Chromatin Proteomics Reveals Variable Histone ... - ACS Publications

17 downloads 0 Views 5MB Size Report
Apr 25, 2016 - modifications in two life cycle stages of Trypanosoma cruzi, the protozoan ..... also analyzed by PEAKS Studio 7.530 software using the same fixed and ..... Furthermore, it is possible that other modifications exist in addition to ...
Article pubs.acs.org/jpr

Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi Teresa Cristina Leandro de Jesus,†,‡,# Vinícius Santana Nunes,§,# Mariana de Camargo Lopes,† Daiana Evelin Martil,‡ Leo Kei Iwai,† Nilmar Silvio Moretti,§ Fabrício Castro Machado,§ Mariana L. de Lima-Stein,§ Otavio Henrique Thiemann,‡ Maria Carolina Elias,† Christian Janzen,∥ Sergio Schenkman,§ and Julia Pinheiro Chagas da Cunha*,† †

Laboratório Especial de Ciclo Celular, Center of Toxins, Immune Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo 05503-900, Brazil ‡ Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo - USP, São Carlos, São Paulo 13563-120, Brazil § Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo 04039-032, Brazil ∥ Department of Cell and Developmental Biology, Theodor-Boveri-Institute at the Biocenter, University of Würzburg, 97070 Germany S Supporting Information *

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 chromatinbased 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, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative 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 to be 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. KEYWORDS: chromatin, histone, Trypanosoma cruzi, acetylation, methylation, cell cycle, life cycle



INTRODUCTION

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

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, and phosphorylation, resulting in modifications on chromatin structure that impact processes like DNA replication and 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. © 2016 American Chemical Society

Received: March 8, 2016 Published: April 25, 2016 2039

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Journal of Proteome Research



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 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 and K5, and mono-, di-, or trimethylated (me1, me2, and 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. Massspectrometry 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 the 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 vary during the different life- and cell-cycle stages. For this, T. cruzi is an interesting model because it alternates between a replicative (epimastigotes and amastigotes in the insect and mammalian host) and a nonreplicative 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.

Article

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 × 107 cells per ml). For differentiation, the epimastigotes were collected by centrifugation (2000g, 5 min) and resuspended in TAU medium (2 mM CaCl2, 2 mM MgCl2, 0.035% NaHCO3, 190 mM NaCl, 17 mM KCl, and pH 6.5) to 2.5 × 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, and 10 mM Lproline to 5 × 106 cells per ml. After 5 days, the equivalent of 5 × 108 parasites was centrifuged at 2000g for 5 min and resuspended in 10 mL of 3 mM KH2PO4, 57.2 mM Na2HPO4, 45 mM NaCl, and 55.5 mM glucose at pH 8.0 and loaded into a 10 mL DEAE−cellulose column (DE-52, Whatman) preequilibrated in the same buffer. Metacyclic trypomastigotes eluted in the flow-through fraction, while epimastigotes and partially differentiated parasites were retained in the column.22 Trypomastigotes forms (Y strain) were collected from the supernatant of infected monolayers of LLC-MK2 cells (from 120 to 144 h after infection) in culture-medium DMEM with 10% FBS at 37 °C and 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 ref 24. Briefly, 5 × 108 epimastigote 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 cOmplete EDTA free protease inhibitor cocktail (Roche)) and then lysed with a potter. Samples were centrifuged at 3000g for 10 min at 4 °C, and the pellet was resuspended in buffer 2 (buffer 1 containing 0.5% saponin). Samples were centrifuged at 3400g 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 cOmplete EDTA free protease inhibitor cocktail). Samples were centrifuged at 12000g 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), the samples were centrifuged at 21000g 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 × 108 epimastigotes or trypomastigotes were extracted with 10 mM Tris−HCl pH 7.4, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 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 for 10 min on ice under agitation. Samples were centrifuged at 3300g for 2 min at 4 °C, and the pellets were once again treated as above. The pellets were treated with DNase (250 U per sample) and incubated for 30 min at 37 °C and then centrifuged, as above. The supernatant was saved, and the pellet was once again 2040

