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Post-translational modifications of Trypanosoma cruzi canonical and variant histones Gisele F. A. Picchi, Vanessa Zulkievicz, Marco Aurelio Krieger, Nilson T. Zanchin, Samuel Goldenberg, and LYRIS M F de GODOY J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00655 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 13, 2017

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Journal of Proteome Research

Post-translational modifications of Trypanosoma cruzi canonical and variant histones Gisele F. A. Picchi1,*, Vanessa Zulkievicz1, Marco A. Krieger1, Nilson T. Zanchin1, Samuel Goldenberg1 and Lyris M. F. de Godoy1,* 1

Instituto Carlos Chagas, Fiocruz Parana, Curitiba, Paraná, Brazil

Keywords: histone, post-translational modifications, mass spectrometry-based proteomics, Trypanosoma cruzi

1

ACS Paragon Plus Environment


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ABSTRACT

Chagas disease, caused by Trypanosoma cruzi, still affects millions of people around the world. No vaccines nor treatment for chronic Chagas disease are available, and chemotherapy for the acute phase is hindered by limited efficacy and severe side effects. The processes by which the parasite acquires infectivity and survives in different hosts involve tight regulation of gene expression, mainly post-transcriptionally. Nevertheless, chromatin structure/organization of trypanosomatids is similar to other eukaryotes, including histone variants and post-translational modifications. Emerging evidence suggests that epigenetic mechanisms also play an important role in the biology/pathogenesis of these parasites, making epigenetic targets suitable candidates to drug discovery. Here, we present the first comprehensive map of post-translational modifications of T. cruzi canonical and variant histones and show that its histone code can be as sophisticated as that of other eukaryotes. A total of 13 distinct modifications were identified, including rather novel and unusual ones such as alternative lysine acylations, serine/threonine acetylation, and N-terminal methylation. Some histone marks correlate to those described for other organisms, suggesting that similar regulatory mechanisms may be in place. Others, however, are unique to T. cruzi or to trypanosomatids as a group and might represent good candidates for the development of anti-parasitic drugs.

2


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INTRODUCTION Chagas disease1 is predominantly endemic in Latin American, where it is estimated to affect 8 million people and to cause over 20,000 deaths each year 2. In this region, triatomine insects are the main source for transmission of Trypanosoma cruzi, the Chagas disease causative agent to humans. However, due to population mobility, it has also been increasingly detected in North America, Europe, and the Western Pacific Region, where transmission can take place by blood transfusion, oral infection, organ donation or congenitally 3. The disease manifests initially in an acute phase characterized by high parasitemia, which lasts for about two months and can be multi or asymptomatic. Patients can then evolve to a long-lasting chronic phase that can either remain asymptomatic or manifest in severe clinical forms, depending on the target tissues mainly cardiac and digestive muscle - where the parasites hide. The symptoms often occur in early adulthood, making Chagas disease a serious health problem due not only to its mortality rates but also to its debilitating effects

2,4

.

Eventually, chronic patients can die of

cardiomyopathy, digestive mega syndromes or a combination of both. The current therapeutic approach for Chagas disease aims at controlling parasitemia in the acute phase, with chemotherapy that can cause severe side effects in the patients. Unfortunately, efficient treatments for the chronic forms of the disease, as well as prophylactic drugs and vaccines, are not available. The life cycle of T. cruzi involves invertebrate (triatomine insects) and vertebrate (mammals, including humans) hosts. During differentiation, the parasite presents four main morphological stages, being two replicative (epimastigotes in the insects and amastigotes in the mammalian cells) and two non-replicative, infective forms (metacyclic trypomastigotes in the insects and bloodstream trypomastigotes in mammals)

1,5

. In order to adapt to these distinct environments 3



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and to survive inside different hosts, the parasite tightly regulates its gene expression 6–8 which, in contrast to other eukaryotes, occurs mainly at the post-transcriptional level 8–11. Despite having divergent mechanisms for gene expression control, T. cruzi and the other trypanosomatid parasites (e.g. Trypanosoma brucei, Leishmania spp.) have a chromatin structure and organization quite similar to other eukaryotes. The DNA is organized into nucleosomes, and all core canonical (H2A, H2B, H3, and H4), some variants (H2A.Z, H2B.V, and H3.V) and a linker histone (H1) are present

12–14

. Furthermore, genome sequencing has shown that

trypanosomatids possess all the machinery necessary to modify chromatin and the presence of histone post-translational modifications (PTMs) has already been observed. T. cruzi histones present some peculiarities, such as sequence divergence of canonical histones compared to other organisms, especially in their N-terminal tails, the presence of variants that are specific to Kinetoplastida and the lack of the globular domain of histone H1

15,16

. Nevertheless, several

residues within histone N-terminal tails, which are substrates for modifications, as well as some PTM marks, seem to be conserved not formed

21

17–20

. Furthermore, even though metaphase chromosomes are

, trypanosomatids regulate their chromatin compaction state according to different

circumstances, such as cell cycle progression

22

and differentiation

23–25

, with higher chromatin

condensation usually being accompanied by a reduction in total mRNA levels. The occurrence of different chromatin compaction and histone modification states argues in favor of epigenetic regulation in trypanosomatids

16,26

. Indeed,

increasing evidence suggests that epigenetic

mechanisms are essential for these organisms 19–30. Epigenetic marks represent a second layer of inheritable information above the genome, which affects the organization of chromatin and DNA accessibility, having great influence on gene activity. Particularly, the “Histone Code”, constituted of a wide variety of histone PTMs, acts as 4


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a dynamic mechanism to direct specific genetic programs. In humans and other eukaryotes, over 20 distinct histone modifications are present, including acetylation, phosphorylation, methylation (mono, di- and tri-), propionylation, butyrylation, crotonylation, formylation, hydroxylation, citrullination,

malonylation,

succinylation,

2-hydroxylisobutyrylation,

glutarylation,

ubiquitination, SUMOylation, O-GlcNAcylation, ADP-ribosylation, and proline isomerization 31. Also, hundreds of histone PTM marks have been reported and proven to be involved in various epigenetic regulatory processes, both under normal physiological conditions and in disease 32–35. Therefore, the search for the so-called "epigenetic drugs" has been pursued for decades and some, such as histone deacetylase (HDAC) inhibitors, had already hit the market, especially for the treatment of cancer and neurological disorders 36–40. Aiming to decrypt the “Trypanosomatid Histone Code”, some functional studies were successfully performed, but the role of histone PTMs in chromatin biology and epigenetic signaling in these parasites is just beginning to be uncovered. As of this writing, only three histone chemical modifications (acetylation, methylation, and phosphorylation) have been reported and the number of known PTM sites is still far smaller than that of humans and other model organisms. For T. brucei, about 30 acetylation and methylation marks on the canonical histones are described

