Proteomic Analysis of Histones H2A/H2B and Variant Hv1 in ...

Proteomic Analysis of Histones H2AH2B and Variant Hv1 inTetrahymena thermophila Reveals an Ancient Network ofChaperones

Kanwal Ashrafdagger1 Syed Nabeel-ShahdaggerDagger2 Jyoti Garg1 Alejandro Saettone2 Joanna Derynck2

Anne-Claude Gingras34 Jean-Philippe Lambert56 Ronald E Pearlman1 and Jeffrey Fillingham2

1Department of Biology York University Toronto ON Canada2Department of Chemistry and Biology Ryerson University Toronto ON Canada3Department of Molecular Genetics University of Toronto Toronto ON Canada4Lunenfeld-Tanenbaum Research Institute Mount Sinai Hospital Toronto ON Canada5Department of Molecular Medicine and Cancer Research Centre Universite Laval Quebec QC Canada6CHU de Quebec Research Center CHUL Quebec QC CanadadaggerThese authors contributed equally to this workDaggerPresent address Donnelly Centre University of Toronto Toronto ON Canada Department of Molecular Genetics University ofToronto Toronto ON Canada

Corresponding authors E-mails jeffreyfillinghamryersonca ronpyorkuca

Associate editor Amanda Larracuente

Abstract

Epigenetic information which can be passed on independently of the DNA sequence is stored in part in the form ofhistone posttranslational modifications and specific histone variants Although complexes necessary for deposition havebeen identified for canonical and variant histones information regarding the chromatin assembly pathways outside ofthe Opisthokonts remains limited Tetrahymena thermophila a ciliated protozoan is particularly suitable to study andunravel the chromatin regulatory layers due to its unique physical separation of chromatin states in the form of twodistinct nuclei present within the same cell Using a functional proteomics pipeline we carried out affinity purificationfollowed by mass spectrometry of endogenously tagged T thermophila histones H2A H2B and variant Hv1We identifieda set of interacting proteins shared among the three analyzed histones that includes the FACT-complex as well as H2A-or Hv1-specific chaperones We find that putative subunits of T thermophila versions of SWR- and INO80-complexes aswell as transcription-related histone chaperone Spt6Tt specifically copurify with Hv1 We also identified importin b6 andthe T thermophila ortholog of nucleoplasmin 1 (cNpl1Tt) as H2AndashH2B interacting partners Our results further implicatePoly [ADP-ribose] polymerases in histone metabolism Molecular evolutionary analysis reciprocal affinity purificationcoupled to mass spectrometry experiments and indirect immunofluorescence studies using endogenously tagged Spt16Tt

(FACT-complex subunit) cNpl1Tt and PARP6Tt underscore the validity of our approach and offer mechanistic insightsOur results reveal a highly conserved regulatory network for H2A (Hv1)ndashH2B concerning their nuclear import andassembly into chromatin

Key words histone FACT histone chaperone histone H2A histone H2B

Introduction

The eukaryotic genome is packaged in the form of a nucleo-protein complex called chromatin The primary repeatingunit of chromatin is the nucleosome which is formed when146 bp of DNA is wrapped around a core histone octamerconsisting of two histone H2AndashH2B heterodimers and oneH3H4 tetramer (Luger et al 1997) Chromatin structureinfluences all DNA-mediated cellular processes includinggene transcription replication recombination and repair(reviewed by Venkatesh and Workman [2015]) Histonescarry posttranslational modifications which have importantroles in gene expression regulation For example an

enrichment of histone H3 trimethylated at lysine (K) 9 orK27 (H3K9me3 or H3K27me3) has been associated with het-erochromatic chromatin regions whereas H3K4me3 andH3K36me3 posttranslational modifications have been linkedwith transcriptional activity (Allshire and Madhani 2018)

Variant histones have been described across species thatdiffer in their primary amino acid sequences (Talbert et al2012) Canonical histones are only expressed during S-phaseand are deposited onto chromatin in a DNA replication-de-pendent (RD) manner whereas variants are expressedthroughout the cell cycle and are deposited onto chromatinin a replication-independent (RI) manner (Mendiratta et al2018) Interestingly RI variants including H33 and H2AZ for

Article

The Author(s) 2019 Published by Oxford University Press on behalf of the Society for Molecular Biology and EvolutionThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License(httpcreativecommonsorglicensesby-nc40) which permits non-commercial re-use distribution and reproduction in anymedium provided the original work is properly cited For commercial re-use please contact journalspermissionsoupcom Open AccessMol Biol Evol 36(5)1037ndash1055 doi101093molbevmsz039 Advance Access publication February 22 2019 1037

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bearticle36510375362030 by guest on 15 March 2022

histones H3 and H2A respectively have been described tohave nonrandom distribution along the chromatin For ex-ample H33 is enriched in the euchromatic regions associatedwith transcriptionally active genes (Goldberg et al 2010Ray-Gallet et al 2011) Genome-wide studies have indicatedthe enrichment of H2AZH33 double variants within regionsof high chromatin accessibility such as active promotersenhancers and insulator regions (Jin et al 2009) highlightingtheir role in gene expression regulation

Chromatin assembly is a fundamental process that mayaffect a broad range of gene regulatory processes such as DNArepair DNA replication and progression through the cell cy-cle (Mendiratta et al 2018) Protein factors known as histonechaperones are thought to have key roles in regulation ofchromatin assembly (Grover et al 2018) For examplechromatin assembly factor-1 and histone-regulator-A havebeen shown to mediate RD and RI chromatin assembly pro-cesses to deposit either H3ndashH4 or H33ndashH4 (Hoek andStillman 2003 Tagami et al 2004 Jullien et al 2012) respec-tively whereas the SWR-complex specifically targets H2AZndashH2B onto chromatin (Gerhold et al 2015) Histonechaperones have specific preferences for binding to eitherH3ndashH4 or H2AndashH2B (Keck and Pemberton 2012 Groveret al 2018) For example nucleosome assembly protein 1(Nap1) and nucleoplasmin 1 (Npm1) are both H2AH2B-specific chaperones (Straube et al 2010 Hammond et al2017) whereas antisilencing factor 1 (Asf1) is an H3H4-specific chaperone (English et al 2006 Mendiratta et al2018) It is currently unclear how chaperones target a certainhistone variant to a specific genomic region and how mech-anistically this task is achieved Several previous studies haveutilized functional proteomics approaches to identify andexamine the role of histone-binding proteins (Tagami et al2004 Latreille et al 2014 Hammond et al 2017) Howeverconsidering the complexity of chromatin assembly and geneexpression regulatory layers it is conceivable that many yet tobe identified chaperones might have roles in these processesComparative proteomics is a powerful tool that has beenwidely employed to study the evolution and functional con-servation of proteins (Boekhorst et al 2008 Lotan et al 2014)However the extent to which the role of previously identifiedhistone chaperones is conserved across the eukaryotic speciesremains unexplored (Grover et al 2018)

The unicellular ciliate protozoan Tetrahymena thermo-phila provides an excellent experimental system to studychromatin dynamics and identify new factors involved inthese processes The T thermophila genome is amenable totractable alterations enabling the endogenous tagging ofgenes of interest Ciliates are considered evolutionarily diver-gent organisms (Orias et al 2011 Gao et al 2016) and aretherefore well-suited to examine the functional conservationof known histone chaperones The T thermophila single cellfeatures a physical separation of two structurally and func-tionally distinct chromatin states in the form of a germ-linediploid micronucleus (MIC) and a polyploid somatic macro-nucleus (MAC) Functionally the MAC regulates gene expres-sion whereas the MIC ensures stable genetic inheritance(Martindale et al 1982) The two nuclei originate from the

same zygotic nucleus during sexual development (conjuga-tion) of the cell and subsequently embark on unique devel-opmental pathways leading toward distinct chromatinorganization within each nucleus (Martindale et al 1982)The alterations in the chromatin states including DNArearrangements and removal of internally eliminatedsequences during T thermophila development (Yao et al1984 1990 2003 Mochizuki and Gorovsky 2004) share sim-ilarities with epigenetic changes that occur to mammalianchromatin during development

The T thermophila genome encodes two major histoneH2A genes (HTA1 and HTA2) which at the protein level arenearly identical with only three amino acid differences in thecentral core region (Liu et al 1996) Furthermore neitherHTA1 nor HTA2 alone is essential for T thermophila vegeta-tive growth suggesting that the function of the encodedproteins is redundant (Liu et al 1996) However the C-terminiof the two proteins differ significantly from each other asH2A1 (encoded by HTA1) has an additional five residues(Liu et al 1996) These additional five residues include anSQ motif which is conserved across species (as in mammalianH2AX) and provides a target site for phosphorylation by aspecific protein kinase family (Song et al 2007) The SQ motifphosphorylation has been shown to function in double-strand break repair during mitosis meiosis and amitosis inT thermophila (Song et al 2007) Thus T thermophila H2A1can be considered an H2Ax ortholog although it differs frommammals where the H2AX histone variant is a quantitativelyminor component (Rogakou et al 1998) Tetrahymena ther-mophila H2B1 and H2B2 encoded by HTB1 and HTB2 re-spectively are nonallelic variants of H2B and only differ atthree positions Similar to H2A T thermophila cells lackingeither HTB1 or HTB2 alone are viable and do not exhibit anygrowth defects indicating the functional redundancy of H2Bs(Wang et al 2009)

The T thermophila H2A variant Hv1 (H2AZ and Htz1 inhumans and yeast respectively) has been found to be essen-tial for growth (Liu et al 1996) Hv1 localizes to the transcrip-tionally active MAC during vegetative growth and is found inthe MIC only during early conjugation events (Stargell et al1993) prior to the stage when MIC becomes transcriptionallyactive (Martindale et al 1985) Thus the localization patternsof Hv1 suggest a role in transcription regulation The mech-anistic details of how Hv1 is targeted to the MAC (and MICduring early conjugation) remain elusive

In this study we employed a functional proteomics work-flow to examine the histone-interactome for the first time inT thermophila Affinity purifications of T thermophila H2A1(HTA1) H2B1 (HTB1) and Hv1 followed by mass spectrom-etry analysis (AP-MS) revealed both new histone-interactingfactors as well as a set of chaperones that have been previ-ously identified only in Opisthokonts indicating the evolu-tionarily conserved histone metabolism regulatory networksSpecifically we identified T thermophila FACT- SWR- andINO80-complexes suggesting an ancient origin for these pro-teins We carried out detailed molecular evolutionary analysesof several histone-interacting proteins which further rein-forced the idea that dedicated chaperones arose very early

Ashraf et al doi101093molbevmsz039 MBE

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during eukaryotic evolution to regulate histone metabolismWe validated several of the identified interactions by recipro-cal affinity purification coupled to mass spectrometry(AP-MS) analyses and indirect immunofluorescence (IF)studies

Results

Identification of T thermophila H2AH2B-InteractingProteome

We generated stable T thermophila lines expressing H2A1(TTHERM_00790790) (H2A hereafter) and H2B1(TTHERM_00633360) (H2B hereafter) with a C-terminalFZZ epitope tag from their native MAC chromosomal lociThe FZZ epitope tag contains 2 protein A moieties and a3xFLAG separated by a TEV cleavage site permitting affinitypurification of the fusion protein and analysis of thecopurifying proteins by Western blotting andor mass spec-trometry To accomplish this we engineered constructs thatincluded1 kb of DNA sequence upstream and downstreamof the predicted stop codons of HTA1 and HTB1 The engi-neered FZZ constructs (supplementary fig 1 SupplementaryMaterial online) were used to transform growing T thermo-phila cells using biolistic transformation Homologous recom-bination mediates the gene replacement of the wild type(WT) HTA1 and HTB1 loci by FZZ constructs (Cassidy-Hanley et al 1997) The polyploid MAC divides amitoticallyand does not afford an equal segregation of alleles (reviewedby Karrer [2012]) Homozygocity in the polyploid MAC of thetransformed cells can be achieved through ldquophenotypicassortmentrdquo (reviewed by Karrer [2012]) Western blottinganalysis using anti-FLAG antibody demonstrated successfulexpression of the epitope-tagged proteins in whole-cellextracts (WCEs) from H2A- and H2B-FZZ-expressing strainscompared with the WCEs prepared from untagged controlcells (fig 1A left panels) To test the possibility that the pres-ence of the FZZ tag might interfere in the localization of thetagged histones we carried out indirect IF analysis on H2A-and H2B-FZZ in growing T thermophila cells Previously H2Aand H2B have been shown to localize to both the MAC andMIC (Song et al 2007 Wang et al 2009) Our IF analysisindicated that H2A- and H2B-FZZ also localize to both theMAC and MIC (fig 1A right panels) supporting that the FZZtag does not interfere with their function

We performed affinity purification in biological replicateson H2A- and H2B-FZZ expressing strains The recovery of thebaits was confirmed by Western blotting using the affinity-purified material from either the untagged WT cells or H2A-and H2B-FZZ cells (fig 1B) To define H2AH2B proteinndashpro-tein interaction (PPIs) networks a gel-free liquid chromatog-raphy coupled to tandem mass spectrometry (LCndashMSMS)analysis was carried out using the affinity purified materialThe mass spectrometry data were evaluated withSAINTexpress which uses semiquantitative spectral countsfor assigning a confidence value to individual PPIs (Teo et al2014) Application of SAINTexpress to the AP-MS data fortwo biological replicates of H2A- and H2B-FZZ affinity puri-fications from growing T thermophila cells scored against

numerous control AP-MS experiments revealed several inter-action partners that pass the cutoff confidence value(Bayesian FDR 1) (supplementary file 2 SupplementaryMaterial online)