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research

MaxQuant27 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 histone 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. 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 (Nterminal and K), methylation (KR), dimethylation (KR), trimethylation (K), and phosphorylation (STY) as variable modifications; maximal number of modification per peptide of five; maximal missed cleavages of two; MS1 tolerance of 6 ppm; MS2 of 0.5 Da; and 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.27 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 MaxQuant29 using a minimum ratio count of two. Protein quantitation was 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 samples). The raw data was also analyzed by PEAKS Studio 7.530 software using the same fixed and variable modifications as for MaxQuant; maximal number of modification per peptide of three, maximal missed cleavages of three; MS1 tolerance of 10 ppm; and MS2 of 0.5 Da. FDR for peptides of −10 log p value ≥15, and for proteins, ≥20. De novo sequencing using PEAKS included ALC (average local confidence) score higher than or equal to 50% and FDR lower or equal to −10 log p value = 15. Relative quantitation using PEAKS Studio was 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.

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 18500g for 30 min at 4 °C, and the pellets were washed three 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

After protein digestion, the peptides were cleaned up for detergent removal by hydrophilic interaction chromatographyHILIC (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 chromatography as described in ref 26 with some modifications. A total of three layers of a 3 M 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 three 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 reverse-phase precolumn (inner diameter of 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 reverse-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 datadependent 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 a mass resolution of 30 000. MS/MS spectra were acquired at normalized collision energy of 35%. Singly charged and charge-unassigned precursor ions were excluded. The dynamic exclusion parameters included an exclusion duration of 45 s, an exclusion list size of 500, and a repeat duration of 30 s. The isolation width for precursor ion selection (in m/z) was two. The raw data were processed in software environment

Prediction of 3D Structure of H3

The H3 sequence was submitted to the Robetta server for fullchain structure prediction using default settings.31,32 The homology models of H2A, H2AZ, H2B, H2Bv, H3v, and H4 were built by MODELER (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 2041

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research Immunoblotting

using two different search engines (Andromeda-MaxQuant and PEAKS Studio software), as described in the Experimental Procedures 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). 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 chromatinenriched extracts with a T. cruzi database as well as with a T. cruzi histone-only database built in-house. Histone PTMs were considered for further investigation only if they were identified exclusively in the latter database. All positive spectra were evaluated by de novo sequencing (Figure S1) and thus considered as a false or a true PTM (see the Experimental Procedures section for details). After filtering (including de novo sequencing analysis), MaxQuant (MQ) analysis retrieved 76 peptides including 16 modified peptides (Table S3), and PEAKS Studio retrieved 233 peptides including 24 modified peptides (Table S4). 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. On the basis of the 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).

Total protein extract was prepared from 1.5 × 10 cells obtained by centrifugation at 2000g for 5 min at 4 °C and washed once with PBS prior to be resuspended in SDSpolyacrylamide gel electrophoresis (SDS-PAGE) sample buffer 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 for 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, anti-H3K76me2, 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 1:2000. 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 antirabbit IgG antibody (Li-Cor) at a 1:20 000 dilution. Bounded antibodies were detected and quantified by using a LICOR Odyssey imaging apparatus. 7

Immunofluorescence

Culture-cell-derived and metacyclic trypomastigotes were incubated with 4% paraformaldehyde in PBS at room temperature for 5 min, centrifuged for 2 min at 2000 g, washed in PBS, permeabilized for 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 and then 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 s. 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 antiH3K76me3 were used at 1:1000. The monoclonal antibodies 2F6 and 3F6 antibodies (respectively, antiflagellum17 and antiglycoprotein 8236) were used at a 1:1000 dilution. The slides were washed with PBS, incubated 1 h at room temperature with Alexa Fluor 594 or 488 goat antirabbit or antimouse IgG (Invitrogen) diluted to 1:2000, 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 and emission filters. Blind deconvolution was performed by employing the AutoQuant X2.2 software (Media Cybernetics).



Figure 1. Epimastigote level of histone-modified peptides compared to that of cell-derived trypomastigotes. MS/MS counts for modified versus unmodified histone peptides are shown in light and dark colors, respectively.