20,29,41–45

. For T. cruzi, close to 50 PTM sites of acetylation, methylation

and phosphorylation are known, mostly located on canonical histones, some of which seem to be regulated across the differentiation from the replicative to the infective form of the parasite 18,46– 51

. In trypanosomatids, histone modifications (H4K4ac, H4K10ac and H4K14ac) seem to be

essential for chromatin assembly and/or remodeling required in transcription and replication 49,52. Also, the histone code potentially affects transcriptional regulation, as suggested by the fact that 5



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the boundaries of polycistronic transcription units are marked by specific patterns of modified histones and/or histone variants in T. cruzi (acetylated H3/H4 and trimethylated H3) 48, T. brucei (acetylated H4, trimethylated H3 and dimers of H2A.Z/H2B.V)

53,54

and L. major (acetylated

histones) 55. Histone modifications also play important functions during cell cycle control. In T. brucei, silencing of the lysine methyltransferase DOT1A generates a population of cells that contain half the normal amount of DNA, consequence of premature progression through mitosis without DNA replication, suggesting that methylation of H3K76 has an important role in proper cell cycle progression

29,42

. Similarly, silencing of the acetyltransferase HAT1 also generates

cells that undergo mitosis without nuclear DNA replication 56. In addition, histone modifications seem to regulate the expression of variant surface glycoproteins (VSGs) in T. brucei, and histone deacetylases DAC1/DAC3

57

and methyltransferase DOT1B

58

play distinct roles in telomeric

VSG expression site silencing. Chromatin modifications and histones marks are also associated with stress response in trypanosomatids 28. The activity of HDAC inhibitors against apicomplexa, helminths and kinetoplastida parasites has also been recently demonstrated and continues to attract increasing interest

57,59–69

. In P.

falciparum, sirtuins (HDACs class III) regulate the expression of virulence genes and represent potential therapeutic targets to inhibit adhesion to erythrocytes and infection of the host cells. Additionally, anti-malarial HDAC inhibitors results in histone hyperacetylation and large changes in gene expression in the asexual stage of the parasite

70,71

. In T. brucei, histone

deacetylases play an important role in subtelomeric gene silencing, revealing DAC3 as a potential target for the development of new anti-parasitic HDAC inhibitors 57. Altogether, the evidence described above suggests that epigenetic mechanisms constitute an important part of trypanosomatid parasite biology and, consequently, of the pathology of their 6


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respective human diseases. However, studies describing the molecular players involved in such mechanisms are urgently needed to allow further understanding of the epigenetic control in these organisms. For T. cruzi, functional studies have been hindered by the lack of basic information, particularly about the occurrence and site-specific localization of histone PTMs. Nevertheless, due to the high degree of similarity of its orthologous genes, it is very likely that epigenetic components are also essential for the growth and survival of this parasite and can represent important targets for the development of new therapies for Chagas Disease. Here, we apply mass spectrometry-based proteomics to the large-scale identification of PTMs in T. cruzi histones. We provide the first comprehensive map of T. cruzi histone marks, including 9 newly described chemical modifications for trypanosomatids, and identify almost two hundred conserved and unique PTM sites in the tails and globular regions of all canonical and variant histones. The results presented in this paper provide substantial new evidence for a better understanding of epigenetic mechanisms in T. cruzi and, ultimately, serves as a basis for selection of candidates for the development of anti-parasitic epigenetic drugs.

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EXPERIMENTAL PROCEDURES Cell culture and histone extraction T. cruzi Dm28c

72

epimastigotes were cultured in liver infusion tryptose (LIT) medium

73

supplemented with 10% fetal bovine serum without agitation at 28ºC. Histone-enriched extracts were obtained as previously described 49, with some modifications. Briefly, 1 x 109 epimastigote cells were collected by centrifugation (5 minutes, 6000 g at 4°C). Cells were lysed by resuspending the obtained pellet in 1 ml of Buffer A (0.25 M Sucrose; 1mM EDTA; 3 mM CaCl2; 0.01 M Tris-HCl pH 7.4; 0.5% (v/v) Saponin; protease inhibitor cocktail (Complete mini EDTA free, Roche) and centrifuged for 10 minutes at 5000 g, 4°C. The pellet containing cell debris and nuclei was washed once in 1 ml of Buffer B (0.25 M Sucrose; 1 mM EDTA; 3 mM CaCl2; 0.01 M Tris-HCl pH 7.4; protease inhibitor cocktail (Complete mini EDTA free, Roche). Soluble nuclear proteins were extracted by treating the obtained pellet in 1 ml of Buffer C (1% (v/v) Triton X-100; 0.15 M NaCl; 0.025 M EDTA; 0.01 M Tris-HCl pH 8; protease inhibitor cocktail (Complete mini EDTA free, Roche). Subsequently, the material was centrifuged for 20 minutes at 10000 g, 4°C, washed 3 times in 100 mM Tris-HCl pH 8, resuspended in 1 ml of 0.4 N HCl and incubated on a rotator for 2 h at 4°C. Acid soluble proteins were recovered in the supernatant after sample centrifugation for 15 minutes at 10000 g, 4°C. The supernatant was transferred to a clean tube; acetone (8 x the initial volume) was added and the sample was incubated overnight at -20°C. The sample was centrifuged for 15 minutes at 3100 g, 4°C. Acetone was removed carefully and the pellet was washed 3 times with 1 ml of acetone. The protein pellet was carefully dried at 37°C and then resuspended in 50 µl of water. Preparation of Histone-enriched extracts for LC-MS/MS 8


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Histone-enriched extracts were digested in-solution and all procedures were carried out at room temperature. For tryptic digestion, enriched histones were denatured in 6M urea/2M thiourea, reduced with 1 mM dithiothreitol, alkylated with 5.5 mM iodoacetamide in the dark, diluted with 4 volumes of 25 mM ammonium bicarbonate and incubated with trypsin (Sequencing Grade Modified, Promega) at a protease/protein ratio of 1/50 for 2 h or 16 h. For Arg-C (Sequencing Grade, Promega) digestion, enriched histones were diluted in Arg-C digestion buffer (150 mM ammonium bicarbonate, 15 mM CaCl2, 10 mM DTT) and incubated with Arg-C at a protease/protein ratio of 1/20 for 2 h or 16 h. Peptide digests were desalted using RP-C18 StageTip columns 74,75 prior to LC/MS/MS. Preparation of isolated histones for GeLC-MS/MS Histone-enriched extracts were separated by 15% SDS-PAGE and stained with coomassie blue. Histone bands were excised, destained and in-gel digested for 16 h at 37ºC as previously described 76, using different enzymes. For in-gel tryptic digestion, histone bands were submitted to in vitro propionylation