This analysis revealed that H2A- and H2B-FZZ copurifywith 14 and 17 significant interacting partners respectively(fig 1C) Three interaction partners TTHERM_00283330TTHERM_00049080 and TTHERM_00726470 copurifiedwith both H2A and H2B TTHERM_00283330 andTTHERM_00049080 proteins are the orthologs of yeastSpt16 (suppressor of Ty 16 SUPT16H in humans) and Pob3(Pol1-binding protein SSRP1 in humans) subunits of theFACT-complex respectively The FACT-complex is a well-characterized transcriptional regulator that functions as anH2AndashH2B dimer chaperone (Belotserkovskaya et al 2003)The third H2AH2B shared interacting partnerTTHERM_00726470 is T thermophila Poly [ADP-ribose] po-lymerase 2 (PARP2Tt) PARPs are functionally diverseproteins with critical roles in chromatin architecturemRNA processing and histone ADP-ribosylation (Hassa andHottiger 2008)

We also identified TTHERM_00429890 as an interactionpartner for H2A-FZZ TTHERM_00429890 shares sequencesimilarity with a known human H2AH2B chaperone proto-oncogene NPM1 (Okuwaki et al 2001) suggesting aconserved histone-binding function for this protein Theremaining H2A-FZZ copurifying proteins include aTetrahymena-specific protein TTHERM_00242240 whichdoes not have an identifiable ortholog in any other organisma DNA-binding AT-Hook domain protein a VWA domain-containing protein a MutS family protein which shares se-quence similarity with yeast MSH6 a POZ domain proteinhistones H2B H3 H4 and two PARPs including PARP6Tt andPARP3Tt (fig 1C)

The T thermophila genome encodes at least 13 importin(imp) a- and 11 impb-like proteins (Malone et al 2008) OurSAINTexpress analysis indicated that H2B copurifies withTTHERM_00962200 which encodes an Impb6 protein Wepreviously have shown that Impb6 interacts with Asf1and likely functions in the H3H4 transport pathway(Garg et al 2013) Among the interacting partnersdetected for H2B-FZZ were three hypotheticalproteins TTHERM_00532520 TTHERM_00657290 andTHERM_00648920 TTHERM_00532520 is a ciliate-specificprotein with an ortholog in Paramecium tetrarelia withoutany recognizable domains whereas TTHERM_00657290appears to carry an SMC-N terminal domainsuggesting a role in chromatin structural maintenanceTHERM_00648920 is a predicted 32 kDa Tetrahymena-specific protein which has several stretches of acidic residuessimilar to other histone chaperones for example NPMs Wenamed THERM_00648920 as ldquohistone-interacting acidicprotein 1rdquo (Hiap1) Additionally other notable H2B-FZZcopurifying proteins include a Basic Leucine ZipperDomain-transcription factor (bZIP1) an Alba2-domainDNA-binding protein DExDH box RNA helicase Drh29Mak21 an apoptosis-antagonizing transcription factorAATF an ARM-repeat protein and an MutS family protein

Proteomic Analysis of Histones H2AH2B and Variant Hv1 in T thermophila doi101093molbevmsz039 MBE

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MSH2 Yeast MSH2 and MSH6 proteins are known to inter-act with each other and have critical roles in DNA-mismatchrepair (Studamire et al 1998) (fig 1C supplementary file 2Supplementary Material online for details on all H2B inter-action partners)

We utilized publicly available microarray expression datato compare gene expression profiles of H2A and H2B withthose of the genes encoding their copurifying proteins Asexpected H2A H2B H3 and H4 cluster together due to theirsimilar expression profiles in S-phase (supplementary fig 2A

FIG 1 H2A-FZZ and H2B-FZZ expression and affinity purification (A) Left panels Expression analysis of H2A-FZZ (H2A1477 kDathorn FZZ18kDa) and H2B-FZZ in comparison to the untagged controls by Western blotting using WCEs Blots were probed with anti-FLAG antibody for FZZdetection whereas anti-Actin and anti-Brg1 (146 kDa) were used as loading controls Right panels H2A- and H2B-FZZ localize to both MAC andMIC Note For H2A-FZZ IF images the lower panel demonstrates dividing cells DAPI was used to stain the nuclei and the position of the MAC andMIC is indicated with arrows and arrow-heads respectively (B) Western blotting analysis indicating the recovery of the affinity purified (AP) H2A-FZZ (left) and H2B-FZZ (right) The top panels were probed with anti-FLAG antibody to examine the recovery of the baits No signal was detectedin the WT Anti-Actin and anti-Brg1 were used as loading controls Two bands in the H2A-FZZ input likely represent dimers (C) Networkrepresentation of H2A- and H2B-FZZ copurifying proteins Node border legend is provided The MS data were searched against the TetrahymenaGenome Database (wwwciliateorg last accessed September 24 2018) (TGD) Full-length protein sequences were retrieved from TGD andsearched against yeast or human proteins to annotate them (see Materials and Methods for details)


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and B Supplementary Material online) Spt16Tt and Pob3Tt

also clustered with H2A and H2B consistent with a role of theFACT-complex in histone metabolism In addition PARP6TtPARP3Tt Poz Msh2 Hiap1 and Impb6 also exhibited simi-larities in their expression profiles with those of the H2A andH2B (supplementary fig 2A and B Supplementary Materialonline) suggesting functional linkage of these proteins withhistones The observation that putative T thermophilaFACT-complex subunits PARPs and NPM1-like proteinscopurify with H2AndashH2B suggests an evolutionarily conservedrole of these proteins in histone metabolism Consideringtheir central role in a number of chromatin-related processesand relevance to human diseases we further characterizedthese proteins

The FACT-Complex Is Conserved across EukaryotesThe FACT-complex is a critical transcription regulator and anH2AH2B chaperone (Mason and Struhl 2003 Hsieh et al2013) The copurification of the putative FACT-complex sub-units with H2A- and H2B-FZZ in an evolutionarily divergenteukaryote highlights the conserved nature of its role inchromatin-related processes However evidence regardingthe origin of the FACT-complex is currently lacking To gaugethe evolutionary history of the FACT-complex we carried outextensive database searches and identified the putative ortho-logs of the FACT subunits that is Spt16 and Pob3 through-out the eukaryotic supergroups including the basal eukaryoteGiardia lamblia (supplementary file 1 SupplementaryMaterial online) This suggests that Spt16 and Pob3 werealready present in the last eukaryotic common ancestorGiven that FACT subunits were likely present in the last eu-karyotic common ancestor we wanted to further examinetheir evolutionary patterns and reconstructed the phyloge-netic trees of Spt16 and Pob3 Clustering in the resultingphylogenetic trees (fig 2A) appears highly similar to that ofthe eukaryotic classification system (Adl et al 2012) Bothproteins that is Spt16 and Pob3 follow nearly identical phy-logenetic paths with a few minor exceptions For example theamoebozoan lineages corresponding to Pob3 form a mono-phyletic group below the metazoans whereas Spt16 amoebo-zoans take a basal position below the opisthokonts (fig 2A)Such differences likely represent the isolated cases wherelineage-specific functional constraints might have been oper-ating on both proteins independently of each otherNevertheless similarities in the phylogenetic histories stronglysuggest that both proteins together experienced strong pu-rifying selection to retain their structural and functional fea-tures To examine the selective constraints operating on theFACT-complex we used nucleotide coding sequences ofSpt16 and Pob3 from the representative lineages and carriedout codon-based Z-test of selection by comparing synony-mous and nonsynonymous variations We found extensivesynonymous variations that were considerably higher thannonsynonymous variations (Plt 0001) in all comparisonsfor both Spt16 and Pob3 indicating the presence of purifyingselection (supplementary file 3 Supplementary Material on-line) Extensive silent variations that we observed at the nu-cleotide level also resulted in a subsequent overall decrease in

codon usage bias (supplementary file 3 SupplementaryMaterial online) consistent with the idea of strong functionalconstraints operating at the protein level Previous high-throughput studies have reported the phosphorylation ofhuman and mouse Spt16 and Pob3 at highly conserved serineresidues (supplementary file 3 Supplementary Material on-line) Interestingly we found that the serine residues in Spt16are preferentially encoded by the codon UCU across all thetaxa For Pob3 serine residues ldquoAGCrdquo is the preferred codonwithin Opisthokonts whereas UCU and AGU are preferen-tially used in plants and protist lineages (supplementary file 3Supplementary Material online) These results indicate thestrong purifying selection operating not only at the proteinlevel to maintain the structural features but also by the usageof preferred codons for functionally important positions

Spt16 contains a signature Spt16_domain (SMART acces-sion SM001286) an N-terminal lobe (SM001285) a peptidase(pfam PF00557) and an Rtt106 domain (SM001287) which isalso found in Pob3 (fig 2B left) The ldquopeptidaserdquo and Rtt106domains are known to function as histone-binding modules(Stuwe et al 2008 Zunder et al 2012) We examined thestructural features of the Spt16Tt and Pob3Tt We alignedSpt16Tt and Pob3Tt against budding yeast and human homo-logs and observed that the domain organization in bothproteins is highly conserved (fig 2B left) In fact Spt16Tt

and Pob3Tt respectively exhibit more than 30 and 20sequence identities to their homologs both in the buddingyeast and humans Of note Pob3 in tetrapods has gained ahigh-mobility group (HMG) domain whereas unicellulareukaryotes for example budding yeast FACT-complex inter-act with an HMG protein Nhp6 to provide the same activity(Formosa et al 2001) Ciliates and humans diverged 1781Ma (Kumar et al 2017) and such a degree of sequence andstructural conservation points toward possible functionalsimilarities that might exist among the distant homologsTo further investigate this possibility we used the strategydescribed above to engineer T thermophila cells stablyexpressing C-terminally epitope tagged Spt16Tt-FZZ from itsnative chromosomal locus (fig 2B right) As shown infigure 2B (lower panel) Spt16Tt-FZZ localizes to both theMAC and MIC in growing T thermophila cells Affinity puri-fication on growing Spt16Tt-FZZ strains and SAINTexpressanalysis of the LCndashMSMS data confirmed the copurificationof Pob3Tt with Spt16Tt-FZZ (supplementary file 2Supplementary Material online) We also detected two sub-units of RNA polymerase I and III (RNAP) Rpac1 and Rpa2consistent with a role in transcription regulationAdditionally a T thermophila-specific TTHERM_01046850protein also copurified with Spt16Tt TTHERM_01046850 enc-odes a predicted 53 kDa protein and does not have anyidentifiable domains We named this protein as ldquoFACT-inter-acting mysterious protein 1rdquo (Fimp1) (supplementary file 2Supplementary Material online for all Spt16Tt interactions)Consistent with their copurification Spt16Tt and Pob3Tt sharenearly identical gene expression profiles Similarly Fimp1 alsoclusters along with the FACT-complex (supplementary fig 3Supplementary Material online) Further analysis will be re-quired to understand the mechanistic details of Fimp1


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interaction with the FACT-complex We conclude thatSpt16Tt and Pob3Tt constitute the T thermophila FACT-com-plex with possible roles in histone H2AH2B chaperoning andtranscription regulation

PARP Proteins in T thermophilaThe observation that certain PARPs copurified with histones(fig 1C) prompted us to examine the full repertoire of PARP

proteins in T thermophila Our query against the T thermo-phila genome database using human PARP1 identified at least11 proteins with a PARP-catalytic (PF0064) domain (fig 3Aright) Multiple sequence alignment indicated that catalyticresidues (HYE) within PARP-catalytic domains are highly con-served with the exception of PARPs7ndash9 where the third res-idue aspartic acid (E) has been mutated (fig 3B) Theseobservations suggest that at least some of these PARPs might

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Nematoda

TunicataCCiiooonnaaa innttesstintt allill ssii

CCCiooonnna saavviggnyi

1000TunnicataTT

AArthropoda

AAAApiss mmmeellllifeeraraaa

TTribboolliiiuummcaaastannett um

DDroossssoopphhilaaaaii virirr llisii

DDrrooosssoopphiillaameelanogasteer

DDrooosssrr ooophho illaaaii grimmii shawiww

1100

8866

11100

Caenorhabddidd titt ssii briggsae

Caennorharr bddititt ssii remrr anei

Caenoorhrr abdittdd itt sii eelegans

Caeenorhrr abbdittdd itttt sii elegans

1000

10000

NNematttodda

FFFuuunngggi

CCrryyptttocoocccuus gatttii

Crrryppptocoocccuus neeoformans vaar neoformans

SScchhizzooosaacccchhharoomyycces ppombe

SSSchhhizooosaacccchaaarommyycces japponicus

SSSaccchaaroommycess cerevisiaee

TetTT rrtttt arr ppiisii isii poos rarr phaffff iff iii

CCanndiddaggllall bratatt

NNeeurrossporra ccrrassa

AAAspps eeerrggrrrr iillii lll usssoryyrr zyy aaee

AAAsppeeergrgiilllusss flavuss

11000

1000

1100

11000

1000

999

AApicompplexa

ToxxTT oplasmaaggondiiii

NNeospora caaninum Liverpool

PPlasmoddiuum falciparum

Plasmoddiuum vivax

Plasmoddium yoelii yoelii

100

100

100

EExcavvaataTrichomonassvaginii alis

Tryrypanosoma cruzi

PPlaaanntttaeee

Viiitis vininiffera

AAraarr bidoopssisii thalill aii nna

AArrarabiddoppsis lyrarr ta

1000

AAAmoeebboozzzoaaDiiccttyoostteeliium discooideuumm

Diicctttyoossteeliium purpureuumm

11000

85

A

B

FIG 2 Phylogenetic analysis of the FACT-complex Spt16 and Pob3 subunits (A) Protein phylogenies representing the evolutionary patterns for Spt16(left) and Pob3 (right) FACT-complex subunits under LGthornG model of evolution Numbers on the left side of each branch represent the confidencevalues based on 1000 bootstrap replicas (only reported when at least 50) Different taxonomic groups are highlighted in different colorsTetrahymena thermophila is indicated in red The scale bar shows the number of substitutions per site (B) Left Comparative domain analysis of Tthermophila Spt16Tt and Pob3Tt against human and budding yeast homologs Right Expression analysis of Spt16Tt-FZZ (Spt16Tt116 kDathorn FZZ18 kDa) in comparison to the untagged controls by Western blotting using WCEs Blot was probed with anti-FLAG antibody for FZZ detection andanti-Actin was used as a loading control Bottom panel Indirect IF analysis of Spt16Tt-FZZ Spt16Tt-FZZ localizes to both the MAC and the MICwhereas no signal was detected in the untagged cells DAPI was used to stain the nuclei Arrows represent MAC whereas arrow heads denote MIC