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 DNA-metabolism 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 histones and in the variants H2BV and H3 V (Table 1 and Figure 2). In total, 44 PTMs were identified; including previously described PTMs

RESULTS

Global Histone PTM Analysis of T. cruzi

To identify PTMs in T. cruzi, we prepared chromatin extracts from epimastigotes and trypomastigotes 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 2042

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research

Figure 2. Sequence alignment of histones from T. cruzi, T. brucei, and 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. A total of three different histones (H1) are shown for T. cruzi. The first methionine was removed; therefore, the following amino acid was assigned at the 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; H2Bv, TcCLB.506779.150; H3, TcCLB.509471.86; H3v, TcCLB.506503.150; and H4, TcCLB.507943.40. Antibodies against H3K76 PTMs were prepared using a peptide sequence (underlined) conjugated to KLH by a cysteine residue at peptide C-terminus as described in refs 11 and 14.

Table 1. Histone PTMs Identified by MS in T. cruzi histone H1 TcCLB.510225.10 TcCLB.509837.40 TcCLB.506369.70 H2A H2AZ (possible) H2B H2Bv H3 H3v H4

acetylation

methylation

K90, K91 K51, K55, K63, S1a S63a K116, K120, K121, K126 K54, K58

dimethylation

phosphorylation

K58, K64, K67

S66, S11b S73b

K4

S63

K23, K76 K94

T29

R94

K90, K94 K127, R129 R10, K11

K32 K76, K23, K19

trimethylation

K23, K76 K94

K34 K23, K76, R80 K94

K10, K14

a

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.

2043

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research

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. 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 (monomers 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.

H2BK12Ac is found in differentially methylated regions of imprinted genes.38,39 Novel acetylations and di- and trimethylations were also detected in histone H1. It is important to highlight that the previous analysis of full-length histone H1 by MS suggested multiple methylations 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, and Glu-C digestions were employed earlier.

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, and H3R80me2 (Table 1). 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, 2044

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research

at active genes and are important to generate an open chromatin state.45,46 H3VK94 was found for the first time as mono, di-, and trimethylated. The 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.

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 trimethylated. 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 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 methyltransferases.14 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, and 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. What is noteworthy, although the histone sequence differs from their human counterparts, is that it seems that some T. cruzi PTMs are conserved, suggesting that these sites are localized at the histone 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 of the 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 the 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 considerably from humans, Xenopus laevis, Drosophila melanogaster, and Saccharomyces 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 MODELER showed that all the identified modified residues are also located at protein surface (Figure S2).

Histone PTM 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 and Tables

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.

2, 3, and S5). As pointed out above, T. 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 3. 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 trypomastigotes19 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

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 2045

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research Table 2. Quantitation of Modified Histones Peptides in T. cruzi According to MaxQuant histone PTM

mean epia

SD epi

mean trypo

SD trypo

H2AK116ac H2AK120acK121ac H2BK4me3R10me1K11me1 H2BvK32acK34me2 H3K23me1 H3K23me2 H3K76me1 H3K76me2 H3K76me3 H3vK94me3 H4K10acK14ac

0.0425 0 0.0020 0.0038 0.0019 0 0.0220 0.0321 0.2180 0 0

0.0093 0 0.0011 0.0007 0.0007 0 0.0118 0.0080 0.2152 0 0

0.1121 0.0013 0 0 0.0046 0.0047 0.0002 0.0039 0.1266 0.3072 0.0001

0.0760 0.0006 0 0 0.0008 0.0014 0.0002 0.0021 0.0660 0.3022 0

a

Modified T. cruzi histones peptides of epimastigotes (epi) and cellderived trypomastigotes (trypo) were quantitated by label-free-based on XIC. The mean of three biological replicates (*) and the standard deviation (SD) are showed. Abbreviations: ac, acetylation; me1, methylation; me2, dimethylation; me3, trimethylation.

Table 3. PTMs Identified Mainly at One T. cruzi Life Forma histone H3

H3v H4 H2A

H2B H2Bv H1

PTM

life form

H3K19acK23ac H3K23me2 H3T29p H3K94me2* H3K94me3 H4K10acK14ac H2AK120 K121ac H2AK126acK127me1 H2AK126acR129me1 H2BK4me3 H2BK4me3R10me1K11me1 H2BK32acK34me2 H1S1acS11pb H1K90me2b H1K94me2b

epimastigote trypomastigote trypomastigote trypomastigote trypomastigote trypomastigote trypomastigote trypomastigote trypomastigote epimastigote epimastigote epimastigote trypomastigote trypomastigote trypomastigote

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; me3, trimethylated). The ratios were used to estimate, proportionally, the amount of each modification on the indicated residue in A (epimastigote) and B (cellderived trypomastigote). Data from MaxQuant and PEAKS Studio were used.