76,77

prior to digestion and incubated with trypsin (Sequencing

modified, Promega) at a concentration of 0.0125 µg/µL. For Arg-C digestion, gel slices containing histone bands were equilibrated in Arg-C digestion buffer (150 mM ammonium bicarbonate, 15 mM CaCl2, 10 mM DTT) and incubated with Arg-C at a concentration of 0.25 µg/µL. For Asp-N digestion, gel slices containing histone bands were equilibrated in 25 mM ammonium bicarbonate and incubated with Asp-N (Sequencing Grade, Promega) at a concentration of 0.25 µg/µL. After digestion, peptides were extracted from the gel as previously described 78 and desalted using RP-C18 StageTip columns 74,75 prior to LC-MS/MS. NanoLC-ESIMS/MS analysis 9



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Peptide mixtures were separated by online reversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC) and analyzed by electrospray tandem mass spectrometry (ESI MS/MS). The experiments were performed at the mass spectrometry facility RPT02H PDTIS, (Carlos Chagas Institute - Fiocruz Parana), with an EASY nLC 1000 (ThermoFisher Scientific) system connected to an LTQ Orbitrap XL ETD (ThermoFisher Scientific) mass spectrometer equipped with a nanoelectrospray ion source (Phoenix S&T). Chromatographic separation of the peptides took place in a one-column set-up, with a 30-cm analytical column (75 µm inner diameter) in-house packed with reversed-phase C18 resin (ReproSil-Pur C18-AQ1.9 µm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany), kept at a constant temperature of 60ºC. Solvent A was 0.1% formic acid, 5% DMSO in water, and solvent B was 5% DMSO, 0.1% formic acid in acetonitrile. Samples were injected onto the column and subsequently eluted with a flow rate of 250 nL/min and peptide mixtures were separated with a linear gradient from 5% to 40% acetonitrilein120 min or 240 min. The mass spectrometer was operated in data-dependent mode to automatically switch between MS and MS/MS (MS2) acquisition, using single (CID) or multiple complimentary fragmentation methods (CID/HCD/ETD). Survey full scan MS spectra (at 300 – 1600 m/z range) were acquired in the Orbitrap analyzer with resolution R=60,000 at m/z 400 (after accumulation to a target value of 1,000,000 in the linear ion trap), with preview scan enabled. For CID-only fragmentation, the 10 most intense ions were sequentially isolated and fragmented in the linear ion trap, using CID at a target value of 10,000. For the multiple fragmentation method, the three most intense ions were isolated and sequentially fragmented in the ion trap (CID/ETD) or collision chamber (HCD). Singly-charged precursor ions were not selected for fragmentation. Former target ions selected for MS/MS were dynamically excluded for 30 seconds. Total cycle time was approximately three seconds. The general mass 10


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spectrometric conditions were: spray voltage, 2.4 kV; no sheath and auxiliary gas flow; ion transfer tube temperature, 100ºC; collision gas pressure, 1.3 mTorr; normalized collision energy using wide-band activation mode 35% for MS2. The ion selection threshold was 250 counts for MS2. An activation q = 0.25 and activation time of 30 ms was applied in MS2 acquisitions. The “lock mass” option 79, using DMSO peaks

80

, was enabled in all full scans to improve the mass

accuracy of precursor ions. Data analysis Peaklist picking, protein identification, and validation were performed by de novo assisted database search using the complete proteomic computational platform Peaks Studio (version 7.5, Bioinformatics Solutions Inc.)

81,82

. The multi-step sequential analysis started with Peptide De

Novo (de novo sequencing of fragment spectra); followed by Peaks DB (peptide sequence match of the high quality de novo tags, considering frequent modifications for the database search) and then by Peaks PTM (peptide sequence match of the remaining high quality de novo only peptide tags, using a broader set of modifications for the database search). Proteins were searched against a T. cruzi database containing 19,615 protein sequences from the

CL

Brener

strain,

downloaded

on

May

30,

2015

from

(http://www.tritrypdb.org/), plus the sequences of histone H1 from T. cruzi Y

TriTrypDB, 47

and Dm28c

strain (generated by our group, manuscript in preparation). Efficiency of the search was tested against two decoy databases which were prepared by reversing the sequence of each entry and appending them to the forward sequences. These databases were complemented with frequently observed contaminants (e.g. porcine trypsin, Achromobacter lyticus lysyl endopeptidase and human keratins) and their respective reversed sequences. In all PEAKS searches (Peptide De Novo, PEAKS DB, and PEAKS PTM), the precursor mass tolerance was set to 10 ppm and the 11



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fragment ion mass tolerance was set to 0.5 Da (ion trap spectra) or 20 ppm (Orbitrap spectra). The searches required full enzymatic specificity and a minimum of five amino acids for peptide length, allowing for four missed cleavages. For Peptide De Novo and Peaks DB searches, frequent trypanosome histone modifications were set as variable modifications: acetylation [K] (+42.011 Da); monomethylation [K/R] (+14.016 Da); dimethylation [K/R] (+28.031 Da) and trimethylation [K] (+42.047Da). For Peaks PTM search, in addition to the ones used in the previous step, less frequent histone PTMs were included as variable modifications: phosphorylation [S/T/Y] (+79.966 Da); acetylation [S/T] (+42.011 Da); monomethylation [K/R] (+14.016 Da); crotonylation [K] (+68.026 Da); 2-Hydroxyisobutyrylation [K] (+86.037 Da); succinylation [K] (+100.016 Da); formylation [K] (+27.995 Da); ubiquitination [K] (+114.043 Da); citrullination [R] (+0.984 Da); hydroxylation [Y] (+15.995 Da); methylation [protein Nterm] (+14.016 Da) and dimethylation [protein N-term] (+28.031).Peaks PTM searches also included deamidation [N/Q] (+0.984 Da), to help distinguish from citrullination. Oxidation [M] (+15.995 Da) and N-terminal protein acetylation (+42.011 Da), were also included in all searches. Whenever applicable, to account for modifications introduced by experimental manipulation, carbamidomethylation [C] (57.022 Da) was set as a fixed modification; propionylation [K] (56.026Da); monomethylpropionylation [K] (70.042 Da); propionylation [Protein N-term] (56.026 Da) and propionylation [peptide N-term] (56.026 Da) were included as variable modifications. For all searches, the false discovery rate (FDR) was below 1%, as estimated by decoy-fusion method. When transforming peptide identifications into protein identifications, similar protein sequences (e.g. isoforms) present in the database that could not be distinguished by the

12


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experimentally detected peptides were grouped and referred to as protein groups. PTM sites with an Ascore ≥ 20 were automatically validated.

