Chromatin Proteomics Reveals Variable Histone Modifications during

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Chromatin proteomics reveals variable histone modifications during the life cycle of Trypanosoma cruzi Teresa Cristina Leandro de Jesus, Vinicius Santana Nunes, Mariana de Camargo Lopes, Daiana Evelin Martil, Leo Kei Iwai, Nilmar Silvio Moretti, Fabricio Castro Machado, Mariana L de Lima-Stein, Otavio Henrique Thiemann, Maria Carolina Elias, Christian J. Janzen, Sergio Schenkman, and Julia PC da Cunha J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00208 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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Journal of Proteome Research

Chromatin proteomics reveals variable histone modifications during the life cycle of Trypanosoma cruzi

Teresa Cristina Leandro de Jesus1, 2#, Vinícius Santana Nunes3#, Mariana de Camargo Lopes1, Daiana Evelin Martil2, Leo Kei Iwai1, Nilmar Silvio Moretti3, Fabrício Castro Machado3, Mariana L. de Lima-Stein3, Otavio Henrique Thiemann2, Maria Carolina Elias1, Christian Janzen4, Sergio Schenkman3, Julia Pinheiro Chagas da Cunha1*. # These authors contributed equally to this work.

1

Laboratório Especial de Ciclo Celular, Center of Toxins, Immune Response and Cell

Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil. 2

Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade

de São Paulo - USP, São Carlos, SP, 13563-120, Brazil 3

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de

São Paulo, São Paulo, SP, Brazil. 4

Department of Cell and Developmental Biology, Theodor-Boveri-Institute at the

Biocenter, University of Würzburg, Germany. *Corresponding author: Julia P. Chagas da Cunha, email: [email protected], phone: +(55) 11 26279731. Keywords: Chromatin, histone, Trypanosoma cruzi, acetylation, methylation, cell cycle, life cycle. Abbreviations:

PTM,

post-translational

modifications;

ac,

acetylation;

me1,

monomethylation; me2, dimethylation; me3, trimethylation; p, phosphorylation. Running title: Histone modifications during T. cruzi life cycle.

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Abstract Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair and chromosome condensation. In early-branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, 7 monomethylations, 8 dimethylations, 7 trimethylations and 4 phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than non-replicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell cycle regulation in an early divergent organism.


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Introduction Nucleosomes are the basic unit of chromatin and are composed of two copies of each core histones (H2A, H2B, H3 and H4) and approximately 146 bp of DNA. Histone H1 links one nucleosome to each other and is involved in chromatin compaction. Histones are basic small proteins and are some of the most evolutionarily conserved proteins. Their N-termini protrude from nucleosome structure and are targets for posttranslational modifications (PTM) such as acetylation, (mono, di and tri-) methylation, phosphorylation, resulting in modifications on chromatin structure that impact processes like DNA replication, DNA damage repair as well as gene expression 1. Histone variants also play a role in chromatin structure and are associated with DNA-regulatory processes. Their amino acid sequences differ from their canonical counterparts and they are expressed in lower amounts and in specific genomic regions or situations 2. Together, the presence of histone variants and PTMs are important for chromatin structure and gene regulation, and are transmitted during cell division, helping to maintain the differentiation status through generations in what is called epigenetic heritage. Trypanosomes form a group of protozoa that causes several human and animal diseases and diverged early from other eukaryotes during evolution. The histones of trypanosomes are less conserved with respect to other eukaryotes 3. They have peculiar mechanisms of gene expression control. All genes are transcribed in long polycistronic units, in some cases in opposite directions

4, 5

. The resulting pre-mRNAs are then

processed by trans-splicing and polyadenylation generating mature mRNAs that are exported to the cytosol. The levels of resulting mRNA are then controlled mainly by post-transcriptional mechanisms 6. Specific histone PTMs and variants are particularly



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enriched in these divergent transcription initiation and termination sites

7-9

, which also

seem to contain DNA replication origins 10, 11. Compared to other organisms, fewer histone PTMs have been described so far in trypanosomes, most of them in Trypanosoma brucei, the agent of sleeping sickness. Mass spectrometry analysis and Edman degradation revealed that in T. brucei, histone H4 is ~80% acetylated at K4 and ~10% at K10 12. In addition, histone H4 was found to be acetylated (ac) at lysines K2, K5, and mono-, di-, or trimethylated (me1, me2, me3, respectively) at K17 and monomethylated at K2. Methylations of H4K18 were eventually detected

13

. Histone H2A is unusually acetylated at K115, K119, K120,

K122, K125, and K128. Histone H2B contains K4ac, K12ac, and K16ac. Histone H3 contains S2ac and K23ac and it is trimethylated at lysines K4 and K32. Mono-, di- and trimethylations are also detected in the K76 of histone H3, which appear to be involved in replication regulation (H3K76me1/2) and differentiation (H3K76me3) in this parasite 13-15

. Fewer modifications are described for Trypanosoma cruzi, the etiological agent of

Chagas disease. Mass spectrometry analysis revealed that the histone H4 N-terminal tail contains similar acetylated lysines as in T. brucei, with the predominance of K4Ac, which probably is involved in chromatin assembly 16. T. cruzi also contains low levels of K10Ac and K14Ac, proposed to be involved in chaperone mediated interactions with histones during DNA replication and transcription events

17, 18

. Histone H4 is also

methylated at K18 and dimethylated at R53 16 and H1 is phosphorylated at S12 mainly during cell cycle progression

19

. Trypanosomatids also possess genes that code for

histone variants of H2A, H2B, H4 and H3 but their modifications are not characterized, yet. How histones PTMs regulate transcription, replication and DNA repair in trypanosomes is unknown. One way to approach this question is to analyze how PTMs


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vary during the different life and cell cycle stages. For this, T. cruzi is an interesting model, as it alternates between a replicative (epimastigotes and amastigotes in the insect and mammalian host) and a non-replicative form (trypomastigote), which reduces transcription and becomes infective to the mammalian host

20

. Here, we used high-

resolution mass spectrometry to identify PTMs of T. cruzi histones and to compare their abundance in two different life forms, epimastigotes and trypomastigotes. We found several new modifications and observed that some of them were enriched in one of the life forms. H3K76 mono- and dimethylations were enriched in proliferative forms of T. cruzi, whereby; H3K76me1 and H3K76me2 were associated with mitosis and cytokinesis, respectively. Our data will be useful to further understand the functions of each modification in this early divergent organism. Experimental procedures Parasite cultures and differentiation T. cruzi (Y strain) epimastigotes were cultivated at 28°C in liver infusion tryptose (LIT) medium supplemented with 10% fetal bovine serum

