Chaetocin—A histone methyltransferase inhibitor—Impairs proliferation, arrests cell cycle and induces nucleolar disassembly in Trypanosoma cruzi

Accepted Manuscript Title: Chaetocin—A histone methyltransferase inhibitor—Impairs proliferation, arrests cell cycle and induces nucleolar disassembly in Trypanosoma cruzi Authors: Aline Araujo Zuma, Jean de Oliveira Santos, Isabela Mendes, Wanderley de Souza, Carlos Renato Machado, e Maria Cristina M. Motta PII: DOI: Reference:

S0001-706X(16)30600-3 http://dx.doi.org/doi:10.1016/j.actatropica.2017.02.007 ACTROP 4200

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Acta Tropica

Received date: Revised date: Accepted date:

12-8-2016 12-1-2017 6-2-2017

Please cite this article as: Zuma, Aline Araujo, Santos, Jean de Oliveira, Mendes, Isabela, de Souza, Wanderley, Machado, Carlos Renato, Motta, e Maria Cristina M., Chaetocin—A histone methyltransferase inhibitor—Impairs proliferation, arrests cell cycle and induces nucleolar disassembly in Trypanosoma cruzi.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chaetocin, a histone methyltransferase inhibitor, impairs proliferation, arrests cell cycle and induces nucleolar disassembly in Trypanosoma cruzi

Aline Araujo Zumaa, Jean de Oliveira Santosa, Isabela Mendesb, Wanderley de Souzaa, Carlos Renato Machadob, e Maria Cristina M. Mottaa

a

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos

Chagas Filho, Universidade Federal do Rio de Janeiro, 21491-590, Rio de Janeiro, RJ, Brazil. b

Laboratório de Genética Bioquímica, Departamento de Bioquímica e Imunologia,

Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, MG, Brazil

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Graphical abstract

T. cruzi treated with 10 µM for 2 days presented heterochromatin unpacking, nucleolar fragmentation and lower RNAs levels when compared to control cells.

Highlights

1. T. cruzi epimastigote proliferation was inhibited after treatment with chaetocin. 2. Chaetocin promotes irreversible effects and decreases parasite viability. 3. Treated cells showed nucleolar disassembly as never seen before in trypanosomatids. 4. T. cruzi cell cycle was arrested even after using low concentrations of chaetocin. 5. RNA analyses indicate rRNA transcript impairment in treated protozoa.

Abstract

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The Trypanosomatidae family includes pathogenic species of medical and veterinary interest. Chagas disease is endemic in Latin America, and about 8 million people are infected worldwide. There is a need for more effective drugs for the acute, undetermined and chronic phases of the disease that, in addition, do not cause side effects, stimulating the search for identification of new drug targets, as well as new chemotherapeutic targets. Trypanosomatids contain characteristic structures, such as the nucleus that undergoes a closed mitosis without chromosome formation and variations of chromatin packing in the different protozoa developmental stages. The nuclear DNA is condensed by histones that suffer post-translational modifications, such as addition of methyl groups by histone methyltransferases (MHT) and addition of acetyl groups by acetyltransferases. These processes modulate gene expression and chromatin organization, which are crucial to transcription, replication, repair and recombination. In the present study, the effects of chaetocin, a HMT inhibitor, on T. cruzi epimastigote proliferation, viability, ultrastructure and cell cycle were investigated. Results indicate that chaetocin promoted irreversible inhibition of protozoa growth, evident unpacking of nuclear heterochromatin and intense nucleolus fragmentation, which is associated with parasite cell cycle arrest and RNA transcription blockage. Taken together, data obtained with chaetocin treatment stimulate the use of histone methyltransferase inhibitors against pathogenic trypanosomatids.

Key words: Trypanosoma cruzi, chaetocin, histone methyltransferase inhibitor, proliferation, cell cycle, ultrastructure.

1. Introduction

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Trypanosomatids are found in several countries of different continents and are classified as heteroxenic, which are pathogenic; or monoxenic, which only inhabit invertebrate hosts during their entire life cycle. Although pathogenic species are a minority in this family, they raise medical and veterinary interest since they are agents of lethal diseases in men, as well as in animals and plants of economic interest. In Latin America and in other developed countries, millions people are affected by Chagas disease, whose etiological agent is Trypanosoma cruzi; whereas in Africa, diseases like nagana (in animals) and sleeping sickness (in humans) are caused by Trypanosoma brucei. In underdeveloped countries, numerous cases of leishmaniasis are reported and attributed to several species of the Leishmania genus (de Souza 2002; Jensen and Englund 2012). Trypanosomatids contain typical eukaryotic organelles, such as the endoplasmic reticulum, the Golgi complex and the nucleus. The latter presents the heterochromatin close to the nuclear envelope and around the nucleolus, which is localized in the central region of the nucleus. The nucleolus presents characteristic domains, such as the fibrillary center and the granular region, but is less organized when compared to that observed in upper eukaryotes (Motta et al. 2003). Throughout the cell cycle, chromatin organization and distribution is more dispersed during the interphase and becomes more condensed in the beginning of the closed mitosis, when the nucleus is more elongated and the nucleolus disorganizes. At the end of mitosis, chromatin migrates to the polar region and the nucleus divides during cytokinesis, however condensed chromosomes are never observed (Ogbadoyi et al. 2000; Elias et al. 2002, de Souza 2002). Chromatin is constituted by DNA, which is associated to histones and nonhistone proteins, forming repetitive units, the nucleosomes. Each nucleosome is composed by a DNA fragment and an octameric structure containing the histones H2A, 4