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RESULTS AND DISCUSSION Proteomic profiling of T. cruzi histones Histone-enriched extracts were obtained from log-phase T. cruzi epimastigotes by acid extraction using hydrochloric acid. Two parallel MS-based proteomic workflows were followed for extensive proteomic profiling of T. cruzi histones: LC-MS/MS and GeLC-MS/MS (Figure 1, Experimental Procedures). In both strategies, as a means to increase sequence coverage and sensitivity, a set of distinct proteases, optimized digestion protocols, and complimentary MS/MS fragmentation methods were used.

Figure 1. Workflow for proteomic profiling of T. cruzi histone PTMs. T. cruzi epimastigotes were cultured in vitro until they reached log-phase. After cell lysis, histone-enriched extracts were obtained by acid extraction using hydrochloric acid and samples were processed by two parallel MS-based proteomic strategies. In the LC-MS/MS approach, histone-enriched extracts were digested in-solution with either trypsin (2h and 16h) or Arg-C (2h and 16h). For GeLC14


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MS/MS analysis, histone-enriched extracts were separated by SDS-PAGE and individual histone bands were digested in-gel for 16h with either trypsin (following in vitro propionylation), Arg-C or Asp-N. Peptides generated from both strategies were desalted using StageTips

74,75

, and

analyzed by nanoscale ultra-performance liquid chromatography (nanoUPLC) coupled to highaccuracy tandem mass spectrometry (MS/MS) on a hybrid linear ion trap–Fourier Transform Orbitrap instrument (LTQ-Orbitrap XL ETD). Three complimentary and high-resolution MS/MS fragmentation techniques (Collision-induced dissociation, CID; Higher-energy collisional dissociation, HCD, and Electron transfer dissociation, ETD) were sequentially performed on each precursor ion. Proteins and post-translational modification sites were identified by de novo assisted database search.

All proteomic approaches provided good identification of the T. cruzi histones, although with different degrees of sequence coverage. Best results were obtained in GeLC-MS/MS experiments, using samples that have been previously submitted to propionylation of (unmodified or mono-methylated) lysines followed by digestion with trypsin

76,77

. This strategy

led to both good sequence coverage of T. cruzi histones and, more importantly, to the identification of the histone tails, which were missed by other experimental setups. Such good performance can be attributed to different reasons. First, lysine propionylation blocks cleavage by trypsin, preventing overcutting of the lysine-rich histone N-terminal tails and, as a consequence, generating peptides with suitable size for identification by LC-MS/MS. More than that, propionylation neutralizes the positive charges of lysine, reducing the total charge state of the peptides and, in turn, increasing LC retention and identification by MS/MS, making this strategy more efficient than short trypsin and Arg-C digestions. Moreover, as we started up with histone-enriched extracts, separation by SDS-PAGE prior to digestion and LC-MS/MS greatly

15



decreased sample complexity, allowing the identification of low abundance peptides and PTM sites. A total of 6,733 peptide features mapping to T. cruzi histones were identified (Table S1). The experimental design adopted in this study led to very complimentary data and deep sequence coverage. Most peptides, including unmodified and differently modified versions of a given sequence, were detected in multiple experiments, adding extra confidence to the results. With our combined proteomic strategy all T. cruzi canonical (H2A, H2B, H3 and H4), variant (H2A.Z, H2B.V, and H3.V) and linker (H1) histones were identified with high sequence coverage (Table 1 and Fig S1). However, differently from what was observed for the other histones, initial searches using the CL Brener reference genome showed very poor sequence coverage for histone H1. To overcome this problem, we added the sequence of H1 from the T. cruzi Y 47 and Dm28c strains (generated in our laboratory) to the database. This fact, together with the comparison with H1 from the T. cruzi Y and Dm28c strains (Fig S2), suggested misannotation of the H1 genes particularly of the start and stop codons - in the CL Brener reference genome.

Table 1 - Sequence coverage of T. cruzi canonical and variant histones.

Core

Canonical

Histone

Variant

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

Linker

H2A H2B H3 H4 H2A.Z H2B.V H3.V H1

Sequence length 134 111 132 99 178 143 150 79

No. amino acids covered 129 105 127 91 154 122 124 79

Sequence coverage (%) 96,3 94,6 96,2 91,9 86,5 85,3 82,7 100,0

16


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T. cruzi histones lose the initial methionine during protein processing, as described for humans and a variety of other eukaryotes. In our data, this was confirmed by the identification of peptides mapping to the N-terminal of H2A, H2B, H4, H1, H2A.Z and H2B.V. For histones H3 and H3.V, the first two N-terminal amino acids were not detected and the removal of the initial methionine could not be determined. T. cruzi histones are heavily modified and support a combinatorial histone code Having identified all T. cruzi histones with high sequence coverage, we next investigated their post-translational modifications by searching for the chemical modifications currently known for humans and other eukaryotes. All T. cruzi canonical and variant histones were found to be modified at many different sites, showing a repertoire of 13 distinct chemical modifications. All types of modifications previously reported (acetylation, monomethylation, dimethylation, trimethylation and phosphorylation) were detected in addition to eight new post-translational modifications types identified for the first time in trypanosomatid histones. The new types include citrullination, ubiquitination, hydroxylation and multiple forms of the recently discovered short-chain lysine acylations (glutarylation, crotonylation, 2-hydroxyisobutyrylation, malonylation, and succinylation). The alternative lysine acylation marks glutarylation, crotonylation, 2-hydroxyisobutyrylation, malonylation, and succinylation are predominantly located in globular regions of T. cruzi histones rarely occuring at N-terminal tails, where acetylation dominates the scenario. Moreover, the majority of these acylation marks share the same modification sites, which are usually also acetylated and, in some cases, modified by possible repressive marks, such as methylation and ubiquitination, a phenomenon also observed in humans 83. Therefore, several questions arise: Are 18


Page 19 of 49

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alternative lysine acylations and acetylation playing competing or redundant roles? How is the choice of a particular acylation type made? How is the interplay between these diverse lysine acylations with acetylation? How is their cross-talk with other modifications influencing epigenetic control mechanisms? Several sites of lysine formylation were also identified, which were present in histones at a frequency much higher than in other proteins identified, suggesting a role as histone marks. However, formylation could be caused by the presence of formic acid in the LC mobile phases, and further investigation to confirm its biological origin is required. Therefore, we report the formylation sites in supplementary files but have chosen not to include them in the compiled map of T. cruzi PTM marks of Figures 2 and 4 neither in discussions throughout this article. Lastly, lysine propionylation was only detected when proteins were incubated with propionic anhydride during sample preparation and, therefore, this modification was not considered in the present work. Annotated spectra supporting the identification of all isobaric modifications identified are provided for the readers in the Supporting Information (Supplemental Results Representative spectra for all the distinct modification types identified in T. cruzi (for isobaric modifications, all spectra are included)). We identified, unambiguously, a total of 119 modification sites distributed across all of T. cruzi canonical, variant and linker histones (Figure 2), 114 of which are novel. Among the modified residues, several were found to be substrates of different modifications, adding up to a total of 176 PTM marks, and expanding the number previously reported for T. cruzi by more than 3-fold. Since log-phase parasites (and not synchronized cells at different time points) were used in our analysis, some less abundant or transient modification events might have been lost, so

we

believe

the

numbers

might

19


be

even

higher.