21

. Metacyclic-

trypomastigotes were purified from epimastigote exponential cultures (1 x 107 cells per ml). For differentiation, the epimastigotes were collected by centrifugation (2000 g, 5 min) and resuspended in TAU medium (2 mM CaCl2, 2 mM MgCl2, 0.035% NaHCO3, 190 mM NaCl, 17 mM KCl, pH 6.5) to 2.5 x 108 cells per ml. After 1 h, the parasites were diluted in TAU containing 10 mM glucose, 2 mM L-aspartic, 50 mM L-glutamic, 10 mM L-proline to 5 x 106 cells per ml. After five days the equivalent of 5 x 108 parasites were centrifuged at 2000 g for 5 min and resuspended in 10 ml of 3 mM KH2PO4, 57.2 mM Na2HPO4, 45 mM NaCl, 55.5 mM glucose, pH 8.0 and loaded into a 10 ml DEAE-cellulose column (DE-52, Whatman) pre-equilibrated in the same buffer. Metacyclic trypomastigotes eluted in the flow-through fraction, while epimastigotes and 5 ACS Paragon Plus Environment


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partially differentiated parasites were retained in the column 22. Trypomastigotes forms (Y strain) were collected from the supernatant of infected monolayers of LLCMK2 cells (from 120 to 144 h after infection) in culture medium DMEM with 10% FBS at 37°C, 5% CO2 and intracellular amastigotes were obtained from the same cells 72 h after infection 23. Chromatin Extraction and histone enriched extracts Protocol 1. Chromatin was extracted using a protocol described in 24. Briefly, 5 x 108 epimastigotes forms of T. cruzi were pelleted and resuspended in buffer 1 (250 mM sucrose, 3 mM CaCl2, 1 mM EDTA, 10 mM Tris-HCl 7.4, 10 mM sodium butyrate, supplemented with phosphatase and protease inhibitors, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and cOmpleteTM EDTA free protease inhibitor cocktail – Roche), and lysed with a potter. Samples were centrifuged at 3000 g for 10 min at 4°C and the pellet was resuspended in buffer 2 (buffer 1 containing 0.5% saponin). Samples were centrifuged at 3400 g for 10 min at 4°C and the pellet was resuspended in buffer 3 (1% Triton X-100, 150 mM NaCl, 25 mM EDTA, 10 mM Tris-HCl pH 8.5, 10 mM sodium butyrate, supplemented with phosphatase and protease inhibitors,1 mM NaF, 1 mM Na3VO4, 1 mM PMSF and cOmpleteTM EDTA free protease inhibitor cocktail – Roche). Samples were centrifuged at 12,000 g for 20 min at 4°C and the pellet was washed three times with 10 mM Tris-HCl pH 8.5 and resuspended in this buffer with 250 U of benzonase (Sigma). The reaction was incubated for 30 min at 37°C under agitation (1400 rpm), then the samples were centrifuged at 21,000 g for 10 min at 4°C and the supernatant was kept for further analysis. Protocol 2. Chromatin was isolated using a protocol described previously with a few modifications 25. Pellets containing 5 x 108 epimastigotes or trypomastigotes were extracted with 10 mM Tris-HCl pH 7.4, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 6 ACS Paragon Plus Environment

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10 mM sodium butyrate, 0.1% Triton X-100, supplemented with phosphatase and protease inhibitors, 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and cOmplete EDTA free protease inhibitor cocktail – Roche for 10 min on ice under agitation. Samples were centrifuged at 3,300 g for 2 min at 4°C and the pellets were once again treated as above. The pellets were treated with DNAse (250 U/sample) and incubated for 30 min at 37°C, then centrifuged, as above. The supernatant was saved and the pellet was once again treated with DNAse. After centrifugation, the new supernatant was mixed with the previous one. Protein precipitation and Lys-C/Trypsin digestion For all samples, 150 µg of protein were precipitated with 20% TCA at 4°C overnight. Samples were then centrifuged at 18,500 g for 30 min at 4°C and the pellets were washed 3 times with cold acetone. The final pellets were dried at room temperature and resuspended in 10 mM Tris-HCl pH 8.5, 8 M urea, and mixed by vortex for 30 s. Protein extracts were reduced with 5 mM of DTT for 30 min at room temperature, alkylated with 14 mM of iodoacetamide in the dark for 30 min and digested with 0.5 µg of Lys-C (Promega) for 4 h at 37°C, under agitation (900 rpm). Sequentially, samples were digested with 0.75 µg of trypsin (Sigma) in the presence of 10 mM of Tris-HCl pH 8 and 2 mM CaCl2 overnight at 37°C under agitation (900 rpm). The reactions were stopped with 5% formic acid and vacuum dried. Peptide clean up and stage tip fractionation (SCX) After protein digestion the peptides were cleaned up for detergent removal by hydrophilic interaction chromatography-HILIC (The Nest Group, Inc.) according to the manufacturer’s instructions. Samples were once again dried and redissolved in 400 µl of 0.1% TFA and desalinated using the Sep-pak Light tC18 column (Waters). After desalination, samples were fractionated using strong cation exchange (SCX) offline 7 ACS Paragon Plus Environment


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chromatography as described in

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with some modifications. Three layers of a 3M

Empore Cation Exchange disks were stacked into a 200 µl micropipette tip and washed sequentially with methanol, 5% ammonium hydroxide/80% acetonitrile and 1% TFA (trifluoracetic acid). The samples were resuspended in 1% TFA and loaded into the tips. The membranes were washed 3 times with 0.3% TFA and the peptides were eluted with ammonium acetate gradient (20, 50, 75, 100, 200 and 500 mM) in 0.3% TFA /20% acetonitrile, followed by 5% and 15% of ammonium hydroxide in 80% acetonitrile. Samples were dried and analyzed by mass spectrometry as described below. Mass spectrometry and data analysis Peptides were resuspended in 0.1% formic acid and injected in an in-house made 5 cm reversed phase pre-column (inner diameter 100 µm, filled with a 10 µm C18 Jupiter resins -Phenomenex) coupled to a nano HPLC (NanoLC-1DPlus, Proxeon). The peptide fractionation was carried on an in-house 10 cm reversed phase capillary emitter column (inner diameter 75 µm, filled with 5 µm C18 Aqua resins-Phenomenex) with a gradient of 2-35 % of acetonitrile in 0.1% formic acid for 52 min followed by a gradient of 35-95% for 5 min at a flow rate of 300 nl/min. The eluted peptides were directly analyzed in LTQ-Orbitrap Velos (Thermo Scientific). The source voltage and the capillary temperature were set at 1.9 kV and 200 ◦C, respectively. The mass spectrometer was operated in a data-dependent acquisition mode to automatically switch between one Orbitrap full-scan and ten ion trap tandem mass spectra. The FT scans were acquired from m/z 200 to 2000 with mass resolution of 30,000. MS/MS spectra were acquired at normalized collision energy of 35%. Singly charged and chargeunassigned precursor ions were excluded. The dynamic exclusion parameters included: exclusion duration of 45 sec; exclusion list size of 500; repeat duration of 30s. The isolation width for precursor ion selection (in m/z) was 2. The raw data were processed 8 ACS Paragon Plus Environment