H2B, H3 and H4 (Monneret 2005; Martínez-Iglesias et al. 2008; Legartová et al. 2013). Histones are basic proteins rich in lysine and arginine residues. They present a Cterminal domain, located inside the nucleosome, and an outside N-terminal tail containing lysine residues (Monneret 2005). Several post-translational modifications are observed in histone tails, playing an important role in epigenetic control of gene expression. Methyl groups added to histones by histone methyltransferases (MHT) may alter chromatin condensation, while addition of acetyl groups to histones by acetiltransferases relaxes the DNA fibrils. Such processes influence the access of proteins to DNA, thus modulating the condensation state of chromatin, which is crucial for transcription, replication, repair and recombination (Monneret 2005; Legartová et al. 2013). Although histones are considered one of the most conserved proteins in eukaryotes, in trypanosomatids they are quite divergent compared to other organisms, especially in the N-terminal region, that contains alternative sites for post-translational modifications (revised by Figueiredo et al. 2009). Lysine acetylation and methylation, arginine methylation and serine phosphorylation have been observed in trypanosomatid histones. Histones H4 and H2A N-terminus are frequently acetylated, while histones H3 and H2B are preferentially methylated. In T. brucei and T. cruzi, histone H4 is acetylated in the N-terminal portion at lysine residues 4, 10 and 14, whereas lysine 18 is methylated (Chagas da Cunha et al. 2006, Janzen et al. 2006, Mandava et al. 2007, Elias et al. 2009). The roles of histone modifications on T. cruzi cellular processes have been elucidated. Histone acetylations in lysines 10 and 14 are required for DNA replication and transcription, as well as for chromatin organization and remodeling (Prata Ramos et al. 2015). Such post-translational modifications may also be involved in trypanosomatid 5

differentiation: in epimastigotes, phosphorylation of serine 23 in H2B and methylations of lysine 76 in histone H3 predominate, while trypomastigotes mainly present lysine acetylations in histone H2A and methylation of lysine 23 in histone H3. Furthermore, the replicative stage contains more histone modifications than the trypomastigote form (de Jesus et al. 2016). Histone modifications and distribution are directly related to the protozoan cycle progression. H1 phosphorylation, for example, is concentrated in the nucleolar region during the G1/S phase, but occupies the entire nuclear space in mitoses, when phosphorylation is maximized (Gutiyama et al. 2008). Moreover, histone H4 acetylated at lysine 4 is found in the condensed chromatin, while histones acetylated at 10 or 14 residues are distributed throughout the nucleus (Nardelli et al. 2009, Elias et al. 2009). Considering that epigenetic regulation in pathogenic trypanosomatids affects parasite life cycle and virulence, drugs that target enzymes involved in histone methylation have been employed to clarify how the post-transcriptional modifications of histones influence gene expression (Marks et al. 2004). Histone methyltransferases inhibitors have been used with success against tumor cells, since they promote proliferation inhibition, cell cycle arrest and apoptosis (Marks and Xu 2009). Chaetocin, which is produced by yeast belonging to the genus Chaetomium, is a histone methyltransferase (HMT) inhibitor (Lai et al. 2015). In tumor cells, this compound promotes changes in nuclear organization, such as strong chromosome condensation, and considerably reduces cellular proliferation and viability (Isham et al. 2007; Illner et al. 2010). In this context, it is important to study the effects of HMTs in lower eukaryotes, such as fungi and protists. In the present study, we showed for the first time the effects of chaetocin in a trypanosomatid species. This inhibitor impaired cell proliferation, reduced cell viability 6

and blocked cell cycle on G2/M phase of T. cruzi epimastigotes. Chaetocin also promoted nucleolar disassembly, which seems to be induced by the reduction in rRNA transcription, an effect that has never been described for an inhibitor that has methyltransferases as a target. Furthermore, reversibility assays showed that parasites were not able to re-stablish proliferation after drug removal, indicating that HMT inhibitors may be exploited in therapeutic treatments against trypanosomatid diseases.

