux. The sediment yield from the combined Middle Mara–Talek catchment is approximately the same as the headwaters, despite receiving less rainfall. There was high monthly variability in suspended sediment fluxes. Although hippopotamus pools are not a major source of suspended sediments under baseflow, they do contribute to short-term variability in suspended sediments. This research identified sources of suspended sediments in the Mara River and important regions of the catchment to target for conservation, and suggests hippopotami may influence riverine sediment dynamics.The Mara River Basin in East Africa is a trans-boundary basin of international significance experiencing excessive levels of sediment loads. Sediment levels in this river are extremely high  and appear to be increasing over time. Large wildlife populations, unregulated livestock grazing, and agricultural land conversion are all potential factors increasing sediment loads in the semi-arid portion of the basin. The basin is well-known for its annual wildebeest ( Excessive sediment loads in rivers are a concern throughout the world as suspended sediments can be a major transport medium for chemicals, contaminants and nutrients –3. HumanSuspended sediments are primarily a mix of organic and inorganic terrestrially derived particles . TurbidiThe Mara River, which is shared by Kenya and Tanzania, is a trans-boundary river of tremendous conservation importance, and sedimentation and non-point source pollution have become issues of major concern in recent years . The MarThe majority of studies on the Mara River have focused on soil erosion in the upper catchment of the river basin as the source for increasing sediment loads in the river , 28–30. Hippopotamus amphibius, hereafter referred to as hippos), due to their high density in the Mara and their direct influence on in-stream processes. Hippos are known as ecosystem engineers, capable of exerting a transformative effect upon their environment [One potentially important, but under-studied, driver of sediment generation processes is large wildlife. There are large numbers of a diversity of wildlife species that use the Mara River, including 1.3 million wildebeest during their annual migration from the Serengeti, in addition to large numbers of livestock, all of which may influence sediment generation and flux in the river. Here, we focus on the influence of hippopotami  (36M 724636E 9828995S) is located at a bridge just upstream of the border between Kenya and Tanzania. This site integrates the influence of Emarti (38% of the NMB catchment), the Talek (41% of the NMB catchment) and the Middle Mara region (21% of the NMB catchment). The Middle Mara region has no other perennial rivers contributing to the Mara. Land use in the Middle Mara region is primarily pastoralism, tourism developments and wildlife conservancies. Approximately half of the Middle Mara region is within the MMNR.At Emarti, we measured stage height every 15 minutes from June 2011 –Dec 2014 using a RuggedTroll 100 pressure transducer . At NMB, stage height was measured every 15 minutes from June 2011 –November 2012 using a RuggedTroll 100 pressure transducer and from December 2012 –December 2014 using a pressure transducer connected to a Eureka Manta2 sonde . All measurements from the pressure transducers were corrected for atmospheric pressure changes with a BaroTroll that was also installed at each site . From April–August 2014, the sonde at the NMB was not operational, so stage height was measured every 30 minutes using an Arduino-based ultrasonic sensor .To develop rating curves for stage-discharge relationships at the two sites, we measured discharge on multiple occasions in 2011  with the area-velocity method using a handheld staff gauge or weighted measuring tape for depth and velocimeter for velocity. In 2014, we measured discharge 3 times at Emarti and once at NMB using either the measuring tape and velocimeter or a HydroSurveyor . Bedrock substrate is present in both channel reaches where rating curves were developed, so we assumed channel geomorphology was consistent over time.2 of 0.82 (2 of 0.95 (We converted stage height to discharge using a rating curve developed for each site in R with the nls function . The rat of 0.82 . Two cur of 0.95 . Outlier of 0.95 . A MonteThe Richards-Baker Flashiness Index (R-B Index) was calculated for Emarti and NMB using mean daily discharge data over each year . The indin situ turbidity at the Emarti and NMB sites using a NEP9500 turbidity sensor  built into a Manta2 water quality data logger . Manta2 water quality data loggers were installed at the Emarti and NMB sites intermittently from June 2011 through December 2014. The sondes were programmed to log every 15 minutes. Prior to taking a measurement, the sonde would wipe the turbidity probe to prevent fouling. Sondes were calibrated according to manufacturer instructions. The turbidity sensor is certified up to 3000 NTU, however, it is capable of providing a reading over 4000 NTU. During several floods, turbidity values exceeded what was readable by the sensor at both sites.We measured We collected 44 water samples at both sites  during dry and wet seasons to calculate suspended sediment concentrations relative to turbidity. In 2011, water samples were collected with a US DH-59 depth integrating suspended sediment sampler suspended from bridges at Emarti and NMB. Samples were taken from the most representative and accessible cross section of each site. Care was taken to ensure an equal transit time throughout the depth of the vertical depth profile. From 2012–2014, water samples were taken with a 1-liter Nalgene bottle from a well-mixed representative reach at each site. All water samples were taken in accordance with USGS methods .We filtered a known volume of water through a pre-weighed cellulose nitrate (CN) filter  immediately after sample collection. We dried CN filters with suspended sediment in a solar oven immediately in the field and sealed the samples for transport to the US. Upon arrival in the US, the filter papers were dried in an oven at 60°C for 24h and re-weighed to calculate the mass of the sediment.