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in software environment MaxQuant 27 version 1.3.0.5 and Andromeda Search engine 28. Proteins were identified by searching against the complete database sequence of T. cruzi-Cl Brener (downloaded at TriTryp DB-4.2- 23311 sequences) and against a database of T. cruzi histones sequences (54 sequences) both together with a set of commonly observed contaminants. The histone database contained all histones sequences of H1, H2A, H2B, H2Bv, H3, H3v and H4 found in TritrypDB (http://tritrypdb.org/tritrypdb/) for T. cruzi Cl Brener (Esmeraldo-like and NonEsmeraldo-like). T. cruzi histones present slight differences in their sequence. In order to group the different isoforms of each histone we aligned them, using Clustal Omega tool (http://www.ebi.ac.uk/Tools/msa/clustalo/). Carbamidomethylation (C) was set as fixed modification while oxidation (M), acetylation (N-terminal and K), methylation (KR), dimethylation (KR), trimethylation (K) and phosphorylation (STY) as variable modifications; maximal number of modification per peptide of 5; maximal missed cleavages of 2; MS1 tolerance of 6 ppm; MS2 of 0.5 Da; maximum false peptide and protein discovery rates of 0.01. For matching between runs, the time window was 2 min. Data were sequentially analyzed by Perseus software

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. Protein matching to the

reverse database or identified only with modified peptides were filtered out. Relative protein quantitation was performed using the LFQ algorithm of MaxQuant

29

using

minimum ratio count of two. Protein quantitation were based on LFQ values of “razor and unique peptides” of unmodified peptides. For relative modified peptide quantitation, “intensity” values (average of three replicates) of the corresponding modified peptide (in epimastigote or trypomastigote sample) were divided by LFQ value of the corresponding total histone content (in epimastigote or trypomastigote sample). The raw data was also analyzed by PEAKS Studio 7.5

30

software using the

same fixed and variable modifications as for MaxQuant; maximal number of



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modification per peptide of 3, maximal missed cleavages of 3; MS1 tolerance of 10 ppm; MS2 of 0.5 Da. FDR for peptides -10 log p-value>=15 and for proteins >=20. De novo sequencing using PEAKS included ALC (Average Local Confidence) score higher or equal to 50% and FDR lower or equal to -10 log p-value =15.Relative quantitation using PEAKS Studio were based on the number of spectra for each PTM in epimastigote and trypomastigote normalized by corresponding number of unmodified peptides. De novo sequencing was manually performed for those spectra that were found to correspond to modified histone peptides.

Prediction of 3D structure of H3 The H3 sequence was submitted to the Robetta server for full-chain structure prediction using default settings

31, 32

. The homology models of H2A, H2AZ, H2B,

H2Bv, H3v and H4 were built by MODELLER (version 9.15)

33

, according to

templates, PDB ID code 2CV5, PDB ID code 4CAY, PDB ID code 2CV5, PDB ID code 2CV5, PDB ID code 3X1S and PDB ID code 3W99, respectively. Homology search (BLAST) against the Protein Data Bank was used to identify template structures. The alignment file between the target and the template was generated using the online program Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). The quality of the models were validated using PROCHECK software (version 3.6.2), part of the PDBSum program suite 34. The models were visualized using PyMOL software (version 1.7.4.5)35. Immunoblotting Total protein extract were prepared from 1.5 x 107 cells obtained by centrifugation at 2,000 g for 5 min at 4°C and washed once with PBS prior to be resuspended in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer 10 ACS Paragon Plus Environment

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and boiled at 95°C for 5 min. These extracts of parasites were submitted to electrophoresis on SDS-PAGE (15%) and transferred to nitrocellulose membranes. Membranes were stained with 0.3% Ponceau S in 10% acetic acid to assess the quality of bands and then treated with 3% nonfat milk in PBS for 120 min. The membranes were incubated 2 h with the primary antibodies in 3% nonfat milk in PBS. The anti-H3 rabbit polyclonal antiserum and both rabbit purified antibodies anti-H3K76me1, antiH3K76me2 and anti-H3K76me3 were prepared as described 11, 14. The anti-H3 was used at 1:20,000 dilution, and the purified antibodies were used at 1:2,000. After primary antibody incubation, the membranes were washed three times with PBS containing 0.1% Tween-20 (10 min each) followed by 1 h incubation with 680RD infrared dye labeled goat anti-rabbit IgG antibody (Li-Cor) at 1:20,000 dilution. Bounded antibodies were detected and quantified by using a LI-COR Odyssey imaging apparatus. Immunofluorescence Culture cell-derived and metacyclic trypomastigotes were incubated with 4% paraformaldehyde in PBS at room temperature for 5 min, centrifuged 2 min at 2,000 g, washed in PBS and permeabilized 5 min in PBS containing 0.1% Triton X100, washed once more in PBS and attached to glass slides pretreated with 0.01% poly-L-lysine for 15 min. For experiments with the intracellular amastigote, infected LLCMK2 cells were washed in PBS fixed and permeabilized as above. Epimastigotes were previously washed in PBS and then attached to glass slides pretreated with 0.01% poly-L-lysine for 15 min. The slides were washed in PBS and dipped in ice-cold 100% methanol for 10 seconds. In all cases, the preparations were incubated for 1 h with PBS containing 1.5% bovine serum albumin at room temperature with the primary antibodies diluted in the same buffer. Anti-H3 was used at 1:10,000, both the anti-H3K76me1, anti-H3K76me2 and anti-H3K76me3 were used at 1:1,000. The monoclonal antibodies 2F6 and 3F6 11 ACS Paragon Plus Environment


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antibodies (respectively, anti-flagellum

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36

) were used at

1:1,000 dilution. The slides were washed with PBS, incubated 1 h at room temperature with Alexa Fluor 594, or 488, goat anti-rabbit or anti-mouse IgG (Invitrogen) diluted 1:2,000, washed once more, and mounted in Prolong Gold Antifade Reagent (Invitrogen) in the presence of 10 µg of DAPI per ml. Images were acquired by using a Hamamatsu Orca R2 CCD camera coupled to an Olympus (BX-61) microscope equipped with a ×100 plan Apo-oil objective (NA 1.4). Acquisitions were at every 0.2 µm for each set of excitation/emission filters. Blind deconvolution was performed by employing the AutoQuant X2.2 software (Media Cybernetics).