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99

99

A

B

TTHERM 00726460 (PARP1)











100

76

98

10066

59

05

Human_Parp1 48 ECQRYKPFKQLHNRRLLWHGSRTTNF--------------AGILSQGLRIAPPEA-PVTGPARP1 47 EKERY--MKQLNNKMLLWHGSRLTNY--------------VGILSQGLRIAPPEA-PANGPARP2 47 ENDRY--LKDIKNKMLLWHGSRLTNF--------------VGILSQGLRIAPPEA-PVTGPARP3 43 EDERY--TKDIGNDTLLWHGSRISNF--------------VGILSQGLRIAPPEA-PVSGPARP4 43 EQERY--SKNIGNDTLLWHGSRISNF--------------VGILSQGLRIAPPEA-PVTGPARP5 48 ESQRFFPFKQLPNQTLLWHGSRISNF--------------VGILSEGLRIAPPEA-PMTGPARP6 48 EAERIKQFSD-HTKKLLWHGSGVQNL--------------LSILNFGLRINGIHA-QKSGPARP7 44 EKETLLQ-KGNASERLLFHGPGVNVNP-ETIYT-------AIEEGFDFRN----DSIQNGPARP8 48 EKQKLVQ-KGNAKELLLFHGTR-NNKP-EMIYN-------GTEEGFDFRL-------SAHPARP9 48 EKQKLKE-KGDATEKWLFHGTR-ATHP-SVIYS-------SPEQGFDFRL-------GQGPARP10 46 ERKKLEE-KGDATEKWLFHGTR-NTDP-SVIYK-------GSEEGFDFRV-------CSGPARP11 61 KVKLFGQ-CGPAYTNFGYHGTKKTCVGFEKICTIKDDCPFCSILTFGFRNAFSGKSMSLI

Human_Parp1 93 YMFGKGIYFADMVSKSANYCHTSQG-DPIGLILLGEVALGNMYELKHA-S------HISKPARP1 90 YMFGKGVYFADMCSKSANYCQANKL-NNTGLMFLCEVALGNTNDLISAGY------NASKPARP2 90 YMFGKGVYFADMCSKSANYCFTNKA-NNTGLMLLCEVALGEMNDKYYADY------YASNPARP3 86 YNYGKGIYLADMFDKSRSYCQGNSQ-G-VNYIMLIQAALGNPNRIERTDY------NASNPARP4 86 YNYGKGIYLADQFTKSCDYCAGNSD-G-IHYIMLIKAALGTPNKIEKTDY------NANNPARP5 93 YMFGKGIYMADVVSKAAGYCHAKLD-SPEGLLVLCEAALGQIYECNKAKS-------FKKPARP6 92 SSLGDGIYFADLFSKASAYANNADVGVESRFLLLCEVAVGKEQQIKTNENFTKFANSNYQPARP7 91 QIFGRGAHFHDQASKANQYAYI-TS--GKRQIIIASVLIGKAFETSSNASYTK-------PARP8 91 GMYGRGTYFHEMASYSDGYAYH-DG--SKKVFFLAQVLVGNYYVGGSS-GYVS-------PARP9 91 GMYGKGTYFHDDASYSHSFKYTTPQ--NKSQMFLAAVLVGRCIAQPPN-AFVA-------PARP10 89 GMYGRGTYFHDMASYSYGFGHN-KG--GKIQLFCAKVLIGKCYATGPNGNLTA-------PARP11 120 LRYGKGTYFSPKLQKALNYCQ---S--DQKIILACKIVMGRVFKPSCIDD----------

Human_Parp1 145 LPKGKHSVKGLGKTTPDPSANIS-LDGVDVPLGT-GISSGV---NDTSLLYNEYIVYDIAPARP1 143 LPYGKYSVRALGQIAPPKNSYINIYDDVTVPIGK-GQVRDYKNRLKTPLLHNEYIVYNVKPARP2 143 LPAGKHSTRGRGKTAPPESSYVTIYDDVQVPVGK-GEPQVFPNGQYGSLLYNEFIVYDIRPARP3 138 LPQGTNSCWGWGTFGPEQ--FIT-HNGVKVPHGKPV-----TTQSKNYMTHNEFIIYKVEPARP4 138 LPKGTHSCWGWGTHGPEE--FIT-FNGVKVPKGQEV-----RTKSKHYMKYNEFIIYDIAPARP5 145 PPQYYHSVKGVGKYKTQSEGIQKI-GTTQCFAGKVVESDENGDGQPKDLVYNEYIIYDTSPARP6 152 LMKGFNSVKLVGKSCPDEKKNLVLPNGTIVPIGPIID-------------FNENL-----PARP7 141 PP-VITEGKEQ-----------------------RYDSVKSNNQEGN----NTYAVYHNSPARP8 140 PP-IIPGTNGL-----------------------RYDSIRSNYNEGQ----NMFIIYHNSPARP9 141 PP-FYNQAKGI-----------------------RYDSVRCMGAYGH----NQYIVYHNSPARP10 139 PP-FIAGSKSI-----------------------RYDSIRSNNAIGQ----NEYVIFNNSPARP11 165 ---YFMQFDGS-----------------------KYDCIDADPQYTIDIRDPEICIKNEK

H

Y

E

FIG 3 Domain analysis of Tetrahymena thermophila PARP proteins (A) Left Protein phylogenetic analysis of putative PARPs using the identifiedPARP-catalytic domain sequences under LGthornG model of evolution Tetrahymena thermophila genome database accession numbers along withprotein names are indicated Tree topology represents ML estimations and confidence values are based on 1000 bootstrap replicas (only reportedwhen at least50) The scale bar indicates the number of substitutions per site Right Domain analysis of the T thermophila PARPs The analysiswas carried out using the SMART database (see Materials and Methods) and numbers represent the amino acid positions for each identifieddomain Domain legend is provided in the box (B) Multiple sequence alignments of PARP-catalytic domains of T thermophila PARPs The humanPARP1 catalytic domain is used as a reference to examine the conservation The catalytic residues are highlighted as red boxes


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FIG 4 Expression analysis of Tetrahymena thermophila PARP proteins and PARP6Tt localization profile during development (A) Left Heat maprepresentation of microarray expression values for PARP1-11Tt Z-scores were calculated across the rows for each PARP to examine its differentialexpression across growth starvation and various developmental stages L1ndashLH represent growth phase S0ndash24 represent starvation for 24 h and Cstands for conjugation where 0ndash18 denote hours postmixing the different mating types PDD1 is used as a conjugation-specific marker Right TopExpression analysis of PARP6Tt-FZZ (PARP6Tt 300 kDa thorn FZZ 18 kDa) in comparison to the untagged controls by Western blotting usingWCEs Blot was probed with anti-FLAG antibody for FZZ detection and anti-Actin was used as a loading control Bottom panel Western blottinganalysis indicating the recovery of the affinity purified PARP6Tt-FZZ in comparison to a control purification The blot was probed with anti-FLAG(B) PARP6Tt-FZZ localizes to both MAC and MIC during vegetative growth and starvation PARP6Tt-FZZ cells were mated with untagged WT cellsof different mating type Nuclear events are depicted above the images taken for conjugating cells during various developmental stages DAPI wasused to stain the nuclei PARP6Tt-FZZ localizes to only MAC during early conjugation events At the onset of new MAC development (anlagen)PARP6Tt-FZZ loses signal in the parental MAC and is found within developing MACs Note The signal observed in both mating pairs (PARP6Tt-FZZand controls) at the anlagen stage indicates mixing of cellular contents between the pairing cells CU428 and B2086 refer to the stock strainnumbers of the different mating types as adopted from the Tetrahymena stock center Cornell University (httptetrahymenavetcornelledulast accessed September 24 2018)


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be catalytically active Based on the domain architecture andphylogenetic analysis (fig 3A left) we assigned these putativePARPs into subgroups and established a systematic nomen-clature Notably PARP1 to PARP5 appear closely related toeach other consistent with their similar domain architectureExpression analysis using publicly available RNA-seq and mi-croarray data showed that the T thermophila PARPs havedistinct expression profiles (supplementary fig 4A and BSupplementary Material online) Most of the PARPs areweakly expressed during vegetative growth with the excep-tion of PARP4 and PARP6 (fig 4A) PARP7 and PARP8 arehighly expressed during starvation whereas PARP1 2 and 4have relatively higher expression levels during late develop-mental stages (14ndash16 h postmixing) (fig 4A supplementaryfig 4A and B Supplementary Material online) This suggeststhat PARP expression levels are tightly coordinated duringgrowth and various developmental stages In vertebratesPARP proteins including human PARP1 also contain PADR(PF08063) and zinc finger (zf)ndashPARP domains (PF00645) ThezfndashPARP domain binds to DNA whereas the function of thePADR1 domain remains unknown (Citarelli et al 2010)Interestingly none of the T thermophila putative PARPscarries any PADR1 and zfndashPARP domains Instead we iden-tified six additional proteins carrying PADR1 and zfndashPARPdomains (supplementary fig 4C Supplementary Material on-line) Thus T thermophila PARPs might require additionalprotein factors for their proper functioning

Among the identified PARP proteins PARP6Tt

(THERM_00502600) which copurified with H2A piquedour interest due to its unique domain architecturePARP6Tt contains 25 tandem ankyrin repeats (ANK) as wellas two DNA binding AT-hook domains in addition to thePARP-catalytic and PARP-regulatory (PF02877) domains(fig 3A) This domain organization is unique to Amoebozoa(Dictyostelium) Opisthokonta (fungi) and Chromalveolates(ciliates) and has been categorized as the PARP1 subfamily(Citarelli et al 2010) Interestingly human PARP5a b (knownas Tankyrase 1 and 2 respectively) also contain tandem ANKrepeats as well as a PARP-catalytic domain but lack PARP-regulatory and AT-hook domains Tankyrase 1 and 2 functionin maintenance of telomeres (Chiang et al 2008) To gainfunctional insights we generated a strain of T thermophilastably expressing PARP6Tt-FZZ from its native MAC locus(fig 4A right) We performed AP-MS analysis on growing cellsto investigate the PARP6Tt-interacting proteins The recoveryof the bait was examined using Western blotting analysis(fig 4A right) The SAINTexpress analysis revealed ninehigh-confidence PARP6Tt-FZZ copurifying proteins includinghistone H2A Additionally H3 and ribosomal proteins wereidentified as PARP6Tt-FZZ copurifying partners (see supple-mentary file 2 Supplementary Material online for details)The copurification of H2A with PARP6Tt-FZZ reciprocallyverifies the interaction between the two proteins PARP6Tt

and histones H2A and H3 cluster together based on theirgene expression profiles further indicating a role of PARP6Tt

in histone metabolism (supplementary fig 4DSupplementary Material online)

PARP6Tt is expressed throughout the T thermophila lifecycle with relatively low expression levels during early conju-gation (1ndash2 h postmixing the cells) as examined by using pre-viously published expression data (fig 4A left) (Miao et al2009 Xiong et al 2012) The expression levels increase between6 and 8 h postmixing a time of new MAC development Weperformed IF staining in growing and conjugating T thermo-phila cells to examine the PARP6Tt-FZZ localization duringdevelopment PARP6Tt-FZZ localized to both the MAC andMIC in growing and starved T thermophila (fig 4B)Interestingly we observed that PARP6Tt-FZZ loses signal inthe MIC and localizes exclusively to the MAC during conjuga-tion when the cells have formed pairs (fig 4B) More specificallyit localizes to the parental MAC during early nuclear develop-mental stages including meiosis before switching to the anla-gen which corresponds to midway through development(fig 4B) The localization of PARP6Tt-FZZ in the parentalMAC is lost at the onset of MAC development a stage wherethe two anterior nuclei (the anlagen) have become visiblylarger than the posterior nuclei (fig 4B) This pattern of local-ization is strikingly similar to that of Ibd1 (Interactive Bromo-Domain protein 1) protein which we recently reported tofunction as a recruitment hub for various transcription regu-lators and chromatin remodeling complexes (Saettone et al2018) The PARP6Tt subcellular localization appears to corre-late with transcriptional activity during nuclear developmentFurther studies will be needed to explore the role of PARP6Tt intranscription regulation and histone metabolism