-1) for Emarti and NMB sites with the lm function in R [2 of 0.98 (-1) by multiplying instantaneous estimated river discharge (m3 s-1) by the instantaneous estimated suspended sediment concentration (mg L-1) obtained from our SSC:turbidity regression. We then calculated the mean per month and mean over the entire period of record. We did not calculate total annual flux, as we were missing months for each year of record. Uncertainty in flux was calculated with the sum in quadrature of the fractional uncertainties in the stage height to discharge rating curve and the turbidity to SSC regression [We used a simple linear regression to relate instantaneous Manta2 turbidity data (NTU) to suspended sediment concentration  was computed by dividing the average instantaneous suspended sediment flux over the record by the catchment area (km2). Yield was also computed for the combined Middle Mara and Talek regions by subtracting the Emarti flux from the NMB flux and dividing by the catchment area of the combined Middle Mara and Talek regions. We did not calculate total annual yield, as we were missing months for each year of record.We estimated catchment area of each sampling location using drainage patterns derived from the 15m ASTER Global DEM dataset, Version 2. Analysis was done with ArcGIS 10 using the Spatial Analyst toolbox and the Arc Hydro extension . SedimenThe focus of this analysis was to compare sediment fluxes between Emarti and NMB, so we restricted analysis to dates when we had data for both sites. Travel time between those two sites is highly variable in this system and is dependent on discharge, thus we did not calculate a general time offset for data between Emarti and NMB. However, travel time between the sites is typically within 1–2 days, which likely had minimal effect on our analysis of monthly comparisons.To aid in visualization, data were divided into 8 blocks of continuous coverage with no data gap greater than one week . The timWe measured turbidity upstream and downstream of four different hippo pools for 24–48 hours  in orderTurbidity from the hippo pools were not normally distributed, and data points within each hippo pool were not independent from one another, so we used non-parametric tests that accounted for sample dependence. Because we were examining fine-scale patterns in suspended sediment concentrations, we accounted for travel time between the upstream and downstream measurements by the sondes. We estimated for each time-series the time lag in one minute increments (between 0 and 18 minutes) as the lag that maximized the Kendall Rank Correlation Coefficient (tau) in turbidity between upstream and downstream time-series . The coeThis study was authorized by the Government of Kenya and the National Council for Science and Technology (Research permits NCST/RRI/12/1/BS-011/25 and NCST/RDB/12B/012/36). Permission to conduct this research within the Masai Mara National Reserve was granted by the Transmara and Narok County Councils.3 s-1  and average discharge from NMB was 12.5 m3 s-1 (- 15%+ 19%). The spikes in discharge from the NMB site that were not present at the Emarti site are largely due to the influence of the Talek River, as there are no other rivers entering the Mara within this reach, although overland flow and seasonal drainages could also be a factor (The hydrographs for the two sites show that the Emarti site has the highest baseflow discharge and that there is often water loss between the Emarti and NMB sites during baseflow conditions . Averagea factor .3 s-1 (3 s-1 and the coefficient of variation was highest at NMB in 17 of the 21 months. Minimum discharge was consistently lower at NMB than Emarti except in two months (November 2012 and December 2014).Monthly mean discharge was highest at Emarti in 14 of 21 months although maximum discharge never exceeded 30 m3 s-1 . MaximumNMB had a Richards-Baker Flashiness Index (R-B Index) score that was on average 3.5 times higher than that Emarti and that exceeded the R-B Index at Emarti in every year . The R-B-1 (- 10%+ 12%) in December 2012 to 812 tonnes day-1 (- 12%+ 17%) in December 2014 (-1 (- 9%+ 11%) in December 2012 to 3100 tonnes day-1 (- 6%+ 6%) in September 2014. Flux was higher at Emarti than NMB during one month, indicating the potential for storage of sediments within the river channel between Emarti and NMB. Over the entire length of record, average suspended sediment flux at Emarti was 220 tonnes day-1 (-11%+ 20%) and at NMB it was 710 tonnes day-1 (-7%+9%). Sediment yield was similar for all three regions  report abstracting water directly from the river for their use, and their water consumption increases during the high season of July through October, when the wildebeest migration is present in this region [During baseflow conditions, discharge is often higher at the Emarti site, with an estimated loss of approximately 1 mi to NMB . Dischars region . Abstracs region .3 s-1, 50% of the time within each year [3 s-1 (- 15%+ 19%). Likewise, a recent study of the historical records for the Amala and Nyangores rivers reported an annual combined mean discharge of 12.45 m3 s-1 [3 s-1 (- 23%+ 32%).NMB had a consistently higher R-B Index, indicative of flashier discharge at this site. We would expect NMB to have a lower R-B Index because it drains a catchment approximately 3 times the size of Emarti so flows should be more stable. In spite of this, the NMB site is in the highest quartile of the R-B Index compared to rivers of a similar drainage while the Emarti site is in the lowest quartile . The R-Bach year . Our calPeriods of data collection with overlap were not available for the typical peak wet season (April-May). The absence of these peak discharge periods in our data could have led to an underestimation of total sediment flux and yield from each portion of the watershed. Additionally, the absence of data in several low flow months (January and February) could lead to an overestimation of the total sediment flux and yield. However, we did capture several seasons of data from the other high and low discharge months for Emarti and NMB, and comparison of our overall discharge data to several other studies gives support to our conclusions.The suspended sediment flux at Emarti is