Results Global histone PTM analysis of T. cruzi In order to identify PTMs in T. cruzi, chromatin extracts from epimastigotes and trypomastigotes were prepared in triplicates. These samples were digested, fractionated using SCX-Stage Tips and analyzed by high-resolution mass spectrometry. To identify the maximal number of PTMs, we processed and analyzed data using two different search engines (Andromeda-MaxQuant and PEAKS Studio softwares), as described in materials and methods section. All histones including their variants were identified. The two methods generated slightly different results, but on average, histone sequence coverage was 69%, 24.2 peptides per protein and 9.35 unique peptides per protein (Table S1 and S2, Supporting Information). From canonical histones, histone H1 had the lowest coverage (on average 29%), most probably due to the high number of lysine and arginine residues compared to other histone sequences. To identify histone PTMs in T. cruzi with maximal confidence, we compared the peptides found in chromatin-enriched extracts with a T. cruzi database, as well as with a T. cruzi histone-only database build-up in house. Histone PTMs were considered for 12 ACS Paragon Plus Environment

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further investigation only if they were identified exclusively in the latter database. All positive spectra were evaluated by de novo sequencing (Figure S1, Supporting Information) and thus considered as a false or a true PTM (see materials and methods for details). After filtering (including de novo sequencing analysis), MaxQuant (MQ) analysis retrieved 76 peptides including 16 modified peptides (Table S3, Supporting Information) while PEAKS Studio retrieved 233 peptides including 24 modified peptides (Table S4, Supporting Information). On average, the mass error was +/- 0.16 and 2.26 ppm for MQ and PEAKS Studio, respectively, which was sufficient to differentiate between modifications such as acetylation and trimethylation. Based on MS/MS counts of unmodified versus modified peptides, it was noticed that epimastigotes histones are more modified than trypomastigotes. About 42% and 22% of MS/MS counts were modified histone peptides from epimastigotes and trypomastigotes, respectively (Figure 1). These life forms have many important phenotypic differences, including the capability of DNA replication (epimastigotes) and infection of human cells (trypomastigotes). Besides that, epimastigotes have higher global transcription rates than trypomastigotes

20

. Taken together, a more complex

histone modification pattern is perfectly compatible with the intense RNA- and DNAmetabolism of epimastigotes. Histone PTM identification The histones of T. cruzi are encoded by different genes, which present slight differences in their sequences. To facilitate our analysis, we grouped the different isoforms of each histone and combined the sequences to one representative sequence of each group (Figure 2). If one PTM was found on a peptide that was shared among the groups, only the representative sequence is presented. We identified PTMs in all core 13 ACS Paragon Plus Environment


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histones and in the variants H2BV and H3V (Table I and Figure 2). In total, 44 PTMs were identified; including previously described PTMs such as acetylations at H4K10, H4K14, H1S1 and phosphorylation at H1S11 16, 19. Additionally, we detected specific PTMs in T. cruzi that were either not reported in T. brucei or not yet detected in other organisms. These newly PTMs were found in all histones, except histone H4, including H2AK127me1, H2AR129me1; H2AZK54ac, H2AZK58ac; H3K19ac, H3K23me1, H3K23me2, H3K23me3, H3T29p, H3K76ac, H3R80me2 (Table I). We also detected K4me3 and K11me1 in H2B of epimastigotes (Figure 2). It could be homologous to human H2BK5, which can be either acetylated or methylated, and to K12, which is acetylated, respectively

37

. Interestingly, H2BK12Ac is found in

differentially methylated regions of imprinted genes 38, 39. Novel acetylations, di- and tri-methylations were also detected in histone H1. It is important to highlight that previous analysis of full-length histone H1 by MS suggested multiple methylations and/or acetylations, although the exact position could not be assigned 19. We confirmed the presence of H4K10ac and H4K14ac, already characterized in T. cruzi and T. brucei. However, we did not detect H4K4ac as found in our previous study 16. This probably occurred because here we digested the proteins with Lys-C and trypsin while Glu-C digestions were employed earlier. Previously identified and unknown modifications were also observed in histone H3. K23 is acetylated, as observed in T. brucei 13, 40, but here, we also found that it can be mono-, di- and tri-methylated. These modifications are probably homologous to human H3K27 PTMs. T. cruzi H3K19 is acetylated and could correspond to human H3K23ac, which is methylated as well. Human H3K23 acetylation is recognized by the


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Tripartite motif containing 24 (TRIM24), which is a chromatin regulator that activates estrogen-dependent genes associated with tumor development and cellular proliferation 41

. H3K23 methylation is involved in blocking DNA damage in pericentric

heterochromatin during meiosis 42. We additionally found a fully modified (acetylation and methylations) lysine 76 of histone H3 (H3K76) similarly to H3K79 in mammals

43

. These modifications have

been already described in T. brucei as the target for the Dot1 methyltransferases14 . The C-terminal of H2A is highly acetylated in T. cruzi, as previously observed for T. brucei

12

. We detected acetylations at K116, K120, K121, K126, as well as

methylations at K127 and R129. Most of these PTMs in H2A are unique to trypanosomes and no function had been attributed to them. Noteworthy, although the histone sequence differs from their human counterparts, it seems that some T. cruzi PTMs are conserved, suggesting that these sites are localized at histones surface structure that are available to enzymatic activity. To check this possibility, we analyzed the tridimensional structure of histone H3. A molecular model of histone H3 was obtained as output from the ROBETTA server, refined and validated using PROCHECK in which 95.0% residue fall in the most favored region Ramachandran plot, thus validating the quality of the model. Figure 3A, shows the 3D structure model, highlighting the modified residues. H3K19, K23, K76 and R80, are located at surface of histone H3 whereas T29 is located inwardly. By structural superposition of T. cruzi histone H3 and the crystal structure of human nucleosome core particle (PDB ID code 2CV5), it can be proposed that K76 interacts with histone H4, suggesting that modifications at this site may interfere with nucleosome structure (Figure 3C). Interestingly, H3R80 from H3 monomer A could be able to interact with DNA. Remarkably, this residues side orientation differs



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considerably from humans, X. laevis, D. melanogaster and S. cerevisiae (Figure 3B). It would be fascinating, if a chemical modification (such as the demethylation found in this work), could adjust the structure to resemble those from other eukaryotes. The homology models of H2A, H2AZ, H2B, H2Bv, H3v and H4 constructed by MODELLER showed that all the identified modified residues are also located at protein surface (Figure S2, Supporting Information). Analyses of PTMs of the histone variants Here, we described for the first time, the presence of histone variants in T. cruzi, some of them with novel PTMs that may add another layer on the regulatory function of histone variants in trypanosomes. We detected histone H2AZ (TcCLB.511323.40) that shares 84.7% identity with T. brucei H2AZ. In T. brucei, this variant dimerizes with H2B variant and these dimers are absent from polycistronic transcription units but enriched at transcription start sites