Nucleoplasmin Has an Ancient OriginNPM-family proteins are histone H2AH2B chaperones withcritical roles in various cellular processes (Box et al 2016)NPM-family proteins have been linked to a number of humandiseases including acute myeloid leukemia and are the sub-ject of anticancer drug development (Box et al 2016)Previous work has shown that among vertebrates theNPM-family has greatly diversified giving rise to three mem-bers (NPM1ndash3) whereas invertebrates such as Drosophilacontain only a single Npm-like protein (NLP) (Eirın-Lopezet al 2006) To date no orthologs have been detected inArabidopsis thaliana Saccharomyces cerevisiae orCaenorhabditis elegans Little is known however about theevolution and origin of NPM proteins and as such theyhave not been studied in unicellular model organismsDeciphering the evolutionary history often provides mean-ingful insights into protein function To trace their evolution-ary origin we carried out database searches and identifiedputative NPM homologs throughout the basal unicellulareukaryotes including chromalveolates and excavates (supple-mentary file 1 Supplementary Material online) We recon-structed a protein phylogeny using the identified homologsand found that these proteins have a monophyletic originand share a common ancestry (fig 5A) Importantly the iden-tification of NPM homologs in the earliest branching eukar-yotes such as kinetoplastids confirms an ancient origin of thisprotein family


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Drosophila NLP (dNLP) also binds H2AH2B dimers andassembles histone octamers (Namboodiri et al 2003) sug-gesting functional conservation among distantly related fam-ily members To gain functional insights we compared thestructural features of the putative T thermophila Npm1 withthose of human NPMs and dNLP We observed thatT thermophila Npm1 domain organization is highly con-served and nearly identical to that of human NPM1 (fig 5B

left) In fact the T thermophila Npm1 predicted N-terminalcore domain can be structurally superimposed to that of thehuman NPM1 (fig 5B right) We named the putative Tthermophila homolog as conserved nucleoplasmin-like 1(cNpl1) We engineered T thermophila cell lines stablyexpressing cNPL1-FZZ from its native chromosomal locusThe expression of the tagged protein was examined byWestern blotting (fig 5C left) and AP-MS experiments which

A

B

C

FIG 5 Phylogenetic relationship among NPM-family proteins (A) Protein phylogeny of NPM-family members in Protista under LGthornG model ofevolution Different taxonomic groups are highlighted in colors Arthropoda NPMs are used to represent the metazoan sequences Tree topologyrepresents the ML estimations based on 1000 bootstrap replicas (confidence value only reported when at least50) The scale bar indicates thenumber of substitutions per site (B) Left Domain organization of cNpl1 in comparison to human and Xenopus laevis NPM1 proteins andDrosophila melanogaster NLP ldquoArdquo represents acidic stretches shown in red triangles and NES and NLS stand for nuclear export and import signalsNucleolar localization signal is denoted as NoLS NPM core N-terminal domain (PF03066) is shown in light blue and the C-terminal region is shownin red accent color Note cNpl1Tt NoLS was predicted using the ldquoNODrdquo web server (httpwwwcompbiodundeeacukwww-nodindexjsp lastaccessed September 24 2018) Right Cartoon diagram shows the predicted structure of the cNpl1 core domain in rainbow color The predictedcNpl1Tt structure shown in rainbow color was superimposed with the human NPM1 crystal structure (PDB ID 2P1B) depicted in violet backboneformat N- and C-termini are indicated (C) Left Expression analysis of cNpl1Tt-FZZ (cNPL1 40 kDa thorn FZZ 18 kDa) in comparison to theuntagged controls by Western blotting using WCEs Blot was probed with anti-FLAG antibody for FZZ detection whereas anti-Actin was used as aloading control Right Indirect IF analysis of cNpl1Tt-FZZ cNpl1Tt primarily localizes to MAC No signal was detected in the untagged control cellsDAPI was used to stain the nuclei Arrows represent MAC whereas arrow heads denote MIC


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successfully recovered the bait (not shown) without anyother significant interaction partners (see Discussion) IF anal-ysis showed that cNpl1Tt primarily localizes to the transcrip-tionally active MAC (fig 5C right) consistent with knownroles of human NPM1 in transcription- and chromatin-related processes We conclude that NPMs are a structur-allyfunctionally conserved family of proteins which arosevery early during the eukaryotic diversification

Identification of T thermophila Hv1-InteractingProteomeWe next focused on delineating the PPIs of transcription as-sociated histone H2A variant Hv1 (H2AZ in humans) in Tthermophila We utilized our above described strategy to gen-erate T thermophila strains stably expressing Hv1-FZZ fromtheir native MAC locus The expression of the tagged proteinwas monitored by Western blotting analysis using WCEs pre-pared from Hv1-FZZ expressing cells in comparison to theuntagged control cell lysates (fig 6A left) Hv1 has previouslybeen reported to exclusively localize to the MAC duringgrowth (Stargell et al 1993) Our IF analysis of the Hv1-FZZexpressing cells showed an exclusive MAC signal indicatingthat the FZZ tag does not interfere with the protein localiza-tion (fig 6B)

We subjected the Hv1-FZZ expressing cells to our AP-MSpipeline Recovery of the bait was monitored by Western blot-ting (fig 6A right) SAINTexpress analysis of the LCndashMSMSdata revealed that Hv1 copurifies with 106 significant interact-ing partners (BFDR 1) We annotated these hits either byhomology searches against the S cerevisiae and humangenomes or by using T thermophila genome database anno-tations (supplementary file 2 Supplementary Material onlinefor annotations and conservation of interaction data fig 6C)ATP-dependent chromatin-remodeling complexes includingSWR- and INO80-complexes are known to antagonisticallymodulate H2AZ (Htz1 in yeast) dynamics The SWR-C is spe-cialized to deposit H2AZ onto chromatin (Krogan et al 2003Kobor et al 2004) whereas INO80-C mediates the reverse ofthis reaction (Papamichos-Chronakis et al 2011) mainly atnonpromoter sites (reviewed by Gerhold and Gasser [2014])Both the SWR-C and INO80-C have several shared as well asdistinct subunits (reviewed by Gerhold and Gasser [2014])Interestingly SAINTexpress analysis of the Hv1-FZZ AP-MSdata revealed the copurification of a set of proteins that basedon similarity to S cerevisiae orthologs comprise the putativesubunits of T thermophila INO80-C and SWR1-C The identi-fied INO80-C putative subunits include Arp8 Actin1 (alsoshared with SWR-C) Yuh1 and Ino80 (fig 6C) In additionwe also identified the RuvB1 (also shared with SWR-C) andIes2 subunits of the INO80-C albeit at a slightly relaxedSAINTexpress value (BFDR 3) We have recently purifiedT thermophila SWR-C via Swc4-FZZ and identified at least 12subunits (Saettone et al 2018) In addition to Actin1 andRuvB1 SAINTexpress identified Swr1 Swc2 and Arp5 subunitsof SWR-C as high confidence interacting proteins (fig 6C)These data indicate that Hv1 deposition and eviction from

the chromatin are tightly regulated by a highly conservednetwork of chromatin-remodeling complexes

Other high-confidence Hv1 copurifying proteins withchromatin-related functions (inferred by sequence similarityto proteins in yeast and humans) could be broadly dividedinto four groups 1) putative transcription and chromatin as-sembly regulators including Spt16 and Pob3 (FACT-complex)Spt6 Cys2-His2 zf transcription factor ZAP1 TAF6 HMG pro-tein Ixr1 transcription factors bZIP1 and bZIP2 2) chromatinremodeling SWISNF complex subunits Swi3 and Snf12 3)PARP proteins including PARP1 PARP2 and PARP5 and 4)proteins with various DNA- and RNA-related functions suchas putative Alba2 DNA-binding protein RNA-helicases andtopoisomerases (fig 6C) Furthermore we also identified aPOZ-domain protein Hiap1 and 8 additional Tetrahymena-specific hypothetical proteins without any recognizabledomains We named these proteins as ldquohypothetical histonecopurifying proteins (HHCP1ndash8)rdquo (fig 6C) (see supplementaryfile 2 Supplementary Material online for conserved and novelinteractions)

We clustered the Hv1-FZZ copurifying proteins based ontheir gene expression profiles (supplementary fig 5Supplementary Material online) Our analysis suggests thatproteins with key roles in histone metabolism such as histonechaperones share highly similar expression profiles and clustertogether with Hv1 whereas factors with diverse functions (asinferred by similarities with yeast or human proteins) such asRNA-helicases topoisomerases and kinases are less likely tohave expression patterns comparable with those of the histo-nes (supplementary fig 5 Supplementary Material online)Notably consistent with their known role(s) in histone me-tabolism INO80-C SWR1-C FACT-complex Spt6 and SWISNF-complex subunits cluster together with Hv1 due to theirvery similar gene expression profiles further reinforcing theidea that these proteins are functionally conserved in T ther-mophila We conclude that variant Hv1 in T thermophilaforms several functional links that might influence the tran-scriptional landscape of the cell and furthermore Hv1 distri-bution along the chromatin is regulated via a highly conservednetwork of chaperones

DiscussionAlthough the deposition complexes for histones H2AH2Band H2A variant H2AZ have been identified (Zhang et al2017) information regarding the histone chaperoningnetwork(s) outside of Opisthokonta remains limitedConsidering the complexity of the histone deposition path-ways new factors are likely to be found to have key roles inthese processes Tetrahymena thermophila is an evolution-arily divergent unicellular eukaryote and is particularly suit-able to study histone dynamics (Orias et al 2011 Gao et al2016) In fact initial clues regarding the transcription-relatedrole(s) of H2A variants emerged from T thermophila follow-ing the observations that Hv1 resides within the transcrip-tionally active nuclei (Martindale et al 1985 Stargell et al1993) As per our ongoing efforts to understand the histonedeposition pathways here we report the first comprehensive


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FIG 6 Hv1-FZZ expression and affinity purification (A) Left Expression analysis of Hv1-FZZ (H2A15 kDathorn FZZ18 kDa) in comparison to theuntagged controls by Western blotting using WCEs Blot was probed with anti-FLAG antibody for FZZ detection whereas anti-Actin was used as aloading control Right Western blotting analysis indicating the recovery of the affinity purified (AP) Hv1-FZZ The blot was probed with theindicated antibodies No signal was detected in the WT lanes Note Two bands in the Hv1-FZZ input lane could represent dimerized histones (B)Indirect IF analysis of Hv1-FZZ Hv1-FZZ exclusively localized to MAC only during growth The lower panel indicates dividing cells No signal wasdetected in the untagged control cells DAPI was used to stain the nuclei Arrows represent MAC whereas arrow heads denote MIC (C) Networkview of Hv1-FZZ PPIs Bait node is shown in yellow Prey node borders are colored according to their putative functions or protein complexesNetwork legend is provided in the box


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PPI network for H2A its variant Hv1 and H2B in Tthermophila

Ancient Histone ChaperonesAn interesting outcome of our work is that T thermophilahistones H2A (Hv1)H2B are connected to a network ofhighly conserved chaperones and karyopherins We have pre-viously reported that Impb6 physically interacts with Asf1both of which localize to both MAC and MIC with a signif-icantly stronger signal in the MIC indicating that Impb6functions in the H3H4 transport pathway (Garg et al2013) The copurification of Impb6 with H2B highlights theidea that it might be a more generalized karyopherin in Tthermophila for core histone transport pathways It will beimportant to test this hypothesis by depleting Impb6 anddetermining whether core histone can enter the MAC orMIC Apart from cNpl1 which was found exclusively in theMAC most of the H2A and H2B interacting partners that wecharacterized in this work localized to both the MAC andMIC We expect RD histone-binding proteins to be found inthe MAC and MIC as core histones are found within bothnuclei (Song et al 2007 Wang et al 2009) The variantHv1 is known to have nuclear-specific functions(Martindale et al 1985 Stargell et al 1993) We found thatanother karyopherin Impb3 (TTHERM_00550700) copurifiedwith Hv1 (though it fell below our stringent confidencethreshold) and like Hv1 it localizes to MAC only (supple-mentary fig 6 Supplementary Material online) consistentwith a functional link between the two proteins We suggestthat transport of T thermophila H2A (Hv1)H2B to the nucleiand their subsequent assembly onto chromatin is mediatedby an interplay among conserved karyopherins histone chap-erones and chromatin-remodeling complexes (fig 7) consis-tent with what has been proposed in humans and yeast It willbe important to determine the complete PPI networks forImpb6 and Impb3 whether by AP-MS or orthogonal

methods such as Bio-ID Future work should focus on under-standing the nuclear-specific replication-independent chro-matin assembly pathways and the role of chaperones such ascNpl1 in these processes

Numerous chaperones such as NASP NPMs and yeastAsf1 possess long acidic stretches consistent with their po-tential to bind basic histones (reviewed by De Koning et al[2007]) Hiap1Tt also possesses several acidic stretches with anoverall net negative charge (not shown) suggesting a possi-bility to function as a histone-binding protein We suggestthat Hiap1 functions as an H2AH2B chaperone in T thermo-phila It is also worthwhile to note here that the T thermo-phila ortholog of Nap1 also copurified with H2B and Hv1(though it fell below our high-confidence threshold) Nap1is a histone chaperone with a known function in H2AH2Btransport (Mosammaparast et al 2002) Further work beyondthe scope of this report will be required to examine the role ofT thermophila Nap1 and Hiap1 proteins in H2AH2B metab-olism It will be important to express Hiap1 as a recombinantprotein and examine whether it binds histones