H3K76me1 and K76me2 Detection during Mitosis in Proliferating Parasites

a

Abbreviations: ac, acetylation; me1, methylation; me2, dimethylation; me3, trimethylation; p, phosphorylation. bData not normalized by total histone content. Cell-derived trypomastigotes were used in this analysis.

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 nonproliferating stages such as cell-derived trypomastigotes or trypomastigotes obtained from the 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 (Figures 7A and S3A) and H3K76me2 in mitosis and cytokinesis of epimastigotes (Figure 7B) and intracellular amastigotes (Figure S3B). 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

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 trimethylated H3K23 is found in lower amounts (2.6%). Similarly, acetylation of the C-terminus of H2A predominates in trypomastigotes (Table 3). In contrast, nonmodified mono- and dimethylated H3K76 (Table 2) is enriched in epimastigotes compared to trypomastigote forms. Interestingly, K76me1 is present in low amounts compared to K76me2 and K76me3 (Figures 4 and 5), although it is more enriched in epimastigotes than trypomastigotes. H3K76me3 is present in both parasite forms. 2046

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research

Figure 6. Expression of histone methylated H3K76 in different T. cruzi forms. Panel A: immunoblotting of total proteins of epimastigotes (E), trypomastigotes (T) derived from infected mammalian cells, intracellular amastigotes (A), and purified metacyclic trypomastigotes (M) probed with anti-H3K76me1, anti-H3K76me2, and anti-H3K76me3. Anti-H3 was used as the loading control. Panel B: quantification of immunoblotting using the ratio of methylation (K76) and histone H3 signals. The upper graphic represents the gel probed with antiH3K76me1; the middle and lower graphics, respectively, represent the anti-H3K76me2 and anti-H3K76me3 gels. The bands were quantified by LI-COR Odyssey imaging software. The values are mean ± standard deviation (n = 3). A single asterisk indicates significant differences compared to epimastigote levels, and two asterisks indicate significant differences compared to trypomastigote levels (p < 0.05).

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), 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 cannot 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) irrespective of the cell cycle stage (Figures 7, 8, S3, and S4). These results are similar to what was observed in T. brucei,14 in which mono- and dimethylation 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 Cterminal; modifications at histone H1, H3T29, H3vK94, and

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 DAPI-stained 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. 2047

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research

catalytic component of the polycomb repressive complex-2 (PRC2), is responsible for methylation of H3K27 and is wellknown for its involvement in repressing gene expression.55 In mice, H3K27me3 contributes to the establishment of “bivalent domains” in embryonic stem 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 Cterminus of H2A could be involved in gene silencing or chromatin organization in trypomastigotes. As was previously shown in T. brucei,14 we detected variable amounts of mono-, di-, or trimethylation of lysine 76 of histone H3 in T. cruzi. The mono- and dimethylated 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 because 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 the late G2 and M phases 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 small number of parasites have these PTMs. Alternatively, the antibodies could be influenced by the presence of other PTMs present in nonreplicative 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 nonproliferating cells is not surprising because these cells differentiate from proliferating parasites and might maintain trimethylated chromatin. The function of K76me3 is unknown, but the 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 the developmental differentiation of Dot1b-depleted parasite.60

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.

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. We cannot exclude that other PTMs sites may exist in T. cruzi because 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 the 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 nonacetylatable at K10 and K14 disturbs the addition and eviction of the 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 nonproliferative stages. This could mark cells with inactive transcription sites. H3K27me1 (a possible human homologue of T. cruzi H3K23) was shown to be located at active promoters, particularly downstream of TSS in eukaryotes,38 and 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



CONCLUSIONS We provide identification of novel histone PTMs and their differential expression in proliferating and nonproliferating the life stages of T. cruzi. Globally, epimastigotes contain more modified histones than trypomastigotes. Some PTMs are preferentially expressed in nonproliferating cells such as the 2048