2. Material and Methods 2.1 Protozoa culture and drug treatment Epimastigote forms of Y strain T. cruzi were cultivated for 24 hours at 28ºC in liver infusion tryptose (LIT) medium (Camargo 1964) supplemented with 10% fetal calf serum. Chaetocin was diluted in dimethyl sulfoxide (DMSO) to a concentration of 10 mM and evaluated in concentrations of 1, 5, 10, and 50 μM. Cells were collected every 24 hours for counting in a Neubauer chamber. To compare the control and the treated groups, paired t-tests were applied to the results using a 95% confidence interval (GraphPad Prism version 5.00 for Windows; GraphPad Software Inc., San Diego, CA, USA). To evaluate the reversibility effect of cell proliferation, parasites were treated for 2 days, cultures were washed with LIT to remove the drug from the medium and subsequently incubated with LIT and fetal calf serum up to 168 hours. 7

2.2 Cell Viability Parasites were analyzed by the MTS/PMS method (Henriques et al. 2011), which is based on mitochondrial dehydrogenase enzyme activity, and also by propidium iodide (PI) incorporation, which is based on plasma membrane integrity. In the MTS/PMS method, parasites were incubated with the MTS/PMS solution for 4 hours. Untreated parasites were fixed with 0.4% formaldehyde for 10 minutes and used as negative control. The percentage of viable parasites was obtained using a spectrofluorometer (Molecular Devices Microplate Reader, SpectraMax M2/M2e, Molecular Devices) at a wavelength of 490 nm. For PI incorporation, parasites were ressuspended in PBS with 10 µg/ml PI. As negative controls, parasites were permeabilized with 1% Triton X-100 for 15 minutes and then incubated with PI. The samples were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences, USA), considering 10.000 events, and the data were analyzed using BD Accuri C6 software.

2.3 Transmission Electron Microscopy Treated and non-treated parasites were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 hour and were washed in the same buffer. The cells were post-fixed for 1 hour in 0.1 M cacodylate buffer containing 1% OsO4 and 0.8% potassium ferricyanide. Parasites were washed in the same buffer, dehydrated in a graded series of acetone, and embedded in Epon (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were stained with uranyl acetate for 45 minutes, then with lead citrate for 5 minutes and observed using a Zeiss 900 transmission electron microscope (Zeiss, Oberkochen, Germany).

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2.4 Transmission Electron Microscopy - Cytochemical analysis with Ethanolic Phosphotungstic Acid (PTA) The cells were fixed in 2.5% glutaraldehyde in cacodylate buffer for 1 hour, incubated in 2% PTA in absolute ethanol for 2 hours and dehydrated in a graded series of ethanol. The samples were embedded in Epon and the ultrathin sections then observed using a Zeiss 900 transmission electron microscope (Zeiss, Oberkochen, Germany).

2.5 Scanning Electron Microscopy Parasites were fixed in 2.5% glutaraldehyde in phosphate buffer and adhered to poly-L-lysine-coated microscope coverslips. The samples were post-fixed in cacodylate buffer containing 1% OsO4 and 0.8% potassium ferricyanide and then washed in the same buffer. Parasites were dehydrated in a graded series of ethanol, critical point dried in CO2 and ion sputtered. The samples were observed using a Quanta X50 scanning electron microscope (FEI Company, Netherlands).

2.6 Cell Cycle Analysis by Flow Cytometry Parasites were washed in PBS and fixed in 0.25% formaldehyde for 5 minutes, followed by cold 70% ethanol for 30 minutes under vortexing. The cells were then incubated with 25 μg/ml RNase and 100 μg/ml propidium iodide (PI) for 30 minutes at 37ºC and analyzed as described previously.

2.7 RNA analyses In this assay, 1x108 epimastigotes of T. cruzi were collected by centrifugation and their total RNA extracted with Trizol (Invitrogen) according to the manufacturer's 9

instructions. The obtained RNA was submitted to purification using the RNeasy MinElute CleanUp Kit (Qiagen) and the quality of the RNA was evaluated through observation in agarose gel stained with Sybr Safe.