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Page 20 of 49

Figure 2. Post-translational modifications identified on T. cruzi canonical and variant histones. A compilation of PTM sites identified in the present work is shown. Light gray rectangles indicate the histone-fold within the globular domain. Numbers below the sequences indicate the amino acid position on mature T. cruzi histones (after removal of the initial methionine). 20


Page 21 of 49

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1

Taking into account the types and number of PTMs identified in T. cruzi histones, chromatin

2

remodeling in this organism should follow a dynamic regulation similar to other eukaryotes.

3

Depending on the type of cell signal, a set of epigenetic “writers” transfer chemical groups to

4

histones whereas a set of epigenetic “erasers” remove chemical groups in such a manner that the

5

proper histone PTM landscape is put in place for the epigenetic “readers” to play their roles. The

6

occurrence of distinct PTM patterns on individual histone molecules (Fig 3 and Tables S2 to S9)

7

indicates that T. cruzi is able to regulate histone modifications in a combinatorial fashion to

8

establish distinct chromatin states, supporting the existence of a sophisticated histone code in

9

trypanosomatids. At this stage, however, further research is required to pinpoint recurring

10

combinatorial histone PTM patterns, to identify the writers and readers related to such patterns

11

and to determine their functional relevance in the biology and pathogenesis of these parasites.

12

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Page 22 of 49

1 2

Figure 3. Example of PTM patterns identified for H3 and H2A.Z. Model sequences are

3

shown in light red, identified peptides are shown in blue and PTMs are indicated in the circles.

4 5 6


Page 23 of 49

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1

T. cruzi histones have distinct modification profiles and present conserved and unique PTM

2

marks

3

A comprehensive map of the post-translational modifications currently known for T. cruzi

4

histones is shown in Figure 4. Most of the PTM marks previously described for T. cruzi were

5

confirmed, although some were not identified in the present study. These differences could

6

reflect the analysis of different life forms and/or T. cruzi strains in the different studies, but could

7

also be due to technical reasons, such as the use of different proteomic protocols and low

8

stoichiometry of the modified residue, among others.

9

Although unusually divergent from other eukaryotes, especially on their N-terminal tails,

10

histones are highly conserved among the trypanosomatid parasites (Figure S3). Therefore, the

11

occurrence of similar histone modifications across trypanosomatids is to be expected. Not

12

surprisingly, our data show that T. cruzi shares a high number of PTM marks previously reported

13

for T. brucei. Despite the overall sequence divergence along evolution, some of the histone

14

amino acids that serve as a substrate for modifications in T. cruzi histones are conserved in most

15

organisms. In this context, a number of T. cruzi PTM marks were found to be homologous to

16

histone PTMs of humans and other eukaryotes, indicating that the biochemical epigenetic

17

mechanisms are similar in all eukaryotes.

18 19



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Page 24 of 49

1 2

Figure 4. Summary of PTM marks described for T. cruzi histones. A compilation of PTM

3

sites identified in the present work and previously reported in the literature (a: da Cunha et al., 24 ACS Paragon Plus Environment

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1

2005 47; b: da Cunha et al., 2006 18; c: Marchini et al., 2011 50 and d: de Jesus et al., 2016 51) is

2

shown.

3 4

The PTM profiles of each histone, with emphasis on trypanosomatid data, are discussed below:

5

H2A

6

Histone H2A is mostly acetylated, as previously demonstrated

18

, but is also modified by

7

methylation (mono-, di- and tri-), citrullination, crotonylation, and 2-hydroxyisobutyrylation. In

8

total, we identified 20 modification sites and 23 PTM marks that occur extensively on, but are

9

not limited to its flexible tails, 19 of which are novel for T. cruzi.

10

The initial methionine is removed during protein processing and the subsequent alanine is

11

modified by protein N-terminal methylation, a trypanosome-specific modification also observed

12

in T. brucei 19. N-terminal acetylation of A1, reported for T. brucei, was not detected. Also K4 is

13

monomethylated in T. cruzi epimastigotes, as opposed to the acetylation found in T. brucei

14

bloodstream trypomastigotes 19,20.

15

One striking aspect of the T. cruzi H2A PTM profile is the presence of a hyperacetylated C-

16

terminal tail, a feature also observed in T. brucei 29 that seems to be specific of trypanosomatids.

17

Although

18

posttranscriptional, hyperacetylation has been postulated as a mechanism to establish long

19

regions of euchromatin and to regulate particular gene clusters under special circumstances, such

20

as during differentiation or in response to environmental stimuli

21

acetylated residues were detected in different number and combinations on single or multiply

22

modified peptides containing up to five simultaneous acetylations. K123 was found to be

gene

expression

control

in

trypanosomatids

is

44

believed

to

be

mainly

. In our data, the C-terminal



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Page 26 of 49

1

acetylated or monomethylated. Also, K127 and R129 methylation have been described to be

2

predominant in trypomastigotes 51, evidencing that the modification state of the C-terminal tail of

3

individual T. cruzi H2A molecules is being regulated.

4

Besides the C-terminal tail acetylation sites, H2A also presents four other sites of acetylation,

5

four of citrullination and one of crotonylation, adding to the set of histone marks that have been

6

previously associated with active chromatin states. Interestingly, lysine 21 can bear acetylation

7

and 2-hydroxybutyrylation, known as active histone marks, as well as methylation, which can be

8

associated with both transcription activation and repression 84.

9

H2A.Z

10

The variant H2A.Z is modified by acetylation and phosphorylation, accounting for a total of 19

11

PTM sites and 20 PTM marks localized exclusively at its N- or C-terminal tails, among which 14

12

are novel for trypanosomatids. It loses the initial methionine and presents acetylation of the N-

13

terminal S1, in contrast to the methylation of A1 observed for H2A. Similar to its canonical

14

counterpart, H2A.Z is mainly acetylated, presenting two hyperacetylated regions: one in the N-

15

terminal tail and another at the C-terminal tail. For both hyperacetylated regions, the multiple

16

acetylation sites were found to occur simultaneously in the same peptide, and unmodified

17

versions were rarely detected (Table S7), suggesting that the state of hyperacetylation of H2A.Z

18

is predominant.