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research

(4) Martinez-Calvillo, S.; Yan, S.; Nguyen, D.; Fox, M.; Stuart, K.; Myler, P. J. Transcription of Leishmania major Friedlin chromosome 1 initiates in both directions within a single region. Mol. Cell 2003, 11 (5), 1291−1299. (5) El-Sayed, N. M.; Myler, P. J.; Blandin, G.; Berriman, M.; Crabtree, J.; Aggarwal, G.; Caler, E.; Renauld, H.; Worthey, E. A.; Hertz-Fowler, C.; Ghedin, E.; Peacock, C.; Bartholomeu, D. C.; Haas, B. J.; Tran, A. N.; Wortman, J. R.; Alsmark, U. C.; Angiuoli, S.; Anupama, A.; Badger, J.; Bringaud, F.; Cadag, E.; Carlton, J. M.; Cerqueira, G. C.; Creasy, T.; Delcher, A. L.; Djikeng, A.; Embley, T. M.; Hauser, C.; Ivens, A. C.; Kummerfeld, S. K.; Pereira-Leal, J. B.; Nilsson, D.; Peterson, J.; Salzberg, S. L.; Shallom, J.; Silva, J. C.; Sundaram, J.; Westenberger, S.; White, O.; Melville, S. E.; Donelson, J. E.; Andersson, B.; Stuart, K. D.; Hall, N. Comparative genomics of trypanosomatid parasitic protozoa. Science 2005, 309 (5733), 404−9. (6) De Gaudenzi, J. G.; Noe, G.; Campo, V. A.; Frasch, A. C.; Cassola, A. Gene expression regulation in trypanosomatids. Essays Biochem. 2011, 51, 31−46. (7) Maree, J. P.; Patterton, H. G. The epigenome of Trypanosoma brucei: a regulatory interface to an unconventional transcriptional machine. Biochim. Biophys. Acta, Gene Regul. Mech. 2014, 1839 (9), 743−50. (8) Wright, J.; Siegel, T.; Cross, G. Histone H3 trimethylated at lysine 4 is enriched at probable transcription start sites in Trypanosoma brucei. Mol. Biochem. Parasitol. 2010, 172 (2), 141−144. (9) Siegel, T. N.; Hekstra, D. R.; Kemp, L. E.; Figueiredo, L. M.; Lowell, J. E.; Fenyo, D.; Wang, X.; Dewell, S.; Cross, G. A. Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei. Genes Dev. 2009, 23 (9), 1063−76. (10) Tiengwe, C.; Marques, C. A.; McCulloch, R. Nuclear DNA replication initiation in kinetoplastid parasites: new insights into an ancient process. Trends Parasitol. 2014, 30 (1), 27−36. (11) Gassen, A.; Brechtefeld, D.; Schandry, N.; Arteaga-Salas, J. M.; Israel, L.; Imhof, A.; Janzen, C. J. DOT1A-dependent H3K76 methylation is required for replication regulation in Trypanosoma brucei. Nucleic Acids Res. 2012, 40 (20), 10302−11. (12) Janzen, C. J.; Fernandez, J. P.; Deng, H.; Diaz, R.; Hake, S. B.; Cross, G. A. Unusual histone modifications in Trypanosoma brucei. FEBS Lett. 2006, 580 (9), 2306−10. (13) Mandava, V.; Fernandez, J. P.; Deng, H.; Janzen, C. J.; Hake, S. B.; Cross, G. A. Histone modifications in Trypanosoma brucei. Mol. Biochem. Parasitol. 2007, 156 (1), 41−50. (14) Janzen, C. J.; Hake, S. B.; Lowell, J. E.; Cross, G. A. Selective dior trimethylation of histone H3 lysine 76 by two DOT1 homologs is important for cell cycle regulation in Trypanosoma brucei. Mol. Cell 2006, 23 (4), 497−507. (15) Figueiredo, L. M.; Cross, G. A.; Janzen, C. J. Epigenetic regulation in African trypanosomes: a new kid on the block. Nat. Rev. Microbiol. 2009, 7 (7), 504−513. (16) da Cunha, J. P.; Nakayasu, E. S.; de Almeida, I. C.; Schenkman, S. Post-translational modifications of Trypanosoma cruzi histone H4. Mol. Biochem. Parasitol. 2006, 150 (2), 268−77. (17) Ramos, T. C.; Nunes, V. S.; Nardelli, S. C.; Dos Santos Pascoalino, B.; Moretti, N. S.; Rocha, A. A.; da Silva Augusto, L.; Schenkman, S. Expression of non-acetylatable lysines 10 and 14 of histone H4 impairs transcription and replication in Trypanosoma cruzi. Mol. Biochem. Parasitol. 2015, 204 (1), 1−10. (18) Pascoalino, B.; Dindar, G.; Vieira-da-Rocha, J. P.; Machado, C. R.; Janzen, C. J.; Schenkman, S. Characterization of two different Asf1 histone chaperones with distinct cellular localizations and functions in Trypanosoma brucei. Nucleic Acids Res. 2014, 42 (5), 2906−18. (19) da Cunha, J. P.; Nakayasu, E. S.; Elias, M. C.; Pimenta, D. C.; Tellez-Inon, M. T.; Rojas, F.; Manuel, M.; Almeida, I. C.; Schenkman, S. Trypanosoma cruzi histone H1 is phosphorylated in a typical cyclin dependent kinase site accordingly to the cell cycle. Mol. Biochem. Parasitol. 2005, 140 (1), 75−86. (20) Elias, M.; Marques-Porto, R.; Freymuller, E.; Schenkman, S. Transcription rate modulation through the Trypanosoma cruzi life