3. Results T. cruzi epimastigote proliferation was intensively affected by chaetocin. After treatment with the lowest concentration (1 µM) for 3 days (4 days of growth), parasite growth was arrested by approximately 40%. This decrease was stronger when higher concentrations of this compound were used, of 83% and 90% when applying 10 µM and 50 µM, respectively. The IC50 is equivalent to 2 µM after 4 days of proliferation in the presence of the drug. In addition to the dose-dependent effect, a plateau in the growth curve was also observed, suggesting cell cycle arrest (Figure 1). T. cruzi proliferation under treatment with chaetocin was investigated for a long period, of up to 10 days, in order to evaluate if parasite growth would improve with time. As the treatment proceeded, a gradual reduction in cell proliferation was observed, however after 10 days of treatment (11 days of growth), the number of parasites was lower when compared to the first day (Figure 1). After evaluating parasite proliferation, T. cruzi viability in the presence of chaetocin was verified by the MTS/PMS method, which evaluates mitochondrial activity. After 1 day in the presence of chaetocin, cell viability was decreased: 80% of the parasites remained viable after treatment with 1 µM, while this was reduced to less than 10% after treatment with 50 µM. According to this method, viability diminished in a concentration dependent manner until 3 days in the presence of the drug. After this period, the intermediate concentrations (5 and 10 µM) promoted a significant effect in cell viability, which was reduced to less than 20% (Figure 2). 10

Cell viability was also checked through incubation with propidium iodide (PI), which indicates plasma membrane integrity, since it only penetrates lysed membranes of dying cells, but not living cells with intact membranes. According to this procedure, reduction of T. cruzi viability was only severely affected after treatment with 50µM chaetocin, since approximately 56% of parasites were dead after 1 day of treatment (Figure 3C). Chaetocin treatment for longer periods, such as 10 days, caused a decrease in the number of living cells of 25% and 40% when 5 and 10 µM were used, respectively (Figure 3D and E). After this long-term treatment using 50µM chaetocin, viable cells were not detected by any of the applied methodologies (data not shown). Considering that chaetocin affected T. cruzi proliferation and viability, it was decided to verify if the ultrastructure of treated parasites was affected. Transmission electron microscopy showed the typical structures of T. cruzi in epimastigote control cells: the nucleus, with the condensed heterochromatin around the nucleolus and close to the nuclear envelope, the bar-shaped kinetoplast, the Golgi complex and the flagellar pocket (Figure 4A). It is interesting to observe that trypanosomatids present nuclear domains, like the fibrillary and granular zones (figure 4B). Parasites cultivated for 3 days in the presence of 5 µM chaetocin presented membrane structures similar to autophagosomes, sometimes involving reservosomes (Figure 4C - arrowhead). Furthermore, after treatment with 10 µM for 2 days, T. cruzi also presented strong unpacking of nuclear heterochromatin (Figure 4D) and a remarkable nucleolar disassembly, which resulted in the appearance of electron dense and round structures, which probably correspond to components of the granular zone that dispersed (figure 4D-F). After 8 days of treatment with chaetocin, even the lowest concentration induced extraction and vacuolization of the parasite cytoplasm (Figure 4 G-H).

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In order to better analyze the ultrastructural modifications caused by chaetocin in the nucleus, the Ethanolic Phosphotungstic Acid cytochemistry technique (EPTA) was employed. This method is based on the fact that, when in ethanol, PTA stains basic proteins rich in histidine, such as histones, thus revealing DNA regions that are more compact, like heterochromatin. In non-treated parasites, it is possible to note the typical distribution of condensed heterochromatin, the nuclear domains and the kinetoplast staining at its periphery (Figures 5A and B). After incubation with chaetocin at 5 µM for 3 days or 10 µM for 2 days (Figures 5C and D, respectively), parasites presented a reduced heterochromatin area restricted to the nuclear periphery (Figure 5C), thus corroborating the idea that the drug promoted the DNA unpacking. The nucleolar disassembly generated rounded electron dense structures that may represent elements of the granular zone, which were seen dispersed through the nucleus (Figures 5C and D). In order to verify if chaetocin promoted alterations on parasite surface and morphology, treated cells were investigated by scanning electron microscopy. Differently from non-treated protozoa, which are elongated and presented typical epimastigote form (Figure 6A), parasites treated with 5 μM for 3 days showed a wrinkled surface (Figure 6B), whereas those treated with 10 µM for 2 days had a rounded or flattened cell body (Figures figures 6C and D). Considering that chaetocin inhibited T. cruzi proliferation and promoted unpacking of nuclear chromatin, investigations of effects on the protozoan cell cycle were also conducted. For this purpose, flow cytometry assays were performed using 1, 5 and 10 µM after different periods of incubation with the inhibitor. The highest concentration (50 µM) was not used in these assays considering the very low number of viable cells that remained in the culture after drug treatment. In the non-treated condition, about 47% of parasites were in the G1 phase and 34% were in G2/M (Figure 12