9, 44

. This variant is acetylated at K54 and K58,

modifications not found in T. brucei. In chickens, H2A.Z acetylated at K4, K7 and K11 are localized at active genes and are important to generate an open chromatin state 45, 46. H3VK94 was found for the first time mono, di- and tri-methylated. Histone H3 variant is detected at T. brucei telomeres, but it is not required for viability or chromosome segregation, although it shares similarities to CenH3

47

. Despite its

localization, parasites lacking H3 variants still have silent VSG expression sites, suggesting that this variant does not play a role on VSG regulation. In addition, we found novel PTMs of H2BV such as K32ac and K34me1. Histone PTMs quantification and comparison between life cycle stages Besides the identification of new histone PTMs, we also quantified some of these modifications and compared their abundance in different T. cruzi life forms (Figure 4, Table II and III, Table S5, Supporting Information). As pointed out above, T. 16 ACS Paragon Plus Environment

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cruzi provides an interesting model to evaluate how global transcription rates and the absence of DNA replication might be reflected in different histone PTM pattern. Interestingly, some modifications were detected mainly in one life form, as shown in Table III. The majority of unique PTMs were found in trypomastigotes. This was probably due to the fact that chromatin extracts from trypomastigotes contain proportionally more histones than other proteins compared to epimastigotes (de Jesus et al, manuscript in preparation). Thus, it is possible that some PTMs that seem to be trypomastigote exclusive could be present in the same amounts in epimastigotes. Nevertheless, all quantitative data were analyzed comparing quantitative ratios of modified peptide versus total histones to compensate these differences. H1S11p was previously found mainly in trypomastigotes

19

and, not

surprisingly, this observation was confirmed. Moreover, we have previously detected very low amounts of H4K10ac and H4K14ac in epimastigotes 16. Our previous results also showed that H4K10ac and K14ac are increased in trypomastigotes 48, which could explain their increased detection in trypomastigotes. We found that the fully methylated state of H3K23 appeared predominantly in trypomastigotes. We estimated the proportion of K23 modification in epimastigotes and trypomastigotes based on MS/MS counts. Epimastigotes contain 60% of unmodified H3K23 in comparison to only 37% in trypomastigotes (Figure 5). Mono- and dimethylation of H3K23 are present in similar amounts (34% and 26%, respectively) in trypomastigotes, but tri-methylated H3K23 is found in lower amounts (2.6%). Similarly, acetylation of the C-terminus of H2A predominates in trypomastigotes (Table III). In contrast, non-modified, mono- and di-methylated H3K76 (Table II) is enriched in epimastigotes compared to trypomastigote forms. Interestingly, K76me1 is



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present in low amounts compared to K76me2 and K76me3 (Figure 4 and Figure 5) although it is more enriched in epimastigotes than trypomastigotes. H3K76me3 is present in both parasite forms. H3K76me1 and K76me2 are detectable during mitosis in proliferating parasites To verify the abundance of H3K76 methylation, we performed western blots using antibodies specific for each of the modifications on total extracts of proliferating parasites obtained from epimastigote or from infected mammalian cells (amastigotes). We also analyzed the expression in non-proliferating stages such as cell-derived trypomastigotes or trypomastigotes obtained from transformation of epimastigotes (metacyclics), which mimics the differentiation process that occurs in the end gut of the insect vector. The sequences of the peptides used to obtain the antibodies were conserved between T. brucei and T. cruzi histone H3 and are indicated in the Figure 2. Western blotting experiments revealed that K76me1 and K76me2 are more abundant in replicative forms than cell-derived trypomastigotes (Figure 6). K76me1 also appears in high levels in metacyclics. In contrast, the level of K76me3 is similar in all different stages relative to total H3. These results are mainly in agreement with a immunofluorescence analysis using the same antibodies. H3K76me1 was only detected in mitosis (Figure 7A and Figure S3A, Supporting Information) and H3K76me2 in mitosis and cytokinesis of epimastigotes (Figure 7B) and intracellular amastigotes (Figure S3B, Supporting Information). Mitosis and cytokinesis in epimastigotes and amastigotes were clearly visible by the presence of two kinetoplasts and one elongated nucleus or two separated nuclei in a single parasite, respectively 49. G2 cells were defined by the presence of two flagella. Importantly, no fluorescence was detected with antibodies specific for H3K76me1 and me2 in trypomastigote (Figure 8) and in metacyclics (Figure S4, 18 ACS Paragon Plus Environment

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Supporting Information), indicating that the detection of H3K76me1 by western blot labeling in metacyclic trypomastigotes could be a cross-reaction with another modification, not seen in T. brucei nor by immunofluorescence. Nevertheless, we can’t exclude the presence of small amounts of these modifications only detectable by Western blot or that the immunofluorescence method used was not sensible enough to detect this PTM. In contrast, K76me3 was found in all parasites (proliferating and nonproliferating), irrespectively of the cell cycle stage (Figures 7, 8 and Figures S3 and S4, Supporting Information). These results are similar to what was observed in T. brucei 14, in which mono- and di-methylation of K76 also occurs during cell division. Surprisingly, the replicative T. cruzi labeling of K76me1 was restricted to mitosis, whereas in T. brucei, it was detectable in both G2/M and M phases 11.

Discussion Here we identify, in a large-scale proteomics analysis, PTMs of all T. cruzi histones, including their variants. We confirmed some previously identified modifications but also found many novel PTMs. Some of the newly identified modifications seem to have homology to what is found in other eukaryotes (H3K19, H3K23, H3K76, and H2BK4), while others seems to be trypanosome specific (multiple modifications at H2A C-terminal; modifications at histone H1, H3T29, H3vK94, H2BS63). Interestingly, some conserved PTMs were found on the histone surface, while a trypanosome-specific PTM (H3T29p) is mainly localized inwardly, which may indicate that it could modulate specific interactions within the nucleosome structure of trypanosomes. Our data also indicate that histone modifications occur to variable extents in different parasite stages, probably reflecting different situations regarding gene transcription and DNA replication activity.