We have previously reported that histone chaperones in-cluding Asf1 and NASP are highly conserved throughout evo-lution (Nabeel-Shah et al 2014) likely representinginnovations to specifically regulate eukaryotic H3H4 dynam-ics Our present study has highlighted several aspects regard-ing the conserved nature of chromatin-remodeling and H2AH2B assembly complexes The FACT-complex is of particularinterest due to its important roles in chromatin- andtranscription-related processes FACT is a histone chaperoneand facilitates transcription elongation by colocalizing withRNAPII (Mason and Struhl 2003) Our evolutionary analysisindicated that FACT was already present in the last commonancestor of all eukaryotes indicating its functional impor-tance The similarities between the FACT evolutionary profileand the species phylogeny highlight the role of histone chap-erones in eukaryotic evolution This hypothesis is consistent

FIG 7 Model for H2A (Hv1)ndashH2B nuclear transport in Tetrahymena thermophila


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with previous work indicating that chromatin architecturalHMG protein (Gonzalez-Romero et al 2015) histones (Eirın-Lopez et al 2012) and their chaperones including Asf1 NASP(Nabeel-Shah et al 2014) NPMs (Eirın-Lopez et al 2006Frehlick et al 2007) might have played critical roles duringeukaryotic evolution Previous work has shown that T ther-mophila Spt16Tt associates with transcriptionally active MACchromatin in vitro (Fujiu and Numata 2004) Consistent witha role in transcription we also found that Spt16Tt stablyinteracts with RNA polymerase subunits and localizes tothe MAC Spt16Tt localization to MIC likely representstranscription-independent function(s) of the FACT-complex Consistent with this hypothesis FACT also func-tions in an array of processes including DNA replicationand repair (Charles Richard et al 2016 Yang et al 2016Kurat et al 2017)

The T thermophila genome encodes TTHERM_00216040which shares sequence similarity to yeast HMG domain-containing protein Nhp6 We did not recover any HMG pro-tein to copurify with Spt16Tt (when enforcing an FDR cut-offof 1) Our comparative analysis indicated that Pob3 withinvertebrates arthropods tunicates and plants carries an HMGdomain whereas lineages representing fungi amoebazoa cil-iates apicomplexa and excavatas do not possess this domainWe suggest that HMG was not present in the ancestral FACT-complex and was later acquired to meet the demands ofcomplex regulatory layers of chromatin

Human NPM1 is known to function in an array of pro-cesses including histone chaperoning chromatin remodelingtranscription regulation genome stability apoptosis and em-bryogenesis (Okuwaki et al 2001 Grisendi et al 2005Swaminathan et al 2005 Box et al 2016) Owing to its lossin widely studied eukaryotic microbial model organisms (egS cerevisiae) previous studies have been restricted to culturedcells Furthermore earlier attempts to decipher the evolution-ary history of the NPMs have been limited to metazoans(Eirın-Lopez et al 2006) Our finding that cNpl1Tt copurifieswith H2A in T thermophila combined with the observationsthat NPMs are highly conserved throughout the basal eukar-yotes paves the way to study their function in easily tractableeukaryotic model organisms Human NPM1 is thought tohave key roles in cell cycle regulation (Zhao et al 2015Pfister and DrsquoMello 2016) Tetrahymena thermophila cellslacking Cyc2 and Cyc17 are arrested at early crescent (2ndash35 h postmixing) and diakinesis-like metaphase I (5 h post-meiotic induction) stages of meiosis respectively (Xu et al2016 Yan et al 2016) Interestingly cNpl1Tt expression levelsare significantly upregulated at these meiotic stages in Cyc2and Cyc17 knockouts as examined using publicly availableRNA-seq data (supplementary fig 7 SupplementaryMaterial online) This suggests a role for cNpl1Tt in cell cycleregulation Our AP-MS experiments using cNpl1-FZZ success-fully recovered the bait however further work is required toreveal the full scope of its interactions and unravel potentialrole(s) during development To this end carrying out BioIDan orthogonal approach to AP-MS that identifies proteinsproximal to the bait in the cell (Kim et al 2016) during growthand development will be informative and is in progress

Role of PARPs in Histone MetabolismOur study also implicates PARPs in histone metabolismPARPs are functionally diverse proteins with critical roles ina number of processes including DNA break repair (Langelieret al 2012) cell cycle regulation (Masutani et al 1995) mRNAbinding (Melikishvili et al 2017) transcription regulation (Koand Ren 2012 Chen et al 2014) and maintenance of chro-matin architecture (for review Bai 2015) The observation thatthe T thermophila genome encodes 11 putative PARPs andtheir expression is temporally regulated suggests that theseproteins might be important for distinct cellular processesduring various stages of the Tetrahymena life cycle Previousstudies have reported that T thermophila histones are highlyADP-ribosylated (Levy-Wilson 1983) It was recently reportedthat in humans newly synthesized histones H3H4 carry poly(ADP-ribosylated) marks (Alvarez et al 2011) In this study itwas proposed that poly (ADP-ribosylation) might help tokeep histones H3 and H4 folded in the absence of the otherhistones (Alvarez et al 2011) The copurification of certainPARPs with histones in T thermophila is consistent with theseearlier findings Another hypothesis is that certain T thermo-phila PARPs might function as well as a histone chaperonesimilar to what has been shown for human PARP1(Muthurajan et al 2014) PARP6Tt is of particular interestdue to its domain architecture and expression patternsThe PARP6Tt contains 25 tandem ANK repeats similar toits distantly related human Tankyrases 1 and 2 which func-tion in telomere maintenance (Chiang et al 2008) ThePARP6Tt localization pattern during early conjugation corre-lates with the transcriptional state of the nuclei suggesting arole in transcription regulation As the human tankyrases areactively being pursued as drug targets it will be informative tofurther examine the PARP6Tt functions through phenotypicanalysis of a PARP6Tt knockout

Conserved Regulatory Network for Variant Hv1The T thermophila H2A variant Hv1 localization profile hasbeen reported to be correlated with the transcriptional stateof the nuclei (Stargell et al 1993) Consistently recentgenome-wide studies reported a strong enrichment of Hv1near the transcription start sites (Wang et al 2017) The SWR-and INO80-complexes are known to function antagonisticallyto regulate the Htz1 (or H2AZ in humans) chromatin occu-pancy (Gerhold and Gasser 2014) We suggest that similar tohumans and yeast T thermophila Hv1 chromatin occupancyis guided by evolutionarily conserved SWR- and INO80-complexes Based on expression profiles the subunits ofSWR- and INO80-complexes cluster with Hv1 supportingtheir functional link Our recent report suggests that abromo-domain protein Ibd1 in T thermophila might be re-sponsible for recruiting SWR-complex to highly expressedgenes (Saettone et al 2018) Tetrahymena thermophila enc-odes at least 14 bromo-domain proteins and it will be inter-esting to examine the potential role of bromo-domainproteins in INO-80 recruitmentfunction

In addition to the FACT-complex Spt6Tt was also recov-ered as a significant interacting protein in Hv1 AP-MS dataSaccharomyces cerevisiae Spt6 has a well-documented role as


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a histone chaperone during transcription (Bortvin andWinston 1996 Hartzog et al 1998) Spt6 physically interactswith RNAPII and functions to reassemble nucleosomes in thewake of RNAPII passage (Kaplan et al 2003) Recent evidenceindicates that the FACT-complex and Spt6 inhibit the wide-spread chromatin incorporation of H2AZ by preventing thepervasive recruitment of SWR-C to gene bodies (Jeronimoet al 2015) The copurification of Spt6Tt with Hv1 suggeststhat Spt6Tt might have similar functions to regulate the tran-scription and safeguard the Hv1 occupancy across chromatinThe observation that Spt6Tt and FACT-complex have verysimilar expression profiles further reinforces the possibilitythat these proteins are functionally linked Spt6Tt knockoutanalysis followed by monitoring SWR-C and Hv1 chromatinoccupancy will be instrumental to test this hypothesis

ConclusionsOur study has provided the first comprehensive view of Tthermophila histones H2A variant Hv1 and H2B protein-interaction networks Providing new insights into ciliatesrsquo his-tone metabolism our study also highlighted the conservednature of chromatin regulatory networks involving H2A(Hv1)ndashH2B-specific chaperones thus underscoring the broadutility of these results Further work is warranted to under-stand the mechanistic details of conserved chaperones andchromatin-remodeling complexes that we have identifiedhere

Materials and Methods

Cell StrainsTetrahymena thermophila strains CU428 [MprMpr (VII mp-s)] and B2086 [MprthornMprthorn (II mp-s)] of inbreeding line Bwere obtained from the Tetrahymena Stock Center CornellUniversity Ithaca NY (httptetrahymenavetcornelledu)Cells cultured in 1 SPP were maintained axenically at 30C as previously described (Fillingham et al 2001)

Bioinformatics and Molecular Evolutionary AnalysesAmino acid sequences for yeast Spt16 Pob3 and humanNPM1 were acquired from the UniprotKB and were usedas a query to search the NCBI nonredundant database usingPSI-BLAST with default parameters Protein sequences re-trieved were analyzed at the Pfam (httppfamsangeracuk last accessed September 24 2018) (Finn et al 2016)and SMART (httpsmartembl-heidelbergde last accessedSeptember 24 2018) (Letunic and Bork 2018) databases toexamine the domain architecture (supplementary file S1Supplementary Material online for accession numbers) Toreconstruct a protein phylogeny we used amino acid sequen-ces of the identified conserved domains (as identified bySMART analysis) present within Spt16 (FACT-Spt16_NlobPeptidase_M24 (PF00557) Spt16 signature and Rtt106domains) and Pob3 (SSrecog [PF03531] and Rtt106 domain)orthologs For the NPM-family phylogeny complete proteinsequences were used For phylogenetic trees we also includedall the paralogous genes that were identified within a givenspecies Multiple sequence alignments were built using

MUSCLE with default parameters All protein phylogeneticanalyses were carried out using the maximum likelihood (ML)method under LGthornG model using MEGA 7 (Kumar et al2016) The reliability of the resulting phylogenetic trees wasassessed using the bootstrap method (1000 replicas for eachtree) cNpl1 structural prediction and superimposition werecarried out using I-TASSER server (Yang et al 2015)Molecular evolutionary analyses were carried out usingMEGA 7 (Kumar et al 2016) To identify putative PARPswe used the human PARP1 catalytic domain amino acid se-quence as a query against the T thermophila genome (Pleaserefer to supplementary methods Supplementary Materialonline for further details on molecular evolutionary analysesfor Spt16 Pob3 and PARPs)

Macronuclear Gene ReplacementEpitope tagging vectors for H2A H2B Hv1 Spt16Tt Parp6TtcNpl1 and Impb3 were constructed by amplifying two sep-arate1-kb fragments up- and downstream of the predictedstop codons using WT T thermophila genomic DNA as tem-plate Upstream and downstream PCR products weredigested with KpnI and XhoI or NotI and SacI respectivelyThe digested products were cloned into the appropriate siteswithin the tagging vector (pBKS-FZZ) provided by DrKathleen Collins (University of California Berkeley CA) Theresulting plasmid was again digested with KpnI and SacI priorto transformation One micrometer gold particles (60 mgmlBio-Rad) were coated with 5 lg of the digested plasmid DNAwhich was subsequently introduced into the T thermophilaMAC using biolistic transformation with a PDS-1000HeBiolistic particle delivery system (Bio-Rad) The transformantswere selected using paromomycin (60 lgml) To achieveMAC homozygousity cells were grown in increasing concen-trations of paromomycin to a final concentration of 1 mgml

Generation of WCEs and Western BlottingWe used 10 trichloroacetic acid to prepare WCEs by incu-bation on ice for 30 min The WCEs were resuspended in100 ll of SDS loading dye To neutralize the solution 10 llof 1 N NaOH was added WCEs were subjected to electro-phoresis through 10 SDS-PAGE The proteins were trans-ferred to nitrocellulose and probed with indicated antibodiesafter blocking in 5 skim milk Antibodies and dilutions usedwere anti-Flag (14000 Sigma) anti-Actin (110000 Abcam)and anti-Brg1 (11000 as described by Fillingham et al[2006])

Experimental Design for Mass SpectrometryExperimentsFor each analysis at least two biological replicates of each baitwere processed independently These were analyzed along-side negative controls in each batch of samples processedTetrahymena cells expressing no tagged bait (ie empty cells)were used as control To minimize carry-over issues extensivewashes were performed between each sample (see details foreach instrumentation type) and the order of sample acqui-sition on the mass spectrometer was reversed for the secondreplicate to avoid systematic bias On the LTQ mass


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spectrometer a freshly made column was used for each sam-ple as described (Saettone et al 2018)

Affinity Purification and Mass Spectrometry SamplePreparationAffinity purification was carried out essentially as described(Garg et al 2013) Briefly T thermophila were grown in500ml of 1 SPP to a final concentration of 3 105 cellsml werepelleted and frozen at 80 C The pellets were thawed onice and resuspended in lysis buffer (10 mM TrisndashHCl pH 751 mM MgCl2 300 mM NaCl and 02 NP40 plus yeast pro-tease inhibitors [Sigma]) Benzonase (Sigma E8263) was added(500 units) and extracts were rotated for 30 min at 4 CWCEs were clarified by centrifugation at 16000g for 30 minand resulting soluble material was incubated with 50 ll ofpacked M2-agarose (Sigma) at 4 C for 3ndash4 h The M2-agarose was washed once with 10 ml IPP300 (10 mM TrisndashHCl pH 80 300 mM NaCl 01 NP40) two times with 5 ml ofIP100 buffer (10 mM TrisndashHCl pH 80 100 mM NaCl 01NP40) and two times with 5 ml of IP100 buffer without de-tergent (10 mM TrisndashHCl pH 80 100 mM NaCl) Five hun-dred microliters of 05 M NH4OH was used to elute theproteins by rotating for 20 min at room temperaturePreparation of protein eluates for mass spectrometryacquisition was essentially as previously described (Saettoneet al 2018) (Please refer to supplementary methodsSupplementary Material online for details)