7A). Parasites incubated for 1 day with 1 µM of chaetocin presented changes in cell cycle progression, since the number of cells in G2/M increased to 53% after treatment. After 3 days in the presence of 5 µM of chaetocin, the number of cells in G1 decreased to 24% and increased to 66% in G2/M (Figure 7B). Curiously, the cell cycle arrest effect was not concentration-dependent, since parasites treated with 10 µM presented 34% of protozoa in G1 and 59% in G2/M (Figure 7B). Interestingly, most parasites were in the G2/M phase when treated with 1 µM up to 3 days. However, 10 days of treatment increased the percentage of cells in G1 (to 70%) and decreased in G2/M (to 18%). On the other hand, treatments for this same period with 5 and 10 µM maintained a large number of protozoa in G2/M (56 and 46%, respectively) when compared to G1 (27 and 35%, respectively) (Figure 7C). It is worth mentioning that, even after 10 days of treatment, the highest percentage of protozoa in G2/M was observed with 5 µM of the inhibitor, and not with 10 µM. During the proliferation assays, the number of protozoa in culture remained low in the presence of the drug, even for longer periods. Thus, to evaluate if parasites were able to recover their proliferation, reversibility assays were performed, removing the drug from the medium after two days of treatment. The result revealed that, after 8 days of drug removal (11 days of growth), parasites were not able to re-stablish proliferation, except those treated with 1 µM. However, in this case, cell growth was reduced in comparison to control cells. At higher concentrations, the number of protozoa remained low (figure 8), as similarly reported in Figure 1. The reversibility test revealed that the percentage of viable cells increased only after 4 days of chaetocin removal (Figure 9) and it is higher when compared to protozoa submitted to long treatment (Figure 2). The increased number of viable parasites was especially observed in cells that were previously treated with 5 and 10 µM of chaetocin 13

(Figures 2 and 9). This phenomenon was also observed when T. cruzi viability was analyzed by PI incorporation: 90 and 76% of parasites treated with 5 and 10 µM, respectively, were viable after 8 days of drug removal (Figure 10), while, in the presence of the inhibitor, these percentages were equivalent to 75 and 40% (Figure 3). Protozoa submitted to the reversibility test presented an altered ultrastructure even after 8 days of drug removal, of cytoplasmic vacuolization, heterochromatin unpacking and nucleolar fragmentation (Figure 11A-C) took place. Interestingly, the presence of several lipid bodies in the cytoplasm was evident (Figures 11B and C), while this effect was not observed in cells submitted to long drug treatment (figure 4). The ultrastructure of T. cruzi observed in the reversibility assays suggested that the chaetocin effect was irreversible in this protozoan species. Based on this, T. cruzi cell cycle was evaluated after removing the inhibitor from the culture medium. After 1 day of drug removal (4 days of proliferation), the group treated with 1 µM presented an increased in the percentage of cells in G1 and a decrease of cells in G2/M (Figure 12A) when compared to cells incubated with 1 µM of the drug that were not submitted to the reversibility test (Figure 7A). In fact, these values are more similar to control protozoa (Figure 7A). On the other hand, parasite groups treated with 5 and 10 µM presented a higher percentage of cells in G2/M than in G1 (Figure 12A). Considering the cell cycle analysis 8 days after drug removal (11 days of proliferation) the percentage of cells treated with 1, 5 and 10 µM became higher for the G1 phase (52, 46 and 61%, respectively) and lower for G2/M (23, 26 and 22%, respectively) (Figure 12B), when compared to cells after one day of drug removal (Figure 12A). This indicates that protozoa were able to recover cell cycle progression, but not proliferation. The main function of the nucleus is the production of ribosomes, which begins with rRNA transcription and processing. Thus, the nucleolar disassembly observed after 14

treatment with chaetocin might be a result of rRNA transcription blockage. In order to test this hypothesis, RNA levels of treated and non-treated parasites were analyzed. Results revealed that after incubation with 1 and 10 µM of the inhibitor for 48 hours, cells presented reduced amounts of rRNA when compared to control cells (Figure 13, lanes A and C). Considering the reversibility assays, RNA synthesis was not reestablished even after 72 hours of drug removal, as observed in parasites previously treated with 1 and 10 µM of chaetocin, that presented lower RNAs levels when compared to control cells (Figure 13, lanes D-F). It is interesting to observe that parasites treated with 10 µM for 48h presented higher RNA amounts (Figure 13, lane C) in relation to protozoa treated with this same concentration and then submitted to the reversibility test (Figure 13, lane F).