acetylations of lysines at the C-terminus of histone H2A and the methylations of lysine 23 of histone H3, while others predominate in proliferative forms, such as the 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, their association with other proteins, and the evaluation of their dynamics in other life forms (as well as during the cell cycle) will be in the focus of our laboratories in the future.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00208. Figures showing MS/MS spectra of modified peptides in T. cruzi histones; molecular models of histones H2A, H2B, H4, H2AZ, H2Bv, and H3v by homology; H3K76 modifications during the cell division cycle of intracellular amastigotes of T. cruzi; and detection of H3K76me3 in metacyclic-trypomastigotes. (PDF) Tables showing protein identification using MaxQuant, protein identification using PEAKS Studio, histone peptides identified using MaxQuant, histone peptides identified using PEAKS Studio, and a list of modified histone peptides. (XLSX)



AUTHOR INFORMATION

Corresponding Author

*Phone: +(55) 11 26279731; e-mail: julia.cunha@butantan. gov.br. Author Contributions #

T.C.L.d.J. and V.S.N. contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors are grateful to Claudio Rogério Oliveira from the Sociedade Paulista para a Medicina (SPDM) and Ismael Feitosa Lima, Ivan Novaski Avino, and Mariana Morone for technical assistance. T.C.L.J. (11/06087-5), V.S.N. (2014/03714-7), M.C.L. (2015/04867-4), N.S.M. (2012/09403-8), and F.C.M. (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.



ABBREVIATIONS: PTM, post-translational modifications; ac, acetylation; me1, monomethylation; me2, dimethylation; me3, trimethylation; p, phosphorylation



REFERENCES

(1) Bannister, A. J.; Kouzarides, T. Regulation of chromatin by histone modifications. Cell Res. 2011, 21 (3), 381−95. (2) Santoro, S. W.; Dulac, C. Histone variants and cellular plasticity. Trends Genet. 2015, 31 (9), 516−27. (3) Alsford, S.; Horn, D. Trypanosomatid histones. Mol. Microbiol. 2004, 53 (2), 365−372. 2049