MS Data Visualization and ArchivingInteraction networks were generated using Cytoscape (V340Cline et al 2007) Individual nodes were manually arranged inphysical complexes The annotation of the copurifying part-ners was carried out using BLAST searches as well as SMARTdomain analysis (httpsmartembl-heidelbergde lastaccessed September 24 2018) of the predicted amino sequen-ces as acquired from the Tetrahymena genome database(wwwciliateorg last accessed September 24 2018) All MSfiles used in this study were deposited at MassIVE (httpmassiveucsdedu last accessed February 15 2018) Additionaldetails (including Mass IVE accession numbers and FTPdownload links) can be found in supplementary table S2FSupplementary Material online For gene expression analysismicroarray data (accession number GSE11300) was acquired(httptfgdihbaccn last accessed September 24 2018) andthe expression values were represented in the heatmap for-mat Hierarchical clustering was performed to assess the sim-ilarities in gene expression profiles

Indirect IFCells were grown and fixed during vegetative growth 24-h starvation and 2 4 6 and 75 h postmixing after starvationto perform indirect IF as previously described (Garg et al2013) (Please refer to supplementary methodsSupplementary Material online for details)

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Dr Takahiko Akematsu for his assistance with mi-croscopy We also thank Anita Samardzic for her technicalassistance with Tetrahymena growth media preparationsWork in the Fillingham and Lambert laboratories was sup-ported by the Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) Discovery Grants RGPIN-2015-06448 and RGPIN-2017-06124 respectively J-PL holds aJunior 1 salary award from the Fonds de Recherche duQuebec-Sante (FRQ-S) and was also supported through aJohn R Evans Leaders Fund from the Canada Foundationfor Innovation (37454) Work in the Pearlman laboratorywas supported by Canadian Institutes of Health Research(CIHR) (MOP13347) and Natural Sciences and EngineeringResearch Council of Canada (NSERC) Discovery Grant539509 Work in the Gingras laboratory was supported bythe Canadian Institutes of Health Research (CIHR)Foundation Grant (FDN 143301) The authors declare noconflict of interest

Author ContributionsKA generated H2A-FZZ Spt16-FZZ PARP6-FZZ and Hv1-FZZ cell lines and performed Western blots affinity purifica-tions IF microscopy data analysis participated in manuscriptdrafting and in overall study design with JF and REPrsquos feed-back SN-S performed evolutionary analysis participated instudy design with feedback from JF REP and KA preparedall the final figures wrote the manuscript and coordinatedthe edits from all the authors JG generated cNpl1-FZZ cellline performed IF analysis on cNpl1-FZZ and affinity purifi-cation on Hv1-FZZ AS generated H2B-FZZ performed IFsand affinity purification on H2B-FZZ JD participated in H2B-FZZ generation J-PL processed and analyzed samples formass spectrometry provided feedback on data figures andedited the manuscript A-CG participated in manuscriptediting and mass spectrometry REP cosupervised the proj-ect provided reagents monitored the overall progress andparticipated in manuscript editing JF envisioned anddesigned the study cosupervised the project coordinatedthe overall progress of the study and edited the manuscriptAll authors have read and approved the final manuscript

ReferencesAdl SM Simpson AGB Lane CE Lukes J Bass D Bowser SS Brown MW

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Alvarez F Mu~noz F Schilcher P Imhof A Almouzni G Loyola A 2011Sequential establishment of marks on soluble histones H3 and H4J Biol Chem 286(20) 17714ndash17721

Bai P 2015 Biology of poly(ADP-ribose) polymerases the factotums ofcell maintenance Mol Cell 58(6) 947ndash958

Belotserkovskaya R Oh S Bondarenko VA Orphanides G Studitsky VMReinberg D 2003 FACT facilitates transcription-dependent nucleo-some alteration Science 301(5636) 1090ndash1093

Boekhorst J van Breukelen B Heck AJ Snel B 2008 Comparative phos-phoproteomics reveals evolutionary and functional conservation ofphosphorylation across eukaryotes Genome Biol 9(10) R144


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Bortvin A Winston F 1996 Evidence that Spt6p controls chromatinstructure by a direct interaction with histones Science 272(5267)1473ndash1476

Box JK Paquet N Adams MN Boucher D Bolderson E OrsquoByrne KJRichard DJ 2016 Nucleophosmin from structure and function todisease development BMC Mol Biol 17(1) 19

Cassidy-Hanley D Bowen J Lee JH Cole E VerPlank LA Gaertig JGorovsky MA Bruns PJ 1997 Germline and somatic transformationof mating Tetrahymena thermophila by particle bombardmentGenetics 146(1) 135ndash147

Charles Richard JL Shukla MS Menoni H Ouararhni K Lone INRoulland Y Papin C Ben Simon E Kundu T Hamiche A et al2016 FACT assists base excision repair by boosting the remodelingactivity of RSC Bianchi M editor PLoS Genet 12(7) e1006221

Chen H Ruiz PD Novikov L Casill AD Park JW Gamble MJ2014 MacroH2A11 and PARP-1 cooperate to regulate transcriptionby promoting CBP-mediated H2B acetylation Nat Struct Mol Biol

Chiang YJ Hsiao SJ Yver D Cushman SW Tessarollo L Smith S Hodes RJ2008 Tankyrase 1 and tankyrase 2 are essential but redundant formouse embryonic development PLoS One 3(7) e2639

Citarelli M Teotia S Lamb RS 2010 Evolutionary history of thepoly(ADP-ribose) polymerase gene family in eukaryotes BMC EvolBiol 10308

Cline MS Smoot M Cerami E Kuchinsky A Landys N Workman CChristmas R Avila-Campilo I Creech M Gross B 2007 Integration ofbiological networks and gene expression data using Cytoscape NatProtoc 2(10) 2366ndash2382

De Koning L Corpet A Haber JE Almouzni G 2007 Histone chaperonesan escort network regulating histone traffic Nat Struct Mol Biol14(11) 997ndash1007

Eirın-Lopez JM Frehlick LJ Ausio J 2006 Long-term evolution and func-tional diversification in the members of the nucleophosminnucle-oplasmin family of nuclear chaperones Genetics 173(4) 1835ndash1850

Eirın-Lopez JM Rebordinos L Rooney AP Rozas J 2012 The birth-and-death evolution of multigene families revisited In GenomeDynamics Vol 7170ndash196

English CM Adkins MW Carson JJ Churchill MEA Tyler JK 2006Structural basis for the histone chaperone activity of Asf1 Cell127(3) 495ndash508

Fillingham JS Bruno D Pearlman RE 2001 Cis-acting requirements inflanking DNA for the programmed elimination of mse29 a com-mon mechanism for deletion of internal eliminated sequences fromthe developing macronucleus of Tetrahymena thermophila NucleicAcids Res 29(2) 488ndash498

Fillingham JS Garg J Tsao N Vythilingum N Nishikawa T Pearlman RE2006 Molecular genetic analysis of an SNF2brahma-related gene inTetrahymena thermophila suggests roles in growth and nuclear de-velopment Eukaryot Cell 5(8) 1347ndash1359

Finn RD Coggill P Eberhardt RY Eddy SR Mistry J Mitchell AL PotterSC Punta M Qureshi M Sangrador-Vegas A et al 2016 The Pfamprotein families database towards a more sustainable future NucleicAcids Res 44(D1) D279ndashD285

Formosa T Eriksson P Wittmeyer J Ginn J Yu Y Stillman DJ 2001Spt16-Pob3 and the HMG protein Nhp6 combine to form thenucleosome-binding factor SPN EMBO J 20(13) 3506ndash3517

Frehlick LJ Eirın-Lopez JM Ausio J 2007 New insights into the nucleo-phosminnucleoplasmin family of nuclear chaperones Bioessays29(1) 49ndash59

Fujiu K Numata O 2004 Identification and molecular cloning ofTetrahymena 138-kDa protein a transcription elongation factor ho-mologue that interacts with microtubules in vitro Biochem BiophysRes Commun 315(1) 196ndash203

Gao F Warren A Zhang Q Gong J Miao M Sun P Xu D Huang J Yi ZSong W 2016 The all-data-based evolutionary hypothesis of ciliatedprotists with a revised classification of the phylum Ciliophora(Eukaryota Alveolata) Sci Rep 624874

Garg J Lambert JP Karsou A Marquez S Nabeel-Shah S Bertucci VRetnasothie DV Radovani E Pawson T Gingras AC et al 2013

Conserved Asf1-importinb physical interaction in growth and sexualdevelopment in the ciliate Tetrahymena thermophila J Proteomics94311ndash326

Gerhold C-B Hauer MH Gasser SM 2015 INO80-C and SWR-C guard-ians of the Genome J Mol Biol 427(3) 637ndash651

Gerhold CB Gasser SM 2014 INO80 and SWR complexes relatingstructure to function in chromatin remodeling Trends Cell Biol24(11) 619ndash631

Goldberg AD Banaszynski LA Noh K-M Lewis PW Elsaesser SJ Stadler SDewell S Law M Guo X Li X et al 2010 Distinct factors controlhistone variant H33 localization at specific genomic regions Cell140(5) 678ndash691

Gonzalez-Romero R Eirın-Lopez JM Ausio J 2015 Evolution of highmobility group nucleosome-binding proteins and its implicationsfor vertebrate chromatin specialization Mol Biol Evol 32(1)121ndash131

Grisendi S Bernardi R Rossi M Cheng K Khandker L Manova KPandolfi PP 2005 Role of nucleophosmin in embryonic develop-ment and tumorigenesis Nature 437(7055) 147ndash153

Grover P Asa JS Campos EI 2018 H3ndashH4 Histone Chaperone PathwaysAnnu Rev Genet 52109ndash130

Hammond CM Stroslashmme CB Huang H Patel DJ Groth A 2017 Histonechaperone networks shaping chromatin function Nat Rev Mol CellBiol 18(3) 141ndash158

Hartzog GA Wada T Handa H Winston F 1998 Evidence that Spt4Spt5 and Spt6 control transcription elongation by RNA polymeraseII in Saccharomyces cerevisiae Genes Dev 12(3) 357ndash369

Hassa PO Hottiger MO 2008 The diverse biological roles of mammalianPARPS a small but powerful family of poly-ADP-ribose polymerasesFront Biosci 133046ndash3082

Hoek M Stillman B 2003 Chromatin assembly factor 1 is essential andcouples chromatin assembly to DNA replication in vivo Proc NatlAcad Sci U S A 100(21) 12183ndash12188

Hsieh F-K Kulaeva OI Patel SS Dyer PN Luger K Reinberg D StuditskyVM 2013 Histone chaperone FACT action during transcriptionthrough chromatin by RNA polymerase II Proc Natl Acad Sci U SA 110(19) 7654ndash7659

Jeronimo C Watanabe S Kaplan CD Peterson CL Robert F 2015 Thehistone chaperones FACT and Spt6 restrict H2AZ from intrageniclocations Mol Cell 58(6) 1113ndash1123

Jin C Zang C Wei G Cui K Peng W Zhao K Felsenfeld G 2009 H33H2AZ double variant-containing nucleosomes mark ldquonucleosome-free regionsrdquo of active promoters and other regulatory regions NatGenet 41(8) 941ndash945

Jullien J Astrand C Szenker E Garrett N Almouzni G Gurdon JB 2012HIRA dependent H33 deposition is required for transcriptionalreprogramming following nuclear transfer to Xenopus oocytesEpigenetics Chromatin 5(1) 17

Kaplan CD Laprade L Winston F 2003 Transcription elongation factorsrepress transcription initiation from cryptic sites Science 301(5636)1096ndash1099

Karrer KM 2012 Nuclear dualism Methods Cell Biol 10929ndash52Keck KM Pemberton LF 2012 Histone chaperones link histone nuclear

import and chromatin assembly Biochim Biophys Acta 1819(3ndash4)277ndash289

Kim DI Jensen SC Noble KA Kc B Roux KH Motamedchaboki K RouxKJ 2016 An improved smaller biotin ligase for BioID proximity la-beling Mol Biol Cell 27(8) 1188ndash1196

Ko HL Ren EC 2012 Functional aspects of PARP1 in DNA repair andtranscription Biomolecules 2(4) 524ndash548

Kobor MS Venkatasubrahmanyam S Meneghini MD Gin JW JenningsJL Link AJ Madhani HD Rine J 2004 A protein complex containingthe conserved Swi2Snf2-related ATPase Swr1p deposits histonevariant H2AZ into euchromatin PLoS Biol 2(5) E131

Krogan NJ Keogh M-C Datta N Sawa C Ryan OW Ding H Haw RAPootoolal J Tong A Canadien V et al 2003 A Snf2 family ATPasecomplex required for recruitment of the histone H2A variant Htz1Mol Cell 12(6) 1565ndash1576


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Kumar S Stecher G Suleski M Hedges SB 2017 TimeTree a resource fortimelines timetrees and divergence times Mol Biol Evol 34(7)1812ndash1819