4. Discussion Changes in chromatin packing are essential for numerous processes that require enzyme access to DNA, such as replication, transcription, repair, and gene expression. These events are modulated by epigenetic changes, which include the addition and removal of acetyl groups from lysine residues in histones (Monneret 2005; Legartová et al. 2013). Based on this, methyltransferases represent important targets in chemotherapy against tumor cells. Chaetocin is a methyltransferase inhibitor of Histone 3 di-and trimethylation in human cells and has been employed to verify heterochromatin remodeling effects on gene expression of cancer cells (Greiner et al. 2005). It has been shown that such inhibitor impairs proliferation and alters the ultrastructure of tumor cells, especially the nucleus (Illner et al. 2010). Chaetocin also interferes with the expression of var genes, which encode the chief antigenic and virulence determinant of Plasmodium falciparum, 15

by inhibiting protozoan methyltransferase (Ukaegbu et al. 2015). These reports stimulate tests of HMT inhibitors in other pathogenic microorganisms, such as fungi and trypanosomatids, since they may interfere in cell differentiation and infectivity. This study investigated, for the first time, the effects of chaetocin in a trypanosomatid protozoan. Results indicate that this inhibitor promoted a considerable decrease on T. cruzi epimastigote proliferation. The effect of this drug was evaluated using different concentrations that promoted a reduction on parasite growth and viability. It is interesting to observe that the number of treated protozoa was very similar, even after prolonged treatment, indicating impairment of the cell cycle. The inhibitory effect of chaetocin on proliferation has already been reported against different tumor cell lines using concentrations lower than 1 µM (Tram et al. 2013; Dixit et al. 2014). Reversibility assays were performed in order to evaluate if the chaetocin effect on T. cruzi proliferation was definitive or transient. Results demonstrated that parasite treatment, even with the lowest concentration (1uM), impaired normal growth recovery, thus characterizing an irreversible effect, which is relevant in chemotherapy. Although T. cruzi proliferation was not reestablished after chaetocin removal, it was noteworthy to observe that parasite viability was recovered, at least in part. Two different approaches, the MTS/PMS method and PI incorporation, revealed that the percentage of viable cells increased up to 76% after 8 days of drug removal in protozoa previously treated with 10 µM. These viability assays are based on mitochondrial dehydrogenase activity and membrane integrity, respectively. Morphological analyses of T. cruzi by scanning electron microscopy showed that chaetocin treatment promoted cell body shrinkage and the appearance of a wrinkled surface. Transmission electron microscopy approaches revealed that this inhibitor 16

promoted remarkable changes on the nuclear ultrastructure, such as the intense unpacking of nuclear heterochromatin and nucleolus fragmentation. The less condensed state of chromatin observed after drug treatment may be related to proliferation inhibition and cell cycle arrest, as reported for T. cruzi treated with camptothecin, a topoisomerase I inhibitor (Zuma et al. 2014). In parallel, the observed nucleolar disassembly may represent the result of RNA transcription blockage, thus interfering with ribosome biogenesis, protein synthesis and, consequently, with the cell cycle progression, which, in turn, impairs protozoan division. Nucleolar fragmentation has never been described before for an inhibitor that has methyltransferases as a target. Previous works that discuss cell death in protozoa, do not indicate nucleolar alterations as a sign of cell death (Jiménez-Ruiz et al, 2010; Proto et al, 2013), thus we may suggest that this is a direct effect of chaetocin. Such a phenomenon presents ultrastructural similarities with nuclear alterations described in Purkinje cells during the neurodegeneration process that occurs in postnatal life (Baltanás et al. 2010). Data obtained from the RNA analyses support the hypothesis that chaetocin alters chromatin organization and, consequently, RNA transcription, since lower amounts of rRNA were detected in treated protozoa in relation to control cells. After treatment with the lowest dose of chaetocin (1 µM), parasites were able to resume the normal ultrastructural pattern and the RNA transcription levels became similar to non-treated cells. Conversely, after using a higher concentration (10 µM) of the inhibitor, parasites were not able to reestablish, neither the typical ultrastructure, nor the RNA transcription levels, that remained lower even after drug removal. These data suggest that in T. cruzi chaetocin has an irreversible effect on rRNA transcription. One of the most common effects on T. cruzi ultrastructural organization after treatment with chaetocin was the autophagosome formation. This phenomenon is well 17

characterized in mammalian cells; autophagosomes are double membrane-bound structures, which enclose the cellular content to be degraded (Ouyang et al. 2012). This cellular process is part of the programmed cell death by autophagy and can be visualized by transmission electron microscopy. In T. cruzi, the formation of autophagosomes has been described after treatment with several classes of compounds, such as naphthoquinones and lysophospholipid analogues (Menna-Barreto et al. 2009). However, it is worth considering that autophagy, as well as apoptosis and necrosis, can occur in parallel or sequentially, thus promoting protozoa cell death (Elmore, 2007; Golstein and Kroemer 2007). As described for T. cruzi proliferation, the effect of chaetocin on parasite ultrastructure was permanent. In this way, loss of heterochromatin condensation, nucleolus fragmentation and the presence of many cytoplasmic vacuoles were still observed in the parasites, even after eight days of drug removal. The maintenance of these abnormal structural characteristics may have contributed for protozoa proliferation impairment. With regard to protozoa ultrastructure after drug removal, an accumulation of lipid bodies was also observed. Initially, lipid storage was not expected, because the lipid synthesis pathway is not described as a direct target of this compound, although this may represent an effect associated to cell cycle arrest. The increase in lipid body number has been extensively reported in trypanosomatids after treatment with lipid biosynthesis inhibitors, such as amiodarone, azoles and azasterols (Magaraci et al. 2003; Macedo-Silva et al. 2011). As mentioned above, proliferation and ultrastructural data indicated cell cycle blockage, that was confirmed by flow cytometry analyses. The T. cruzi cell cycle arrest at G2/M after treatment with 1 and 5 µM is in accordance to the inability of protozoa to reassume proliferation and chromatin compaction. Interestingly, the impairment of cell 18