19

The N-terminal tail of H2A.Z is also marked by a string of six phosphorylated residues,

20

described by our group in a previous phosphoproteomics study 50. Histone phosphorylation is a

21

transient modification known to influence a plethora of nuclear processes by recruiting or

22

changing the affinity of histone readers and writers in eukaryotes. Particularly, H2A

23

phosphorylation is involved in centromere organization and chromatin condensation during 26 ACS Paragon Plus Environment

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1

mitosis and phosphorylated H2A.X participates in the recognition of DNA double-strand breaks

2

during DNA damage response

3

was not performed, this particular region of H2A.Z was only detected as unmodified, suggesting

4

low stoichiometry and a regulatory role of phosphorylation. In T. cruzi, the occurrence of

5

hyperacetylation and hyperphosphorylation regions can suggest the dual role of H2A.Z in gene

6

expression control and DNA repair.

7

H2B

8 9

85,86

. In the present work, where enrichment of phosphopeptides

Histone H2B is modified by methylation (mono, di- tri-), phosphorylation, acetylation, hydroxylation,

citrullination,

crotonylation,

ubiquitination,

glutarylation,

and

2-

10

hydroxyisobutyrylation. We identified a total of 17 modified residues and 31 PTM marks,

11

localized mostly at its globular region, all of which are novel for T. cruzi.

12

Different to the observed for other T. cruzi histones, few modified residues were identified in

13

its N-terminal tail. After removal of the initial methionine, the first alanine is modified by

14

methylation, as observed for T. brucei 20,44, and threonine two is phosphorylated. Methylation of

15

R10 and K11, as well as K4 trimethylation and S63 phosphorylation, previously reported

16

were not detected in this work. The PTM marks A1me2, K4ac, and K12ac described for T.

17

brucei

18

conserved. The majority of H2B PTM marks correspond to different degrees of methylation, as

19

previously suggested 18, but their number is closely followed by the amount of acetylation sites.

20

Interestingly, several residues were found to be substrate of diverse PTM types, which can cause

21

similar or opposite effects in chromatin structure. The most striking example is K104 which can

22

be modified by six different activating and repressive modification types at different times

19,20

51

,

, were not observed in T. cruzi H2B, despite the fact that the modified residues were



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Page 28 of 49

1

(Table S3) and, alone, account for almost twenty percent of the H2B PTM marks, appearing to

2

be an important site for combinatorial PTM regulation.

3

H2B.V

4

The variant H2B.V was found to be modified by acetylation and hydroxylation, comprising 6

5

modification sites localized almost exclusively at its N-terminal tail, all of which are novel for

6

trypanosomatids. It has the initial methionine removed, but no modification was detected on its

7

N-terminal proline. Most of H2B.V PTM marks correspond to acetylated lysines, which are

8

concentrated in a string located at its N-terminal tail and could be related to the establishment of

9

active chromatin states. For instance, H2B.V has been found to be associated with H2A.Z at 87

and to nucleosomes containing H4K10ac in T. brucei 53, as well as to

10

repetitive DNA regions

11

trimethylation of H3K4 and H3K76 (corresponding to K79 in humans), all histone marks

12

associated with transcriptional activation

13

presents a single tyrosine hydroxylation at the beginning of its globular region. K32 and K34

14

have been reported to be acetylated and dimethylated, respectively

15

acetylation sites and the different degrees of hyperacetylation of the N-terminal tail of H2B.V

16

(Table S8) suggest distinct regulation patterns and contrasts to the high methylation profile and

17

the poorly modified N-terminal tail of H2B.

18

H3

19

88

. Additionally to the several acetylations, H2B.V

51

. The predominance of

Histone H3 was detected as highly modified by methylation (mono-, di- and tri-), acetylation,

20

glutarylation,

2-hydroxyisobutyrylation,

citrullination,

crotonylation,

succinylation,

21

ubiquitination and hydroxylation, comprising a total of 25 modified residues and 39 PTM marks,

22

localized at both its N-terminal tail and globular region, 33 of which are novel for T. cruzi. 28 ACS Paragon Plus Environment

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1

Removal of the initial methionine, K19/K23 acetylation, K23 dimethylation/trimethylation and

2

T29 acetylation could not be confirmed. Also, S1ac, K4me, K23ac, and K32me3, described for

3

T. brucei

4

trypanosomatids. T. cruzi H3 is mostly methylated, as previously suggested 18, with the majority

5

of (mono-, di- and tri-) methylation sites being localized at its globular region. Many

6

acetylations, which dominate the N-terminal region of H3, are also observed. Several sites of

7

citrullination and the alternative lysine acylations (glutarylation, 2-hydroxyisobutyrylation,

8

crotonylation, succinylation) are present across the entire sequence of H3.

9

20,44

, were not detected, despite conservation of the modified residues between the two

Noteworthy, H3K76, a target of DOT1 methyltransferases in T. brucei

42

, was found to be

10

mono-, di- or trimethylated in the majority of the identified peptides of T.cruzi. As shown by de

11

Jesus et al.

12

methylation is more abundant in replicative forms of T. cruzi; K76 monomethylation is only

13

detected during mitosis while dimethylation is present during both mitosis and cytokinesis and

14

K76me3 is expressed in all contexts analyzed. This is similar to what has been reported for T.

15

brucei

16

H3K76me2 is a key regulator of cell cycle progression 89.

20,44

51

, these modifications are being differentially regulated: H3K76 mono- and di-

, for which the degree of H3K76me3 is present across the entire cell cycle while

17

Some lysine and arginine residues of H3 are modified by a variety of modification types. For

18

example, K61 is a substrate of several marks (acetylation, crotonylation, succinylation,

19

glutarylation and ubiquitination) which were found alone or in combination (Table S4), not only

20

supporting the existence of a mechanism of combinatorial regulation and cross-talk between

21

PTM marks, but also suggesting that a single residue may be of key importance for these

22

processes. Importantly, histone H3 presents four sites of glutamine monomethylation, a

23

modification type not observed in the other T. cruzi histones.