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We cannot exclude that other PTMs sites may exist in T. cruzi, since some peptides rich in lysines and arginines (mainly from N-termini) were missing in our analysis due to preferential trypsin digestion. This is clearly represented by the absence of H4K4Ac in our list of detected products. Furthermore, it is possible that other modifications exist in addition to the PTMs described in this study. For example, lysines could be decorated by many different types of modifications, including, crotonylation, N-formylation, succinylation, butyrylation, O-GlcNAcylation, 5-hydroxylation and many others 50. Our work opens the possibility to understand the specific function of these modifications. For example, acetylation of the histone H4 N-terminus could be associated with histone deposition during eukaryotic S-phase 51, 52 and whether it plays a similar role in this parasite needs further evaluation. We recently demonstrated that the presence of histone H4 non-acetylatable at K10 and K14 disturbs the addition and eviction of histone H3/H4 dimer through the interaction with their histone chaperones during transcription and replication

17

, also explaining the possible role of these

modifications in the transcription and replication sites in the genome 9, 53, 54. H3K23me1, me2 and me3 are enriched in non-proliferative stages. This could mark cells with inactive transcription sites. H3K27me1 (a possible human homolog of T. cruzi H3K23) was shown to be located at active promoters, particularly downstream of TSS in eukaryotes

38

while H3K27me3 is tightly associated with inactive gene

promoters acting in opposition to H3K4me3, a mark for transcriptionally active chromatin. Moreover, EZH2, a methyltransferase that constitute the catalytic component of the polycomb repressive complex-2 (PRC2), is responsible for methylation of H3K27 and is well known for its involvement in repressing gene expression

55

. In mice,

H3K27me3 contributes to the establishment of 'bivalent domains' in embryonic stem


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cells, which maintain genes essential for development in a poised state for activation or repression 56. In addition, it was observed that H3K27 can also be acetylated acting in antagonism to H3K27me3 in mammals

57

. Therefore, the fact that methylated H3K23

was predominantly found in trypomastigote, which have higher amounts of heterochromatin and low global transcription rates, suggests that H3K23 may have a similar function in trypanosomes. Further investigations are necessary to unravel whether H3K23me3 occurs in special domains in the T. cruzi genome and if it is able to regulate differentiation of this parasite. Similarly, the enrichment of acetylated lysine at the C-terminus of H2A could be involved in gene silencing or chromatin organization in trypomastigotes. As previously shown in T. brucei 14, we detected variable amounts of mono-, di-, or tri-methylation of lysine 76 of histone H3 in T. cruzi. The mono- and di-methylated forms were found mainly in proliferative stages in agreement with the role of these modifications in replication control, which was confirmed by their presence in dividing parasites. These observations agree with the fact that these modifications should be present in mitosis as they are involved in cell progression in several eukaryotes

58

,

probably by acting on a checkpoint through recognition by enzymes and proteins involved in homologous recombination 59. Interestingly, trypanosomes have two Dot1 homologues, Dot1A and Dot1B. Dot1A is essential for growth and catalyzes H3K76me1 and H3K76me2 found during late G2 and M phase of the cell cycle. In contrast, Dot1B catalyzes H3K76me3 and its depletion is tolerated in cell culture, although some parasites showed aneuploidy and cell cycle alterations

14

. The fact that

K76me1 and K76me2 were detected by western blot and were not seen in nonreplicative and infective forms by immunofluorescence in trypomastigotes and metacyclics could also indicate that they are present at very low levels or that only a



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small number of parasites have these PTMs. Alternatively, the antibodies could be influenced by the presence of other PTMs present in non-replicative parasites that could interfere with the detection in immunofluorescence conditions. For example, we found that H3R80 is dimethylated in T. cruzi and because of its proximity to K76 it could interfere with antibodies specific for methylated K76 in immunofluorescence analysis. The presence of H3K76me3 in non-proliferating cells is not surprising because these cells differentiate from proliferating parasites and might maintain trimethylated chromatin. The function of K76me3 is unknown, but depletion of Dot1b, which abolishes K76 trimethylation prevents the differentiation of the human infective form to the insect stage of T. brucei

14

. However, the exact mechanism behind this phenotype is

still unclear although recent studies revealed a karyokinesis defect during developmental differentiation of Dot1b-depleted parasite 60.

Conclusions We provide identification of novel histone PTMs and their differential expression in proliferating and non-proliferating life stages of T. cruzi. Globally, epimastigotes contain more modified histones than trypomastigotes. Some PTMs are preferentially expressed in non-proliferating cells such as acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 while others predominate in proliferative forms, such as methylations of lysine 76 of histone H3. Our studies will serve to better understand the functions of histone PTMs in chromatinassociated biological processes. Further studies on the identification of their genomic location, on their association with other proteins and on the evaluation of their dynamics in other life forms as well as during cell cycle will be in the focus of our laboratories in the future.


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Acknowledgments The authors are grateful to Claudio Rogério Oliveira from the Sociedade Paulista para a Medicina (SPDM) and Ismael Feitosa Lima, Ivan Novaski Avino, Mariana Morone for technical assistance. TCLJ (11/06087-5), VSN (2014/03714-7), MCL (2015/04867-4), NSM (2012/09403-8) and FCM (2014/01577-2) were supported by a fellowship from

FAPESP. This work was supported by funds from FAPESP (08/57910-0, 11/22619-7, 11/51973-3 and 13/07467-1) and CNPq 445655/2014-3.

Supporting Information Supporting information contains 4 figures and 5 tables. Supplemental figures includes: i. all MS/MS spectra that were used for de novo sequencing (including personal annotation), ii.

3D structural models of T.cruzi histones and iii.

immunofluorescence images of different methylated states of H3K76 in intracellular amastigotes and trypomastigotes-metacyclics. Supplemental tables, includes a list of protein and peptides identified using either MaxQuant or PEAKS Studio.

Legend to Supplemental Figures Figure S-1 A-AB. MS/MS spectra of modified peptides in T. cruzi histones. Figure S-2. Molecular models of histones H2A (A), H2B (B), H4 (C), H2AZ (D), H2Bv (E) and H3v (F) by homology. Figure S-3. H3K76 modifications during the cell division cycle of intracellular amastigotes of T. cruzi. Figure. S-4. Only H3K76me3 is detectable in metacyclic-trypomastigotes.

Supplemental Tables Table S-1. Protein identification using MaxQuant. 23 ACS Paragon Plus Environment


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Table S-2. Protein identification using PEAKS Studio. Table S-3. Histone peptides identified using MaxQuant. Table S-4. Histone peptides identified using PEAKS Studio. Table S-5. List of modified histone peptides.

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Dejung, M.; Subota, I.; Bucerius, F.; Dindar, G.; Freiwald, A.; Engstler, M.;

Boshart, M.; Butter, F.; Janzen, C. J., Quantitative Proteomics Uncovers Novel Factors



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Involved in Developmental Differentiation of Trypanosoma brucei. PLoS Pathog 2016, 12, (2), e1005439.