approximately 1/3 of that at NMB. The bulk of the sediment flux appears to be generated in the semi-arid Middle Mara and Talek regions. Since Emarti to NMB is a hydrologically losing reach, our flux calculations may overestimate the contribution of the Middle Mara and Talek regions if previously sequestered in-channel sediments from Upper Mara are remobilized from within the channel during floods. However, previous research during a three-month period in 2011 using a sediment fingerprinting method has supported the finding that 2/3 of the suspended sediments at NMB are coming from the Middle Mara and Talek regions and are not the result of the remobilization of in-channel sediments from the Upper Mara . Further-1, and sediment flux at Emarti was only 81,000 tonnes year-1. Average monthly fluxes were more variable at NMB (higher CV) with an increase as high as 600% between August and September, 2014, due to a marked increase in discharge. Monthly fluxes were much more stable at Emarti. The largest increase in monthly flux at Emarti was approximately 300% over the previous month. Interestingly, fluxes at Emarti dropped 800% between September and November, 2013. This was likely a consequence of the reduction of mean flow from 17 m3 s-1 to 7 m3 s-1 between September and November.Sediment flux at NMB was on average over 253,000 tonnes yearWe found that the sediment yields across the three catchments were not significantly different from one another. It is not possible to separate the Middle Mara and Talek regions in the yield calculations, although previous analyses using a sediment fingerprinting approach have suggested that the Talek has a higher yield than the Middle Mara region . The MidOur findings are contrary to the traditional model of mountain streams having a disproportionately large influence on the sediment yield of a river due to high levels of precipitation and elevation change . AlthougThe Talek region appears to have experienced landscape degradation as early as the 1970s. Between 1980 and 2000, there was a 70% decrease in wildlife on the Mara Rangelands that was attributed in part to the conversion to wheat cultivation in the Loita Plains, which are situated partly within the Talek watershed , 64. RecSuspended sediment loads are highly episodic. Up to 90% of the annual mass of sediments can be transported within just 10% of the time . Sub-daiAlthough we captured a range of hydrological events from all portions of the study area, it is possible that we did not catch sediment transport that would be representative of a full hydrological year. Several months of low flow and high flow were missed each year due to sonde failure and logistical constraints. Our measurements are likely an underestimate of the true flux and yield, as the measurement range on the turbidity sensor was exceeded multiple times. For calculation purposes, SSC, flux and yield were made with the maximum value, which would be an under-representation of the true value during those extreme events.When calculating flux and yield estimates, the propagation of uncertainty throughout each measured value provided cumulative error estimates in the range of 5% to 55%. The largest confidence intervals were for low flux values for the NMB site and high flux values for the Emarti site. Errors inherent in the discharge measurements and in the relationship between stage height and discharge were the primary source of uncertainty for sediment flux and yield estimates. Care had to be taken during collection of discharge measurements due to the high tourism environment, presence of large wildlife (hippos and Nile crocodiles) and the geomorphology of the river. We believe that the sacrifices in accuracy that we made were necessary to the overall success of gathering data in this type of environment.In three out of the four hippo pools, there was an increase in mean turbidity downstream of hippo pools compared to upstream. There was also a large increase in the variance and coefficient of variation in turbidity downstream of all the hippo pools. Hippos likely increase river bed and bank erosion through wallowing behavior within the river and through creation of deeply incised paths through the riparian zone. Hippos also continually deposit significant amounts of new organic material along the bottom of the pool through defecation . We haveOur research shows that the highest sediment flux into the Mara River comes from the Middle Mara and Talek regions of the basin. Our findings are contrary to the generally accepted theory that deforestation in the Upper Mara is currently responsible for most of the suspended sediment loading in the river. The deforestation in the Mau Forest has likely impacted the hydrologic cycle of the Mara River, resulting in lower baseflows and higher peak flows, and also has likely increased sediment flux from that region , 19. HowOur study also suggests that hippos may have complex effects on suspended sediment dynamics in rivers, with hippo pools acting as both sources and sinks for organic material and suspended sediments. Defecation and physical movements of hippos in pools may increase sediment loading and downstream transport, albeit in short pulses. However, hippo pools also reduce water velocity and increase sediment deposition. The net effect of hippo pools on sediment dynamics in rivers is largely influenced by discharge.The drivers of sedimentation rates in the Mara River are an issue of tremendous political and conservation interest. The river is shared by two countries, and it is at the headwaters of the Nile River. In addition to sustaining some of the most famous protected areas in the world, it also provides domestic water sources for over half a million rural poor people. Concerns about water quantity and quality in the Mara have been a long-standing issue in the region, with potential consequences for the region’s economy, wildlife and human health. Furthermore, several dams are potentially planned for the Mara River, which could significantly alter sediment regimes in the basin . Our resS1 FigOutliers that were removed are presented as a triangle. 95% confidence intervals are in red.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.