cycle was not dose-dependent, since the number of protozoa in the G2/M phase after treatment for 3 days with 10 µM chaetocin was lower than that observed for cells treated with 5 µM for the same period. Considering the reversibility tests, after 8 days of drug removal protozoa were able to restore the cell cycle, since the percentage of parasites in G1 increased, being similar to that observed for control cells. Nevertheless, the reestablishment of the cell cycle did not result in the recovery of proliferation, probably because chaetocin promoted a kind of irreversible modification that prevents the synthesis or re-activation of essential factors for parasite growth. This indicate that the protozoa remain alive in a “senescence-like” state, as previously proposed for T. cruzi after treatment with camptothecin, that induced cell cycle arrest and an early apoptosis that did not progressed (Zuma et al. 2014). Chaetocin has been described as an epigenetic agent that affects the methylation status of histone H3K9 by inhibiting histone lysine methyltransferase (Cherblanc et al. 2012). In early-branched organisms, as trypanosomatids, histones are less conserved and appear to contain alternative sites for modifications. Such characteristics can be explored to better understand how histone modifications affect gene expression and other chromatin-based processes in these protozoa. In epimastigotes the most common histone post-translational modifications are phosphorylation in serine 23 of H2B as well as mono- and dimethylations of lysine 76 of histone H3 (de Jesus et al. 2016). The H3K76 modifications are found mainly in cells undergoing mitosis and cytokinesis and can represent a potential target to methyltransferases inhibitors and to the better understanding of how these compounds can bring significant epigenetic changes in T. cruzi. It has been shown that lysine methylation is carried out by enzymes that contain the SET domain, as the mammalian Suv39h1, which adds methyl group to histone H3 at 19

Lys-9 (Rea et al. 2000). In protists, SET domain-containing histone lysine methyltransferases (HKMT), were only described in Plasmodium falciparum and are associated to addition of methyl groups on K4, K9 and K36 of histone H3 and K20 of histone H4. Such modifications change dynamically during the parasite development stages and regulate transcriptional activation on these microorganisms (Bozdech et al. 2003). The tri-methylation of histone H3 lysine 4 (H3K4me3) for example, is associated with transcriptional activation in the conserved histone code and also with actively transcribed genes, as the var genes, which are involved in antigenic variation and pathogenesis of P. falciparum (Lopez-Rubio et al. 2009, Salcedo-Amaya et al. 2009). Protein methyltransferase enzymes (PMTs) are able to methylate multiple protein substrates, including both histones in the nucleus and non-histone proteins in the cytosol (Hamamoto et al. 2015). It is possible that some epigenetic modifications of histones are conserved in T. cruzi, while others are unique to this parasite, but their functions remain to be elucidated. Taken together, the results displayed herein encourage the use of histone methyltransferase inhibitors in T. cruzi chemotherapeutic studies, considering the effects on parasite proliferation, nuclear ultrastructure and cell cycle. Moreover, we suggest that chaetocin can be used as a tool to further comprehend the protozoan cell biology and gene expression. Acknowledgements This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq) and Fundação de Amparo à Pesquisa do estado de Minas Gerais (FAPEMIG).

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7. Legends: Figure 1: T. cruzi epimastigote proliferation after treatment with chaetocin. The asterisk indicates the addition of the drug to the culture medium. The number of cells was similar after using concentrations higher than 5 µM. The IC50 is equivalent to 2 µM for 3 days (4 days of proliferation). The data are the average of three independent experiments. DMSO, dimethyl sulfoxide.

Figure 2: T. cruzi epimastigote viability after treatment with chaetocin by the MTS/PMS method. Note the concentration-dependent effect on the reduction of the number of living parasites, especially until the fourth day of treatment. It is worth noting that chaetocin was added in culture medium after 24 h of growth. The data are the average of three independent experiments.

Figure 3: T. cruzi epimastigote viability verified by PI incorporation in cells treated with chaetocin. (A) Control parasites. (B) Positive control with Triton X-100. (C) Treatment with 50 µM for 1 day, which corresponds to the second day of growth, promoted significant decreases in the number of the viable parasites (46%). (D-E) For longer periods of treatment, such as 10 days, the number of living parasites decreased using lower concentrations, of 5 µM (D) and 10 µM (E), respectively. The data are the average of two independent experiments.