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1

Page 30 of 49

H3.V

2

The variant H3.V is modified by acetylation and methylation (mono-, di- and tri-), totalizing 6

3

modified residues and 9 PTM marks identified, localized predominantly at its N-terminal tail, 6

4

of which are novel for trypanosomatids. The PTM marks of its N-terminal tail include three

5

methylation and two acetylation sites, and the peptides mapping to this region were found to bear

6

different combinations of one or both modifications (Table S9). The only PTM site localized at

7

the histone globular domain is K94, found bearing one, two or three methyl groups in most of the

8

peptides identified, confirming what was previously described 51 and suggesting that this residue

9

is equivalent to H3K76. For instance, K94 trimethylation and perhaps dimethylation seem to be

10

enriched in trypomastigotes 51, demonstrating its regulation during differentiation of the parasite.

11

H4

12

Histone H4 was found to be modified by acetylation, monomethylation, dimethylation,

13

phosphorylation,

14

malonylation and hydroxylation. In total, we describe 28 modified residues and 48 PTM marks,

15

45 of which are novel for T. cruzi. Both the N-terminal tail and the globular region are heavily

16

modified.

17

monomethylation or both, and peptides containing multiple modification sites in different

18

combinations indicate that a combinatorial mechanism of regulation is in place (Table S5). The

19

initial methionine is absent and the N-terminal H4A1 can be either monomethylated

20

characteristic also described for T. brucei

21

modified by methylation and acetylation, respectively

22

HAT-3, is also predominant and occurs after chromatin assembly

The

citrullination,

N-terminal

tail

glutarylation,

contains

44

crotonylation,

several

residues

, or acetylated. 18

28

2-hydroxyisobutyrylation,

substrate

of

acetylation,

18

, a

. Also, K18 and K57 can be

. In T. brucei H4K4ac, catalyzed by 43

. In T. cruzi, H4K10ac and


Page 31 of 49

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1

H4K14ac are required for chromatin assembly/remodeling during transcription and replication90

2

and increased in trypomastigotes 51. Similarly, in T. brucei, H4K10ac is concentrated in probable

3

sites of transcription initiation

4

levels of H4K10ac/H4K14ac and a decrease of H4K4ac are observed, indicating their association

5

with DNA damage response

6

sites bearing diverse modification types. Most of them are concentrated in a string from R65 to

7

K77, which comprises almost half of H4 PTM marks and, as observed for the N-terminal tail, are

8

distributed in differently modified peptides, consistent with epigenetic regulation.

9

H1

10

49

54,56

. Moreover, after exposure to γ-radiation an increase in the

. The globular region of H4 shows a multitude of modification

T. cruzi histone H1 is modified by methylation, acetylation and phosphorylation

46,47,51

. The

11

N-terminal serine is acetylated, after loss of the initial methionine, and followed by

12

phosphorylation on serine 11. 46,47 In T. cruzi, serine 11, which is located within a typical cyclin-

13

dependent kinase site, is phosphorylated in non-replicating infective forms of the parasite

14

Also, the level of H1S11ph is regulated across the cell cycle of T. cruzi 47, occurring when cells

15

progress from the S to M phase, reinforcing its cyclin-dependent nature. Phosphorylation of S11

16

is also believed to decrease the association of H1 with chromatin and, therefore, to influence

17

chromatin structure. Additionally, several other sites of acetylation, methylation and

18

phosphorylation have been recently described

19

trypomastigotes. In the present work, the presence of S1ac was confirmed (Table S6), but the

20

other PTM marks were not detected. Considering the transient nature of phosphorylation, the fact

21

that we used a different strain and analyzed only asynchronous cultures of epimastigotes, the

22

absence of these sites in our comprehensive mapping of T. cruzi histone PTMs could be

51

46

.

, including some that seem predominant in



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Page 32 of 49

1

explained by their low abundance in the cell population analyzed, and argues in favor of its strain

2

and/or stage-specific and time-dependent expression in the parasite.

3

T. cruzi expresses histone isoforms bearing unique PTM marks

4

In the T. cruzi genome, each histone is represented by multiple genes. For a given histone,

5

some nucleotide sequences are identical, some differ from each other by a few amino acids, and

6

others seem to represent truncated versions of the main genes. In our dataset, we identified

7

peptides matching to distinct gene products, demonstrating that T. cruzi expresses sequence

8

divergent histone isoforms. Among the multiple isoforms detected for each histone, the one most

9

frequent and/or presenting the highest sequence coverage (excluding truncated genes) was

10

chosen as the model sequence to be used throughout the article (Fig S1).

11

Alternative peptides bearing substitutions provide experimental evidence for the expression of

12

protein isoforms of histones H2A, H2B and H4 (Figure 5 and Table S10). Interestingly, some of

13

these alternative peptides were also found to bear unique PTM marks for histone H2A

14

(K121me3, K101me2, K120hib) and H4 (Y49ph, K42me2, K43me, and K58me2) isoforms,

15

which were not detected for their model counterparts.


Page 33 of 49

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1 2

Figure 5. Identification of histone isoforms. Model sequences are shown in light red,

3

alternative identified peptides are shown in blue and PTMs are indicated in the circles above

4

modified residues.

5 6

In humans and other eukaryotes, clusters of histone genes also encode isoforms, which can

7

differ in their primary sequence by only a few residues. Even though discrete, these differences

8

can be very significant biologically, since even small changes in amino acid composition have

9

been shown to disrupt nucleosomal organization and/or to alter the functional capabilities of

10

histones in different scenarios, including human disease 91–94. Therefore, the presence of slightly

11

divergent histone isoforms bearing unique PTM marks has the potential to add an extra layer of

12

regulation and to significantly expand the complexity of chromatin structure and epigenetic

13

control in T. cruzi, but their biological relevance needs yet to be determined by further research.

14 15 16 33 ACS Paragon Plus Environment


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1

Page 34 of 49

CONCLUDING REMARKS

2

Increasing evidence has associated a "Trypanosomatid Histone Code" to the regulation of the

3

biology and pathogenesis of these parasites. For example, histone PTMs have been shown to

4

influence important processes such as transcription initiation, replication, and DNA repair,

5

strongly suggesting that epigenetic mechanisms seem to be just as important for trypanosomatids

6

as they are for other eukaryotes. For T. cruzi, functional epigenetic studies have been hindered

7

for years by the lack of basic information, particularly about the occurrence and site-specific

8

localization of histone post-translational modifications, which represent an important starting

9

point in understanding epigenetic control. With our large-scale proteomic analysis, we now fill

10

this gap, by answering the long-asked question: “To which extent, and by which post-

11

translational modifications, are Trypanosoma cruzi histones modified?” The comprehensive map

12

of T. cruzi histone marks, presented herein, shows that post-translational modifications are

13

abundant and diverse across the flexible tails and globular region of all canonical and variant

14

histones. Furthermore, our data show that epigenetic control via histone modifications has the

15

potential to be as complex and sophisticated in this parasite as it is in other eukaryotes, including

16

humans. Thus, this work can greatly contribute to improving the knowledge about epigenetic

17

control in T. cruzi, paving the way for further functional studies that can help to understand key

18

regulatory epigenetic mechanisms on this organism, at the molecular level.