Kumar S Stecher G Tamura K 2016 MEGA7 Molecular EvolutionaryGenetics Analysis version 70 for bigger datasets Mol Biol Evol 33(7)1870ndash1874

Kurat CF Yeeles JTP Patel H Early A Diffley JFX 2017 Chromatincontrols DNA replication origin selection lagging-strand synthesisand replication fork rates Mol Cell 65(1) 117ndash130

Langelier M-F Planck JL Roy S Pascal JM 2012 Structural basis for DNAdamage-dependent poly(ADP-ribosyl)ation by human PARP-1Science 336(6082) 728ndash732

Latreille D Bluy L Benkirane M Kiernan RE 2014 Identification of his-tone 3 variant 2 interacting factors Nucleic Acids Res 42(6)3542ndash3550

Letunic I Bork P 2018 20 years of the SMART protein domain annota-tion resource Nucleic Acids Res 46(D1) D493ndashD496

Levy-Wilson B 1983 Glycosylation ADP-ribosylation and methylationof Tetrahymena histones Biochemistry 22(2) 484ndash489

Liu X Li B Gorovsky MA 1996 Essential and nonessential histoneH2A variants in Tetrahymena thermophila Mol Cell Biol164305ndash4311

Lotan T Chalifa-Caspi V Ziv T Brekhman V Gordon MM Admon ALubzens E 2014 Evolutionary conservation of the mature oocyteproteome EuPA Open Proteomics 327ndash36

Luger K Meuroader AW Richmond RK Sargent DF Richmond TJ 1997Crystal structure of the nucleosome core particle at 28 A resolutionNature 389(6648) 251ndash260

Malone CD Falkowska KA Li AY Galanti SE Kanuru RC LaMont EGMazzarella KC Micev AJ Osman MM Piotrowski NK et al 2008Nucleus-specific importin alpha proteins and nucleoporins regulateprotein import and nuclear division in the binucleate Tetrahymenathermophila Eukaryot Cell 7(9) 1487ndash1499

Martindale DW Allis CD Bruns PJ 1982 Conjugation in Tetrahymenathermophila A temporal analysis of cytological stages Exp Cell Res140(1) 227ndash236

Martindale DW Allis CD Bruns PJ 1985 RNA and protein synthesisduring meiotic prophase in Tetrahymena thermophila J Protozool32(4) 644ndash649

Mason PB Struhl K 2003 The FACT complex travels with elongatingRNA polymerase II and is important for the fidelity of transcriptionalinitiation in vivo Mol Cell Biol 23(22) 8323ndash8333

Masutani M Nozaki T Wakabayashi K Sugimura T 1995 Role ofpoly(ADP-ribose) polymerase in cell-cycle checkpoint mechanismsfollowing gamma-irradiation Biochimie 77(6) 462ndash465

Melikishvili M Chariker JH Rouchka EC Fondufe-Mittendorf YN 2017Transcriptome-wide identification of the RNA-binding landscape ofthe chromatin-associated protein PARP1 reveals functions in RNAbiogenesis Cell Discov 317043

Mendiratta S Gatto A Almouzni G 2018 Histone supply multitieredregulation ensures chromatin dynamics throughout the cell cycleJ Cell Biol 218(1)39ndash54

Miao W Xiong J Bowen J Wang W Liu Y Braguinets O Grigull JPearlman RE Orias E Gorovsky MA 2009 Microarray analyses ofgene expression during the Tetrahymena thermophila life cyclePLoS One 4(2)e4429

Mochizuki K Gorovsky MA 2004 Small RNAs in genome rearrange-ment in Tetrahymena Curr Opin Genet Dev 14(2) 181ndash187

Mosammaparast N Ewart CS Pemberton LF 2002 A role for nucleo-some assembly protein 1 in the nuclear transport of histones H2Aand H2B EMBO J 21(23) 6527ndash6538

Muthurajan UM Hepler MRD Hieb AR Clark NJ Kramer M Yao TLuger K 2014 Automodification switches PARP-1 function fromchromatin architectural protein to histone chaperone Proc NatlAcad Sci U S A 111(35) 12752ndash12757

Nabeel-Shah S Ashraf K Pearlman RE Fillingham J 2014 Molecularevolution of NASP and conserved histone H3H4 transport pathwayBMC Evol Biol 14139

Namboodiri VMH Dutta S Akey IV Head JF Akey CW 2003 The crystalstructure of Drosophila NLP-core provides insight into pentamerformation and histone binding Structure 11(2) 175ndash186

Okuwaki M Matsumoto K Tsujimoto M Nagata K 2001 Function ofnucleophosminB23 a nucleolar acidic protein as a histone chap-erone FEBS Lett 506(3) 272ndash276

Orias E Cervantes MD Hamilton EP 2011 Tetrahymena thermophila aunicellular eukaryote with separate germline and somatic genomesRes Microbiol 162(6) 578ndash586

Papamichos-Chronakis M Watanabe S Rando OJ Peterson CL 2011Global regulation of H2AZ localization by the INO80 chromatin-remodeling enzyme is essential for genome integrity Cell 144(2)200ndash213

Pfister JA DrsquoMello SR 2016 Regulation of neuronal survival by nucleo-phosmin 1 (NPM1) is dependent on its expression level subcellularlocalization and oligomerization status J Biol Chem 291(39)20787ndash20797

Ray-Gallet D Woolfe A Vassias I Pellentz C Lacoste N Puri A SchultzDC Pchelintsev NA Adams PD Jansen LET et al 2011 Dynamics ofhistone H3 deposition in vivo reveal a nucleosome gap-filling mech-anism for H33 to maintain chromatin integrity Mol Cell 44(6)928ndash941

Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM 1998 DNAdouble-stranded breaks induce histone H2AX phosphorylation onserine 139 J Biol Chem 273(10) 5858ndash5868

Saettone A Garg J Lambert J-P Nabeel-Shah S Ponce M Burtch AThuppu Mudalige C Gingras A-C Pearlman RE Fillingham J 2018The bromodomain-containing protein Ibd1 links multiplechromatin-related protein complexes to highly expressed genes inTetrahymena thermophila Epigenetics Chromatin 11(1) 10

Song X Gjoneska E Ren Q Taverna SD Allis CD Gorovsky MA 2007Phosphorylation of the SQ H2AX motif is required for proper mei-osis and mitosis in Tetrahymena thermophila Mol Cell Biol 27(7)2648ndash2660

Stargell LA Bowen J Dadd CA Dedon PC Davis M Cook RG Allis CDGorovsky MA 1993 Temporal and spatial association of histoneH2A variant hv1 with transcriptionally competent chromatin duringnuclear development in Tetrahymena thermophila Genes Dev7(12B) 2641ndash2651

Straube K Blackwell JS Pemberton LF 2010 Nap1 and Chz1 have sep-arate Htz1 nuclear import and assembly functions Traffic 11(2)185ndash197

Studamire B Quach T Alani E 1998 Saccharomyces cerevisiae Msh2pand Msh6p ATPase activities are both required during mismatchrepair Mol Cell Biol 18(12) 7590ndash7601

Stuwe T Hothorn M Lejeune E Rybin V Bortfeld M Scheffzek KLadurner AG 2008 The FACT Spt16 ldquopeptidaserdquo domain is a histoneH3-H4 binding module Proc Natl Acad Sci U S A 105(26)8884ndash8889

Swaminathan V Kishore AH Febitha KK Kundu TK 2005 Human his-tone chaperone nucleophosmin enhances acetylation-dependentchromatin transcription Mol Cell Biol 25(17) 7534ndash7545

Tagami H Ray-Gallet D Almouzni G Nakatani Y 2004 Histone H31 andH33 complexes mediate nucleosome assembly pathways depen-dent or independent of DNA synthesis Cell 116(1) 51ndash61

Talbert PB Ahmad K Almouzni G Ausio J Berger F Bhalla PL BonnerWM Cande W Chadwick BP Chan SWL et al 2012 A unifiedphylogeny-based nomenclature for histone variants EpigeneticsChromatin 5(1) 7

Teo G Liu G Zhang J Nesvizhskii AI Gingras A-C Choi H 2014SAINTexpress improvements and additional features inSignificance Analysis of INTeractome software J Proteomics10037ndash43

Venkatesh S Workman JL 2015 Histone exchange chromatin structureand the regulation of transcription Nat Rev Mol Cell Biol 16(3)178ndash189

Wang Y Chen X Sheng Y Liu Y Gao S 2017 N6-adenine DNA meth-ylation is associated with the linker DNA of H2AZ-containing well-


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positioned nucleosomes in Pol II-transcribed genes in TetrahymenaNucleic Acids Res 45(20) 11594ndash11606

Wang Z Cui B Gorovsky MA 2009 Histone H2B ubiquitylation is notrequired for histone H3 methylation at lysine 4 in Tetrahymena J BiolChem 284(50) 34870ndash34879

Xiong J Lu X Zhou Z Chang Y Yuan D Tian M Zhou Z Wang L Fu COrias E et al 2012 Transcriptome analysis of the model protozoanTetrahymena thermophila using Deep RNA sequencing PLoS One7(2) e30630

Xu Q Wang R Ghanam AR Yan G Miao W Song X 2016 The key roleof CYC2 during meiosis in Tetrahymena thermophila Protein Cell7(4) 236ndash249

Yan G-X Dang H Tian M Zhang J Shodhan A Ning Y-Z Xiong J MiaoW 2016 Cyc17 a meiosis-specific cyclin is essential for anaphaseinitiation and chromosome segregation in Tetrahymena thermo-phila Cell Cycle 15(14) 1855ndash1864

Yang J Yan R Roy A Xu D Poisson J Zhang Y 2015 The I-TASSER Suiteprotein structure and function prediction Nat Methods 12(1) 7ndash8

Yang J Zhang X Feng J Leng H Li S Xiao J Liu S Xu Z Xu J Li D et al2016 The histone chaperone FACT contributes to DNA replication-coupled nucleosome assembly Cell Rep 14(5) 1128ndash1141

Yao M-C Fuller P Xi X 2003 Programmed DNA deletion as anRNA-guided system of genome defense Science 300(5625)1581ndash1584

Yao M-CC Choi J Yokoyama S Austerberry CF Yao C-HH 1984 DNAelimination in Tetrahymena a developmental process involving ex-tensive breakage and rejoining of DNA at defined sites Cell 36(2)433ndash440

Yao MC Yao CH Monks B 1990 The controlling sequence for site-specific chromosome breakage in Tetrahymena Cell 63(4) 763ndash772

Zhang Y Ku WL Liu S Cui K Jin W Tang Q Lu W Ni B Zhao K 2017Genome-wide identification of histone H2A and histone variantH2AZ-interacting proteins by bPPI-seq Cell Res 27(10) 1258ndash1274

Zhao X Ji J Yu L-R Veenstra T Wang XW 2015 Cell cycle-dependentphosphorylation of nucleophosmin and its potential regulation bypeptidyl-prolyl cistrans isomerase J Mol Biochem 495ndash103

Zunder RM Antczak AJ Berger JM Rine J 2012 Two surfaces on thehistone chaperone Rtt106 mediate histone binding replication andsilencing Proc Natl Acad Sci U S A 109(3) E144ndashE153


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histones H3 and H2A respectively have been described tohave nonrandom distribution along the chromatin For ex-ample H33 is enriched in the euchromatic regions associatedwith transcriptionally active genes (Goldberg et al 2010Ray-Gallet et al 2011) Genome-wide studies have indicatedthe enrichment of H2AZH33 double variants within regionsof high chromatin accessibility such as active promotersenhancers and insulator regions (Jin et al 2009) highlightingtheir role in gene expression regulation

Chromatin assembly is a fundamental process that mayaffect a broad range of gene regulatory processes such as DNArepair DNA replication and progression through the cell cy-cle (Mendiratta et al 2018) Protein factors known as histonechaperones are thought to have key roles in regulation ofchromatin assembly (Grover et al 2018) For examplechromatin assembly factor-1 and histone-regulator-A havebeen shown to mediate RD and RI chromatin assembly pro-cesses to deposit either H3ndashH4 or H33ndashH4 (Hoek andStillman 2003 Tagami et al 2004 Jullien et al 2012) respec-tively whereas the SWR-complex specifically targets H2AZndashH2B onto chromatin (Gerhold et al 2015) Histonechaperones have specific preferences for binding to eitherH3ndashH4 or H2AndashH2B (Keck and Pemberton 2012 Groveret al 2018) For example nucleosome assembly protein 1(Nap1) and nucleoplasmin 1 (Npm1) are both H2AH2B-specific chaperones (Straube et al 2010 Hammond et al2017) whereas antisilencing factor 1 (Asf1) is an H3H4-specific chaperone (English et al 2006 Mendiratta et al2018) It is currently unclear how chaperones target a certainhistone variant to a specific genomic region and how mech-anistically this task is achieved Several previous studies haveutilized functional proteomics approaches to identify andexamine the role of histone-binding proteins (Tagami et al2004 Latreille et al 2014 Hammond et al 2017) Howeverconsidering the complexity of chromatin assembly and geneexpression regulatory layers it is conceivable that many yet tobe identified chaperones might have roles in these processesComparative proteomics is a powerful tool that has beenwidely employed to study the evolution and functional con-servation of proteins (Boekhorst et al 2008 Lotan et al 2014)However the extent to which the role of previously identifiedhistone chaperones is conserved across the eukaryotic speciesremains unexplored (Grover et al 2018)