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research cycle occurs in parallel with changes in nuclear organisation. Mol. Biochem. Parasitol. 2001, 112 (1), 79−90. (21) Camargo, E. P. Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Rev. Inst Med. Trop Sao Paulo 1964, 6, 93−100. (22) da Silva Augusto, L.; Moretti, N. S.; Ramos, T. C. P.; de Jesus, T. C. L.; Zhang, M.; Castilho, B. A.; Schenkman, S. A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi. PLoS Pathog. 2015, 11 (2), e1004618. (23) Abuin, G.; Freitas-Junior, L. H. G.; Colli, W.; Alves, M.; Schenkman, S. Expression of trans-sialidase and 85-kDa glycoprotein genes in Trypanosoma cruzi is differentially regulated at the posttranscriptional level by labile protein factors. J. Biol. Chem. 1999, 274 (19), 13041−13047. (24) Toro, G. C.; Galanti, N. Trypanosoma cruzi histones. Further characterization and comparison with higher eukaryotes. Biochem Int. 1990, 21 (3), 481−490. (25) Godoy, P. D.; Nogueira-Junior, L. A.; Paes, L. S.; Cornejo, A.; Martins, R. M.; Silber, A. M.; Schenkman, S.; Elias, M. C. Trypanosome prereplication machinery contains a single functional orc1/cdc6 protein, which is typical of archaea. Eukaryotic Cell 2009, 8 (10), 1592−1603. (26) Rappsilber, J.; Mann, M.; Ishihama, Y. Protocol for micropurification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007, 2 (8), 1896−906. (27) Cox, J.; Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008, 26 (12), 1367−72. (28) Cox, J.; Neuhauser, N.; Michalski, A.; Scheltema, R. A.; Olsen, J. V.; Mann, M. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 2011, 10 (4), 1794−805. (29) Cox, J.; Hein, M. Y.; Luber, C. A.; Paron, I.; Nagaraj, N.; Mann, M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 2014, 13 (9), 2513−26. (30) Ma, B.; Zhang, K.; Hendrie, C.; Liang, C.; Li, M.; DohertyKirby, A.; Lajoie, G. PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17 (20), 2337−42. (31) Kim, D. E.; Chivian, D.; Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 2004, 32, W526−W531. (32) Chivian, D.; Kim, D. E.; Malmstrom, L.; Schonbrun, J.; Rohl, C. A.; Baker, D. Prediction of CASP6 structures using automated Robetta protocols. Proteins: Struct., Funct., Genet. 2005, 61 (7), 157−166. (33) Webb, B.; Sali, A. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc Bioinformatics 2014, 47, 1−32. (34) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 1993, 26 (2), 283−291. (35) Schrödinger LLC. PyMOL Molecular Graphics System, version 1.8; Schrödinger: New York, 2015. (36) Neira, I.; Silva, F. A.; Cortez, M.; Yoshida, N. Involvement of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin and invasion of epithelial cells. Infect. Immun. 2003, 71 (1), 557−561. (37) Golebiowski, F.; Kasprzak, K. S. Inhibition of core histones acetylation by carcinogenic nickel(II). Mol. Cell. Biochem. 2005, 279 (1−2), 133−9. (38) Wang, Z.; Zang, C.; Rosenfeld, J. A.; Schones, D. E.; Barski, A.; Cuddapah, S.; Cui, K.; Roh, T. Y.; Peng, W.; Zhang, M. Q.; Zhao, K. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat. Genet. 2008, 40 (7), 897−903. (39) Singh, P.; Cho, J.; Tsai, S. Y.; Rivas, G. E.; Larson, G. P.; Szabo, P. E. Coordinated allele-specific histone acetylation at the differentially methylated regions of imprinted genes. Nucleic Acids Res. 2010, 38 (22), 7974−90.