Figure 4: T. cruzi epimastigote ultrastructure observed by TEM after treatment with chaetocin. (A) Non-treated T. cruzi, showing the nucleus with condensed heterochromatin (ht) around the nucleolus (nu) or juxtaposed to the nuclear envelope, and the kDNA (k). (B) Parasite nucleolar domains: the fibrillary (fz) and granular zones 29

(gz), ht - heterochromatin. (C) T. cruzi treated with 5 µM for 3 days. Note the presence of structures similar to autophagosomes (arrowhead) surrounding a reservosome (r). (DF) T. cruzi treated with 10 µM for 2 days presenting heterochromatin unpacking and nucleolar fragmentation (black arrow). (F) The granular structures (black arrows) probably correspond to components of the granular zone that dispersed. (G-I) Treatment with lower doses of chaetocin (1 µM) for 8 days promoted cytoplasm extraction (arrows) and vacuolization (v).

Figure 5: T. cruzi epimastigote ultrastructure after treatment with chaetocin observed by TEM after using the EPTA cytochemistry technique. (A) and (B) Non-treated T. cruzi, showing staining at the nuclear condensed heterochromatin (ht) around the nucleolus (nu) and also in the kinetoplast (k) periphery. (B) The fibrillar and granular zones of the nucleolus. (C) T. cruzi treated with 5 µM for 3 days and (D) with 10 µM for 2 days presented an heterochromatin unpacking and nucleolar fragmentation. The granular structures (arrow) probably correspond to elements of the granular zone that dispersed.

Figure 6: T. cruzi epimastigote morphology after treatment with chaetocin observed by SEM. A - Non-treated parasite, showing the typical elongated cell body of the epimastigote form. B - T. cruzi treated with 5 µM for 3 days presented plasma membrane wrinkling. C and D - T. cruzi treated with 10 µM for 2 days presented a rounded shape and sometimes a flattened cell body.

Figure 7: T. cruzi cell cycle after treatment with chaetocin. (A) and (B) After 1 and 3 days of treatment, there was an increase in the number of cells in G2/M. (C) After 10 days, the percentage of parasites in G1 phase increased after treatment with 1 µM, while 30

the decrease for G2/M phase was observed, when compared to cells treated with 5 and 10 µM. The highest percentage of parasites in G2/M was observed after treatment with 5 µM for 3 days. The data are the average of two independent experiments. Figure 8: Reversibility test after treatment with chaetocin. The asterisk and the arrow indicate the addition and removal of the drug, respectively. Treated parasites were not able to re-stablish their proliferation, except for those treated with 1 µM. The data are the average of three independent experiments.

Figure 9: T. cruzi epimastigote viability after the reversibility test applying the MTS/PMS method. After seven days of proliferation (four days after drug removal), the percentage of living parasites began to increase for cells treated with 5 and 10 µM chaetocin. The asterisk indicates the drug removal. The data are the average of three independent experiments.

Figure 10: T. cruzi epimastigote viability after the reversibility test for chaetocin was measured considering PI incorporation. (A) and (B) Parasites previously treated with 5 and 10 µM, respectively. After the removal of the drug from the medium, the number of viable cells increased, with the highest percentage after eight days after drug removal (eleven days of proliferation). The data are the average of two independent experiments.

Figure 11: T. cruzi epimastigote ultrastructure of cells submitted to the reversibility test. (A) Protozoa previously treated with 5 µM showed intense cytosolic vacuolization. (BC) Cells initially treated with 5 and 10 µM, respectively, presented higher amounts of lipid bodies (lb). (C) Even after drug removal parasites presented an altered nuclear structure, such as nucleolar fragmentation. 31

Figure 12: T. cruzi cell cycle during the reversibility test with chaetocin after one or eight days of drug removal. (A) After 1 day of drug removal, the percentage of protozoa in the G1 phase was higher for cells treated with 1 µM. (B) After eight days of drug removal, the number of parasites in the G1 phase was higher. The data are the average of two independent experiments.

Figure 13: Ribosomal RNA (rRNA) quantification after treatment with chaetocin. The T. cruzi epimastigote form was treated with 0 µM (NT), 1 µM or 10 µM of chaetocin for 2 days. In the reversibility test, parasites were incubated with culture medium for 3 days. Upper panel: Graphic representation of the rRNA quantification from cells after treatment with chaetocin. A value of 1 was arbitrarily assigned to the non-treated control. Bottom panel: Agarose 1% gel showing the bands for the rRNA of the analyzed samples. * P >0.05; ***P >0.001 One-way ANOVA with Bonferroni post test.

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