The unicellular ciliate protozoan Tetrahymena thermo-phila provides an excellent experimental system to studychromatin dynamics and identify new factors involved inthese processes The T thermophila genome is amenable totractable alterations enabling the endogenous tagging ofgenes of interest Ciliates are considered evolutionarily diver-gent organisms (Orias et al 2011 Gao et al 2016) and aretherefore well-suited to examine the functional conservationof known histone chaperones The T thermophila single cellfeatures a physical separation of two structurally and func-tionally distinct chromatin states in the form of a germ-linediploid micronucleus (MIC) and a polyploid somatic macro-nucleus (MAC) Functionally the MAC regulates gene expres-sion whereas the MIC ensures stable genetic inheritance(Martindale et al 1982) The two nuclei originate from the

same zygotic nucleus during sexual development (conjuga-tion) of the cell and subsequently embark on unique devel-opmental pathways leading toward distinct chromatinorganization within each nucleus (Martindale et al 1982)The alterations in the chromatin states including DNArearrangements and removal of internally eliminatedsequences during T thermophila development (Yao et al1984 1990 2003 Mochizuki and Gorovsky 2004) share sim-ilarities with epigenetic changes that occur to mammalianchromatin during development

The T thermophila genome encodes two major histoneH2A genes (HTA1 and HTA2) which at the protein level arenearly identical with only three amino acid differences in thecentral core region (Liu et al 1996) Furthermore neitherHTA1 nor HTA2 alone is essential for T thermophila vegeta-tive growth suggesting that the function of the encodedproteins is redundant (Liu et al 1996) However the C-terminiof the two proteins differ significantly from each other asH2A1 (encoded by HTA1) has an additional five residues(Liu et al 1996) These additional five residues include anSQ motif which is conserved across species (as in mammalianH2AX) and provides a target site for phosphorylation by aspecific protein kinase family (Song et al 2007) The SQ motifphosphorylation has been shown to function in double-strand break repair during mitosis meiosis and amitosis inT thermophila (Song et al 2007) Thus T thermophila H2A1can be considered an H2Ax ortholog although it differs frommammals where the H2AX histone variant is a quantitativelyminor component (Rogakou et al 1998) Tetrahymena ther-mophila H2B1 and H2B2 encoded by HTB1 and HTB2 re-spectively are nonallelic variants of H2B and only differ atthree positions Similar to H2A T thermophila cells lackingeither HTB1 or HTB2 alone are viable and do not exhibit anygrowth defects indicating the functional redundancy of H2Bs(Wang et al 2009)

The T thermophila H2A variant Hv1 (H2AZ and Htz1 inhumans and yeast respectively) has been found to be essen-tial for growth (Liu et al 1996) Hv1 localizes to the transcrip-tionally active MAC during vegetative growth and is found inthe MIC only during early conjugation events (Stargell et al1993) prior to the stage when MIC becomes transcriptionallyactive (Martindale et al 1985) Thus the localization patternsof Hv1 suggest a role in transcription regulation The mech-anistic details of how Hv1 is targeted to the MAC (and MICduring early conjugation) remain elusive

In this study we employed a functional proteomics work-flow to examine the histone-interactome for the first time inT thermophila Affinity purifications of T thermophila H2A1(HTA1) H2B1 (HTB1) and Hv1 followed by mass spectrom-etry analysis (AP-MS) revealed both new histone-interactingfactors as well as a set of chaperones that have been previ-ously identified only in Opisthokonts indicating the evolu-tionarily conserved histone metabolism regulatory networksSpecifically we identified T thermophila FACT- SWR- andINO80-complexes suggesting an ancient origin for these pro-teins We carried out detailed molecular evolutionary analysesof several histone-interacting proteins which further rein-forced the idea that dedicated chaperones arose very early


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Results









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Homo sapiens

Bos taurus

Mus musculus

Rattus norvegicus

Felis catus

Gorilla gorilla

Danio rerio

Takifugu rubripes


Vertebrates

Apis mellifera

Tribolium castaneum




Ciona intestinalis





Arabidopsis lyrata

Vitis vinifera


Plantae

Cryptococcus gattii




Candida glabrata



Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Fungi









Trypanosoma cruzi


100

100

100

100

100

100

100

100

61100

100

10067

100

97100

100

100

100

9960

100

100

100

98

100

67

95

100

100

100

9893

78

60

85

Vertebrates

Fungi

Ciliates

Apicomplexa

Plantae

Arthropoda

Amoebozoa

Excavata




98








100

100

1100

Trypanosoma cruzzi


11000











FFuuunnnggii

11000

10000

661110000

1000

1000

1000

55



VViVitis vvinifera



11000667




11000






101 0

998888

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BBos ttaauuruuss



FFellis ccaatuuss


DDaaniioo rrerrioo



VVertebraatees

1100

999600

11000

110000

10000



1100Tunicata

85

Veerrtebrates

Mus musculus

Felis catus

Rattus norvegicus

Gorilla gorilla

Homo sapiens

Bos taurus

Danio rerio

Danio rerio


Takifugu rubripes

Ciona intestinalis

Ciona savignyi

Apis mellifera

Tribolium castaneum

Drosophila virilis









Vitis vinifera


Arabidopsis lyrata

Cryptococcus gattii






Candida glabrata

Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Toxoplasma gondii



Plasmodium vivax







Trypanosoma cruzi

100

100

100

100

96

100

100

100

100

100

100

100

100

100

100

86

100

96

100

99

78

74

98

79

69

100

98

99

74

100

99

CCCiiliattees





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1100

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1000

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11000

1000

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11000

1000

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AApicompplexa




Plasmoddiuum vivax


100

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1000



11000

85

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Results









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Homo sapiens

Bos taurus

Mus musculus

Rattus norvegicus

Felis catus

Gorilla gorilla

Danio rerio

Takifugu rubripes


Vertebrates

Apis mellifera

Tribolium castaneum




Ciona intestinalis





Arabidopsis lyrata

Vitis vinifera


Plantae

Cryptococcus gattii




Candida glabrata



Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Fungi









Trypanosoma cruzi


100

100

100

100

100

100

100

100

61100

100

10067

100

97100

100

100

100

9960

100

100

100

98

100

67

95

100

100

100

9893

78

60

85

Vertebrates

Fungi

Ciliates

Apicomplexa

Plantae

Arthropoda

Amoebozoa

Excavata




98








100

100

1100

Trypanosoma cruzzi


11000











FFuuunnnggii

11000

10000

661110000

1000

1000

1000

55



VViVitis vvinifera



11000667




11000






101 0

998888

10000


BBos ttaauuruuss



FFellis ccaatuuss


DDaaniioo rrerrioo



VVertebraatees

1100

999600

11000

110000

10000



1100Tunicata

85

Veerrtebrates

Mus musculus

Felis catus

Rattus norvegicus

Gorilla gorilla

Homo sapiens

Bos taurus

Danio rerio

Danio rerio


Takifugu rubripes

Ciona intestinalis

Ciona savignyi

Apis mellifera

Tribolium castaneum

Drosophila virilis









Vitis vinifera


Arabidopsis lyrata

Cryptococcus gattii






Candida glabrata

Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Toxoplasma gondii



Plasmodium vivax







Trypanosoma cruzi

100

100

100

100

96

100

100

100

100

100

100

100

100

100

100

86

100

96

100

99

78

74

98

79

69

100

98

99

74

100

99

CCCiiliattees





996110000

9966

0505

Nematoda



1000TunnicataTT

AArthropoda






1100

8866

11100





1000

10000

NNematttodda

FFFuuunngggi











11000

1000

1100

11000

1000

999

AApicompplexa




Plasmoddiuum vivax


100

100

100


Tryrypanosoma cruzi

PPlaaanntttaeee

Viiitis vininiffera



1000



11000

85

A

B



1042

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100

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Homo sapiens

Bos taurus

Mus musculus

Rattus norvegicus

Felis catus

Gorilla gorilla

Danio rerio

Takifugu rubripes


Vertebrates

Apis mellifera

Tribolium castaneum




Ciona intestinalis





Arabidopsis lyrata

Vitis vinifera


Plantae

Cryptococcus gattii




Candida glabrata



Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Fungi









Trypanosoma cruzi


100

100

100

100

100

100

100

100

61100

100

10067

100

97100

100

100

100

9960

100

100

100

98

100

67

95

100

100

100

9893

78

60

85

Vertebrates

Fungi

Ciliates

Apicomplexa

Plantae

Arthropoda

Amoebozoa

Excavata




98








100

100

1100

Trypanosoma cruzzi


11000











FFuuunnnggii

11000

10000

661110000

1000

1000

1000

55



VViVitis vvinifera



11000667




11000






101 0

998888

10000


BBos ttaauuruuss



FFellis ccaatuuss


DDaaniioo rrerrioo



VVertebraatees

1100

999600

11000

110000

10000



1100Tunicata

85

Veerrtebrates

Mus musculus

Felis catus

Rattus norvegicus

Gorilla gorilla

Homo sapiens

Bos taurus

Danio rerio

Danio rerio


Takifugu rubripes

Ciona intestinalis

Ciona savignyi

Apis mellifera

Tribolium castaneum

Drosophila virilis









Vitis vinifera


Arabidopsis lyrata

Cryptococcus gattii






Candida glabrata

Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Toxoplasma gondii



Plasmodium vivax







Trypanosoma cruzi

100

100

100

100

96

100

100

100

100

100

100

100

100

100

100

86

100

96

100

99

78

74

98

79

69

100

98

99

74

100

99

CCCiiliattees





996110000

9966

0505

Nematoda



1000TunnicataTT

AArthropoda






1100

8866

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1000

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NNematttodda

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11000

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Plasmoddiuum vivax


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Homo sapiens

Bos taurus

Mus musculus

Rattus norvegicus

Felis catus

Gorilla gorilla

Danio rerio

Takifugu rubripes


Vertebrates

Apis mellifera

Tribolium castaneum




Ciona intestinalis





Arabidopsis lyrata

Vitis vinifera


Plantae

Cryptococcus gattii




Candida glabrata



Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Fungi









Trypanosoma cruzi


100

100

100

100

100

100

100

100

61100

100

10067

100

97100

100

100

100

9960

100

100

100

98

100

67

95

100

100

100

9893

78

60

85

Vertebrates

Fungi

Ciliates

Apicomplexa

Plantae

Arthropoda

Amoebozoa

Excavata




98








100

100

1100

Trypanosoma cruzzi


11000











FFuuunnnggii

11000

10000

661110000

1000

1000

1000

55



VViVitis vvinifera



11000667




11000






101 0

998888

10000


BBos ttaauuruuss



FFellis ccaatuuss


DDaaniioo rrerrioo



VVertebraatees

1100

999600

11000

110000

10000



1100Tunicata

85

Veerrtebrates

Mus musculus

Felis catus

Rattus norvegicus

Gorilla gorilla

Homo sapiens

Bos taurus

Danio rerio

Danio rerio


Takifugu rubripes

Ciona intestinalis

Ciona savignyi

Apis mellifera

Tribolium castaneum

Drosophila virilis









Vitis vinifera


Arabidopsis lyrata

Cryptococcus gattii






Candida glabrata

Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Toxoplasma gondii



Plasmodium vivax







Trypanosoma cruzi

100

100

100

100

96

100

100

100

100

100

100

100

100

100

100

86

100

96

100

99

78

74

98

79

69

100

98

99

74

100

99

CCCiiliattees





996110000

9966

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Nematoda



1000TunnicataTT

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1100

8866

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1000

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Plasmoddiuum vivax


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Homo sapiens

Bos taurus

Mus musculus

Rattus norvegicus

Felis catus

Gorilla gorilla

Danio rerio

Takifugu rubripes


Vertebrates

Apis mellifera

Tribolium castaneum




Ciona intestinalis





Arabidopsis lyrata

Vitis vinifera


Plantae

Cryptococcus gattii




Candida glabrata



Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Fungi









Trypanosoma cruzi


100

100

100

100

100

100

100

100

61100

100

10067

100

97100

100

100

100

9960

100

100

100

98

100

67

95

100

100

100

9893

78

60

85

Vertebrates

Fungi

Ciliates

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Plantae

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Excavata




98








100

100

1100

Trypanosoma cruzzi


11000











FFuuunnnggii

11000

10000

661110000

1000

1000

1000

55



VViVitis vvinifera



11000667




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101 0

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BBos ttaauuruuss



FFellis ccaatuuss


DDaaniioo rrerrioo



VVertebraatees

1100

999600

11000

110000

10000



1100Tunicata

85

Veerrtebrates

Mus musculus

Felis catus

Rattus norvegicus

Gorilla gorilla

Homo sapiens

Bos taurus

Danio rerio

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Ciona intestinalis

Ciona savignyi

Apis mellifera

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Arabidopsis lyrata

Cryptococcus gattii






Candida glabrata

Neurospora crassa

Aspergillus oryzae

Aspergillus flavus

Toxoplasma gondii



Plasmodium vivax







Trypanosoma cruzi

100

100

100

100

96

100

100

100

100

100

100

100

100

100

100

86

100

96

100

99

78

74

98

79

69

100

98

99

74

100

99

CCCiiliattees





996110000

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1000TunnicataTT

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1100

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100

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100


Tryrypanosoma cruzi

PPlaaanntttaeee

Viiitis vininiffera



1000



11000

85

A

B



1042

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99

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100

76

98

10066

59

05




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1045

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1053

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1054

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1055

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99

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100

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10066

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Proteomic Analysis of Histones H2A/H2B and Variant Hv1 in ...

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