(40) Horn, D. Introducing histone modification in trypanosomes. Trends Parasitol. 2007, 23 (6), 239−242. (41) Tsai, W. W.; Wang, Z.; Yiu, T. T.; Akdemir, K. C.; Xia, W.; Winter, S.; Tsai, C. Y.; Shi, X.; Schwarzer, D.; Plunkett, W.; Aronow, B.; Gozani, O.; Fischle, W.; Hung, M. C.; Patel, D. J.; Barton, M. C. TRIM24 links a non-canonical histone signature to breast cancer. Nature 2010, 468 (7326), 927−32. (42) Papazyan, R.; Voronina, E.; Chapman, J. R.; Luperchio, T. R.; Gilbert, T. M.; Meier, E.; Mackintosh, S. G.; Shabanowitz, J.; Tackett, A. J.; Reddy, K. L.; Coyne, R. S.; Hunt, D. F.; Liu, Y.; Taverna, S. D. Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis. eLife 2014, 3, e02996. (43) Nguyen, A. T.; Taranova, O.; He, J.; Zhang, Y. DOT1L, the H3K79 methyltransferase, is required for MLL-AF9-mediated leukemogenesis. Blood 2011, 117 (25), 6912−22. (44) Lowell, J. E.; Kaiser, F.; Janzen, C. J.; Cross, G. A. Histone H2AZ dimerizes with a novel variant H2B and is enriched at repetitive DNA in Trypanosoma brucei. J. Cell Sci. 2005, 118 (24), 5721−30. (45) Bruce, K.; Myers, F. A.; Mantouvalou, E.; Lefevre, P.; Greaves, I.; Bonifer, C.; Tremethick, D. J.; Thorne, A. W.; Crane-Robinson, C. The replacement histone H2A.Z. in a hyperacetylated form is a feature of active genes in the chicken. Nucleic Acids Res. 2005, 33 (17), 5633− 9. (46) Ishibashi, T.; Dryhurst, D.; Rose, K. L.; Shabanowitz, J.; Hunt, D. F.; Ausio, J. Acetylation of vertebrate H2A.Z and its effect on the structure of the nucleosome. Biochemistry 2009, 48 (22), 5007−17. (47) Lowell, J. E.; Cross, G. A. A variant histone H3 is enriched at telomeres in Trypanosoma brucei. J. Cell Sci. 2004, 117 (24), 5937−47. (48) Nardelli, S. C.; da Cunha, J. P.; Motta, M. C.; Schenkman, S. Distinct acetylation of Trypanosoma cruzi histone H4 during cell cycle, parasite differentiation, and after DNA damage. Chromosoma 2009, 118 (4), 487−99. (49) Elias, M. C.; da Cunha, J. P.; de Faria, F. P.; Mortara, R. A.; Freymuller, E.; Schenkman, S. Morphological events during the Trypanosoma cruzi cell cycle. Protist 2007, 158 (2), 147−57. (50) Arnaudo, A. M.; Garcia, B. A. Proteomic characterization of novel histone post-translational modifications. Epigenet. Chromatin 2013, 6 (1), 24. (51) Vogelauer, M.; Rubbi, L.; Lucas, I.; Brewer, B. J.; Grunstein, M. Histone acetylation regulates the time of replication origin firing. Mol. Cell 2002, 10 (5), 1223−33. (52) Doyon, Y.; Cayrou, C.; Ullah, M.; Landry, A. J.; Cote, V.; Selleck, W.; Lane, W. S.; Tan, S.; Yang, X. J.; Cote, J. ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation. Mol. Cell 2006, 21 (1), 51−64. (53) Respuela, P.; Ferella, M.; Rada-Iglesias, A.; Aslund, L. Histone acetylation and methylation at sites initiating divergent polycistronic transcription in Trypanosoma cruzi. J. Biol. Chem. 2008, 283 (23), 15884−92. (54) Thomas, S.; Green, A.; Sturm, N.; Campbell, D.; Myler, P. Histone acetylations mark origins of polycistronic transcription in Leishmania major. BMC Genomics 2009, 10, 152. (55) Kuzmichev, A.; Nishioka, K.; Erdjument-Bromage, H.; Tempst, P.; Reinberg, D. Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev. 2002, 16 (22), 2893−2905. (56) Bernstein, B. E.; Mikkelsen, T. S.; Xie, X.; Kamal, M.; Huebert, D. J.; Cuff, J.; Fry, B.; Meissner, A.; Wernig, M.; Plath, K.; Jaenisch, R.; Wagschal, A.; Feil, R.; Schreiber, S. L.; Lander, E. S. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006, 125 (2), 315−26. (57) Tie, F.; Banerjee, R.; Stratton, C. A.; Prasad-Sinha, J.; Stepanik, V.; Zlobin, A.; Diaz, M. O.; Scacheri, P. C.; Harte, P. J. CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing. Development 2009, 136 (18), 3131−41. (58) Kim, W.; Choi, M.; Kim, J. E. The histone methyltransferase Dot1/DOT1L as a critical regulator of the cell cycle. Cell Cycle 2014, 13 (5), 726−38. 2050

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051

Article

Journal of Proteome Research (59) Chernikova, S. B.; Dorth, J. A.; Razorenova, O. V.; Game, J. C.; Brown, J. M. Deficiency in Bre1 impairs homologous recombination repair and cell cycle checkpoint response to radiation damage in mammalian cells. Radiat. Res. 2010, 174 (5), 558−65. (60) Dejung, M.; Subota, I.; Bucerius, F.; Dindar, G.; Freiwald, A.; Engstler, M.; Boshart, M.; Butter, F.; Janzen, C. J. Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei. PLoS Pathog. 2016, 12 (2), e1005439.

2051

DOI: 10.1021/acs.jproteome.6b00208 J. Proteome Res. 2016, 15, 2039−2051