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Table I. Histones PTMs identified by MS in T. cruzi Histone

Acetylation

H1

K90, K91

Methylation

Dimethylation

Trimethylation

Phosphorylation

K58, K64, K67

S66, S11b

R94

TcCLB.510225.10 TcCLB.509837.40

K51, K55, K63, S1a

TcCLB.506369.70 S63a

H2A

K116, K120, K121, K126

H2AZ (possible)

K54, K58

H2Bv

K32

H3

K76, K23, K19

H3v H4

K127, R129

R10, K11

H2B

S73b

K90, K94

K4

S63

T29

K34 K23, K76

K23, K76, R80

K23, K76

K94

K94

K94

K10, K14

Histones modified residues are indicated. Three different histones H1 isoforms are shown. a and b represent the same PTM in a different protein isoform. H3v-histone H3 variant; H2Bv-histone H2B variant.



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Table II. Quantitation of modified histones peptides in T. cruzi according to MaxQuant Histone PTM

Mean Epi* SD Epi Mean Trypo*

SD Trypo

0,0425

0,0093

0,1121

0,0760

0

0

0,0013

0,0006

H2BK4me3R10me1K11me1

0,0020

0,0011

0

0

H2BvK32acK34me2

0,0038

0,0007

0

0

H3K23me1

0,0019

0,0007

0,0046

0,0008

H3K23me2

0

0

0,0047

0,0014

H3K76me1

0,0220

0,0118

0,0002

0,0002

H3K76me2

0,0321

0,0080

0,0039

0,0021

H3K76me3

0,2180

0,2152

0,1266

0,0660

H3vK94me3

0

0

0,3072

0,3022

H4K10acK14ac

0

0

0,0001

0

H2AK116ac H2AK120acK121ac

Modified T. cruzi histones peptides of epimastigotes (Epi) and cell-derived trypomastigotes (Trypo) were quantitated by label free based on XIC. Mean of three biological replicates (*) and the standard deviation (SD) are showed. ac-acetylation, me1-methylation, me2-dimethylation, me3-trimethylation.


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Table III. PTMs identified mainly at one T. cruzi life form. Histone

PTM Life Form H3K19acK23ac epimastigote H3 H3K23me2 trypomastigote H3T29p trypomastigote H3K94me2* trypomastigote H3v H3K94me3 trypomastigote H4 H4K10acK14ac trypomastigote H2AK120K121ac trypomastigote H2A H2AK126acK127me1 trypomastigote H2AK126acR129me1 trypomastigote H2BK4me3 epimastigote H2B H2BK4me3R10me1K11me1 epimastigote H2Bv H2BK32acK34me2 epimastigote H1S1acS11p* trypomastigote H1 H1K90me2* trypomastigote H1K94me2* trypomastigote ac-acetylation, me1- methylation,me2-dimethylation,me3-trimethylation,p-phosphorylation. *data not normalized by total histone content. Cell-derived trypomastigotes were used in this analysis.



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Page 36 of 48

LEGEND OF FIGURES Figure 1. Epimastigotes have more histone-modified peptides than cell-derived trypomastigotes. MS/MS counts for modified versus unmodified histone peptides are shown in light and dark colors.

Figure 2. Sequence alignment of histones from T. cruzi, T. brucei, Homo sapiens and their PTMs. The color-coded circles indicate the modified residues identified in T. cruzi and the known PTMs in T. brucei and human histones. Three different histones H1 are shown for T. cruzi. The first methionine was removed; therefore the following amino acid was assigned at first position. The sequences correspond to the following accession numbers: H1 (a- TcCLB.510225.10, b- TcCLB.506369.70 and c- TcCLB.509837.40); H2A- TcCLB.510525.80; H2AZ- TcCLB.511323.40; H2B- TcCLB.511635.10; H2BvTcCLB.506779.150;

H3- TcCLB.509471.86;

H3v- TcCLB.506503.150;

H4-

TcCLB.507943.40. Antibodies against H3K76 PTMs were prepared using peptide sequence underlined conjugated to KLH by a cysteine residue at peptide C-terminus as described in 11, 14.

Figure 3. Overall structure of histone H3 dimer. A. Structural model of H3 generated by ROBETTA. The left-hand monomer is shown as a cartoon representation. Each monomer of the model share a highly similar structural motif among histones constructed from three α-helices connected by two loops, denoted as α1-L1-α2-L2-α3. The right-hand monomer is shown with its electrostatic potential surface (blue: negatively charged; red: positively charged). Modified residues are labeled and shown as stick models. The majority of them are located at the protein surface, except T29. B. Superposition of H3 (monomer A and B) and its modified residues of T. cruzi (pink), H. 36 ACS Paragon Plus Environment

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sapiens (magenta-PDB ID code 2CV5), X. laevis (cyan- PDB ID code 1F66), D. melanogaster (yellow-PDB ID code 4X23) and S. cerevisiae (gray- PDB ID code 4KUD).

C. Structural superposition of the histone H3 (monomer A and B) modified

residues of T. cruzi (pink), H. sapiens (magenta- PDB ID code 2CV5), H. sapiens histone H4 (cyan- PDB ID code 2CV5) and H. sapiens DNA (slate- PDB ID code 2CV5). The superposition suggest that K76 could interact with histone H4, whereas R80 from monomer A could be able to interact with DNA. The structures were formatted, oriented and rendered in PyMOL.

Figure 4. Quantification of histone PTMs in T. cruzi. Ratios of quantitative values of modified histones detected in epimastigotes (E) and cell-derived trypomastigotes (T) forms. Ratios were obtained using LFQ values of MaxQuant or MS/MS counts using PEAKS Studio as described in materials and methods. ac-acetylation, me1-methylation, me2-dimethylation, me3-trimethylation, p-phosphorylation.

Figure 5. Estimation of H3K23 and H3K76 site occupancy. Number of MS/MS of peptides APKAPGAATGVK (for K23) and EVSGAQKEGLR (for K76) unmodified (unMod) or modified as indicated (me1-methylated, me2-dimethylated, me3trimethylated). The ratios were used to estimate, proportionally, the amount of each modification on the indicated residue in A (epimastigote) and B (cell-derived trypomastigote). Data from MaxQuant and PEAKS Studio were used.

Figure 6. Expression of histone methylated H3K76 in different T. cruzi forms. A. Immunoblotting of total proteins of epimastigotes (E), trypomastigotes (T) derived from infected mammalian cells, intracellular amastigotes (A) and purified metacyclic



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Page 38 of 48

trypomastigotes (M) probed with anti-H3K76me1, anti-H3K76me2 and antiH3K76me3. Anti-H3 was used as the loading control. B. Quantification of immunoblotting using the ratio of methylation (K76) / histone H3 signals. Upper graphic represents the gel probed with anti-H3K76me1, middle and lower graphics, respectively, represent to anti-H3K76me2 and anti-H3K76me3 gels. The bands were quantified by LI-COR Odyssey imaging software. The values are mean ± standard deviation (n = 3). One asterisk indicates significant differences compared to epimastigote levels and two asterisks to trypomastigote levels (p < 0.05).