19

Despite the sequence divergence of trypanosomatid histones as compared to most eukaryotes,

20

some of the found PTM marks correlate to those described for humans and other model

21

organisms, suggesting that similar regulatory mechanisms may be in place. On the other hand,

22

others seem to be unique to T. cruzi or to trypanosomatids as a group and could be partially

23

responsible for the unusual and singular mechanisms of nuclear organization, transcription


Page 35 of 49

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1

control, replication and DNA repair existing in these parasites, as well as represent important

2

targets for the development of new anti-parasitic epigenetic drugs.

3

To the best of our knowledge, as of this writing our dataset represents, by far, the most

4

comprehensive map of histone modifications provided for any member of the Trypanosomatidae

5

family. The original results presented herein are of general interest for the parasitology field and,

6

to make it accessible to the scientific community and enable data mining for further studies, all

7

the data will be freely available online through TriTrypDB (http://tritrypdb.org).

8 9

ASSOCIATED CONTENT

10

Supporting Information

11

Figure S1. Sequence coverage for T. cruzi canonical and variant histones.

12

Figure S2. Sequence analysis for T. cruzi linker histone.

13

Figure S3. Sequence alignment between histones of trypanosomatids and other eukaryotes.

14

Table S1. Combined list of identified histone peptides from all experiments.

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Table S2. Differentially modified peptides identified for T. cruzi H2A.

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Table S3. Differentially modified peptides identified for T.cruzi H2B.

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Table S4. Differentially modified peptides identified for T.cruzi H3.

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19


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Table S7. Differentially modified peptides identified for T.cruzi H2A.Z.

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Table S8. Differentially modified peptides identified for T. cruzi H2B.V.

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Table S9. Differentially modified peptides identified for T. cruzi H3.V.

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Table S10. Differentially modified peptides identified for T. cruzi histone isoforms.

24 25

Supplemental Results - Representative spectra for all the distinct modification types identified in T. cruzi (for isobaric modifications all spectra are included).

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This material is available free of charge on the ACS Publications website at http://pubs.acs.org.



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1

AUTHOR INFORMATION

2

Corresponding Author

3

*[email protected]; [email protected]. Phone: 55 41 33133230 / Fax: 55 41 33163267.

4

Author Contributions

5

The manuscript was written through contributions of all authors. All authors have given approval

6

to the final version of the manuscript.

7

Funding Sources

8

This work was partially funded by the 7th Framework Programme of the European Commission,

9

project number EC-GA 602080 (A-PARADDISE - Anti-Parasitic Drug Discovery in

10

Epigenetics).

11

ACKNOWLEDGMENTS

12

We thank Dr. Christian M. Probst for providing the sequence of T. cruzi Dm28c H1. We also

13

thank Dr. Fabricio K. Marchini and Dr. Michel Batista for technical advice and operation of the

14

mass spectrometry facility. The authors declare not to have any financial/commercial conflicts of

15

interest related to this work.

16

ABBREVIATIONS

17

PTM, post-translational modification; HDAC, histone deacetylase; Me, methylation; Me2,

18

dimethylation; Me3, trimethylation; Ac, acetylation; Cr, crotonylation; Hib, 2-

19

hydroxyisobutyrylation; Ma, malonylation; Su, succinylation; Ub, ubiquitination; Cit,

20

citrullination; Ph, phosphorylation; OH, hydroxylation; Gl, glutarylation; Fo, formylation; ESI,

21

electrospray ionization; ETD, electron transfer dissociation; CID, collision‐induced

22

dissociation; and HCD, Higher-energy collisional dissociation; GeLC-MS/MS, in-gel digestion

23

followed by liquid chromatography-tandem mass spectrometry. 36 ACS Paragon Plus Environment

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GRAPHICAL ABSTRACT (for TOC only)

Image courtesy of Gisele F. A. Picchi and Wagner Nagib, Copyright 2016.



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Figure 1. Workflow for proteomic profiling of T. cruzi histone PTMs. T. cruzi epimastigotes were cultured in vitro until they reached log-phase. After cell lysis, histone-enriched extracts were obtained by acid extraction using hydrochloric acid and samples were processed by two parallel MS-based proteomic strategies. In the LC-MS/MS approach, histone-enriched extracts were digested in-solution with either trypsin (2h and 16h) or Arg-C (2h and 16h). For GeLC-MS/MS analysis, histone-enriched extracts were separated by SDS-PAGE and individual histone bands were digested in-gel for 16h with either trypsin (following in vitro propionylation), Arg-C or Asp-N. Peptides generated from both strategies were desalted using StageTips 74,75, and analyzed by nanoscale ultra-performance liquid chromatography (nanoUPLC) coupled to high-accuracy tandem mass spectrometry (MS/MS) on a hybrid linear ion trap–Fourier Transform Orbitrap instrument (LTQ-Orbitrap XL ETD). Three complimentary and high-resolution MS/MS fragmentation techniques (Collision-induced dissociation, CID; Higher-energy collisional dissociation, HCD, and Electron transfer dissociation, ETD) were sequentially performed on each precursor ion. Proteins and posttranslational modification sites were identified by de novo assisted database search. 82x96mm (150 x 150 DPI)


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Figure 2. Post-translational modifications identified on T. cruzi canonical and variant histones. A compilation of PTM sites identified in the present work is shown. Light gray rectangles indicate the histonefold within the globular domain. Numbers below the sequences indicate the amino acid position on mature T. cruzi histones (after removal of the initial methionine). 229x153mm (300 x 300 DPI)


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Figure 3. Example of PTM patterns identified for H3 and H2A.Z. Model sequences are shown in light red, identified peptides are shown in blue and PTMs are indicated in the circles. 75x154mm (300 x 300 DPI)



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Figure 4. Summary of PTM marks described for T. cruzi histones. A compilation of PTM sites identified in the present work and previously reported in the literature (a: da Cunha et al., 2005 47; b: da Cunha et al., 2006 18; c: Marchini et al., 2011 50 and d: de Jesus et al., 2016 51) is shown. 154x208mm (300 x 300 DPI)


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Figure 5. Identification of histone isoforms. Model sequences are shown in light red, alternative identified peptides are shown in blue and PTMs are indicated in the circles above modified residues. 82x94mm (300 x 300 DPI)



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Graphical Abstract (for TOC only). Image courtesy of Gisele F. A. Picchi and Wagner Nagib, Copyright 2016. 169x91mm (300 x 300 DPI)


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Post-translational Modifications of Trypanosoma ... - ACS Publications

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