Figure 7. H3K76me1 and me2 are enriched in the mitosis of epimastigote forms. Immunofluorescences of epimastigotes stained with (A) anti-H3K76me1 antibodies (red); (B) anti-H3K76me2 antibodies (green), or (C) anti-H3K76me3 antibodies (green). Cell cycle stages (G1/S, G2, M and C) are defined by the position of the DAPIstained kinetoplast (k) and nucleus (n) in blue. Bars = 5 µm. The images also show the corresponding field marked with dotted squares in the respective DIC images (bars = 5 µm) and the merged immunofluorescences of the antibody with DAPI.

Figure

8.

Only

H3K76me3

is

detectable

in

trypomastigotes.

Indirect

immunofluorescences of trypomastigote forms using H3K76me1, H3K76me2 and H3K76me3 (green) antibodies. DAPI-stained kinetoplasts (k) and nucleus (n) are indicated in blue (bars = 1 µm). The images also show the corresponding field marked with dotted squares in the respective DIC images (bars = 5 µm) and the merged immunofluorescences of antibodies with DAPI.


Page 39 of 48


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Epimastigote

Page 40 of 48

Trypomastigote 22%

42%

58% 78%

unModified peptides

Modified peptides

Fig 1

ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H1 T. cruzi a

63 SDAA

71KASP

87TAAKK--T-ARKPAVKK

112KPAAAKKAVTKSA--KKHAAKK-APK

134

T. cruzi b

63 SDAA

71KASP

82PAAKK--T-AKKPAAKK

107KPAAAKKAVNKSA--KKHAAKK-APK

129

T. cruzi c T. brucei

1 SDAA 5 ATVK

13KVAA

9KASP

21PAAKK--T-AKKPAMKK 29AAPKK--AVAKKPLAKK

54--AVAKK-----AAPKKVAPKKVAGK

H. sapiens

62 KKAL

109KAAS

125AKAKKPAGAAKKPKKAT

153TPKKAKKPAAAAGAKKAKSPKKAKAA

178

45KLAAAKKAVNKSA--KQPDQKK-ASK

67 72

H2A T. cruzi

8 K---KA

77KPKRLTPRTVTLAVRHDDDLGTLLKDVTLSRGGVMPSLNKALAKKHKSSKKAR

129

T. brucei

8 K---KA

76KTKRLTPRTVTLAVRHDDDLGALLRNVTMSRGGVMPSLNKALAKKQKSGKHAK

128

H. sapiens

9 KARAKA

76KKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPK--KTESHH

124

H2AZ T. cruzi T. brucei H. sapiens

54 KTGGKA--122KAQKTERIKPRHLLLAIRGDEELNQIV-NATIARGGVVPFVHKSLEKKIIKKS 173 53 KTGGKA--121KAQKTERIKPRHLLLAIRGDEELNQIV-NATIARGGVVPFVHKSLEKKIIKKS 172 4 KAGKDS

74KDLKVKRITPRHLQLAIRGDEELDSLI-KATIAGGGVIPHIHKSLIGKKGQQK

125

H2B T. cruzi

1 ATP-KSSSANRK-----------KGGKRSHRKPKR

41SMSGRTMKIVNSFVNDLFERIASEA

65

T. brucei

1 ATP-KSTPAKTR-----------KEAKKTRRQRKR

41SMTSRTMKIVNSFVNDLFERIAAEA

65

H. sapiens

1 PEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKE

52GISSKAMGIMNSFVNDIFERIASEA

76

H2Bv T. cruzi T. brucei

1 PPTKGGKRPLPMGGKGKGKRPASATRKASGGKKKGGARRGKKQQRWDLYIHRTLRQVYKRGTL 63 1 PPTKGGKRPLPLGGKGKGKRPPGQTTKSSSSRKKSGARRGKKQQRWDLYIHRTLRQVYKRGTL 63

H3 T. cruzi

19 KKAPKAPGAATGVKHAQRRWRPGTVALREIRQFQRSTDLLLQKAPFQRLVREVSGAQKEGLRF 81

T. brucei

19 KKASKGSDAASGVKTAQRRWRPGTVALREIRQFQRSTDLLLQKAPFQRLVREVSGAQKEGLRF 81

H. sapiens

23 KAARKSAPATGGVKKPHR-YRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRF 84

H3v T. cruzi T. brucei

60 TVVLREVRRYQSSTEFLIAKAPFRRLVREIVSNLKDSFRMSATCVEALQESTELYVTSVLADA 122 48 TVALREIRRLQSSTDFLIQRAPFRRFLREVVSNLKDSYRMSAACVDAIQEATETYITSVFMDA 110

H4 T. cruzi

1 AKGKKSGEA--KGTQKRQKKILRENVRGITRGSIRRLARRGGVKRISGIIYDEVRGVIKSFVE 61

T. brucei

1 AKGKKSGEA--KGSQKRQKKVLRENVRGITRGSIRRLARRGGVKRISGVIYDEVRGVLKSFVE 61

H.sapiens

1 SGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLE 63

Fig 2


Acetylation Methylation Dimethylation Trimethylation Phosphorylation


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Page 42 of 48

A.

B.

Monomer A

Monomer B

C.C.

Monomer A Fig 3

Monomer B ACS Paragon Plus Environment

Page 43 of 48

8 Log2 E/T (arbitrary units)

6 4 2 0 -2

Fig 4


H3K76me3

H3K76me2

H3K76me1

H3K23me1

H2BS63p

H2AK54acK58ac

H2AK120acK121ac

H2AK116acK120acK121ac

-4 H2AK116ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A.

Page 44 of 48

Epi

100 80 60 40 20 0

H3K23 unMod

B.

H3K76 me1

me2

me3

Trypo

100 80 60 40 20 0

H3K23 unMod

H3K76 me1

me2

me3

Fig 5


Page 45 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B E

T

A

M

1.0

0.5 H3K76me1

H3k76me2

0 1.0

0.5 H3k76me3 0 H3 total

1.0

0.5 0

Fig 6.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

DIC

DNA

Page 46 of 48

H3K76me1

merge

H3K76me2

merge

H3K76me3

merge

k N

G1/S

k N

G2

N

k k

M

N

k

N k

C

B

DIC

DNA k N

G1/S

N k

G2

k k N

M

k N

C

C

DIC

k N

DNA N

k

G1/S N k

G2

N k

M

k k

N

C

k N

Fig 7 ACS Paragon Plus Environment

Page 47 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DIC

DNA

H3K76me1

merge

H3K76me2

merge

H3K76me3

merge

N k

DIC

DNA N k

DIC

DNA k

N

Fig 8



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

for toc only 89x60mm (300 x 300 DPI)


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Chromatin Proteomics Reveals Variable Histone Modifications during

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