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Molecular & Biochemical Parasitology xxx (2005) xxx–xxx

Trypanosoma cruzihistone H1 is phosphorylated in a typical cyclindependent kinase site accordingly to the cell cycle

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Julia Pinheiro Chagas da Cunhaa, Ernesto S. Nakayasub, Maria Carolina Eliasa,1,Daniel C. Pimentac, Maria Teresa Tellez-Inond, Federico Rojasd,

Munoz Manueld, Igor C. Almeidab, Sergio Schenkmana,∗

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a Departamento de Microbiologia, Imunologia e Parasitologia, R. Botucatu 862-8a, EPM-UNIFESP, S˜ao Paulo, SP 04023-062, Brazil8b Departamento de Parasitologia, ICB, USP, S˜ao Paulo, Brazil9

c Centro de Toxinologia Aplicada, CAT/CEPID, Instituto Butantan, S˜ao Paulo, SP, Brazil10d Instituto de Investigaciones en Ingenieria Gen´etica y Biologia Molecular (INGEBI, CONICET), Vta de Obligado 2490, 1428 Buenos Aires, Argentina11

Received 16 September 2004; received in revised form 20 December 2004; accepted 21 December 2004

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chromatins horter whenc oneH thath correlationb sites at theG s TzCRK3,a treating thep lind hatases.

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bstract

Histone H1 of most eukaryotes is phosphorylated during the cell cycle progression and seems to play a role in the regulation oftructure, affecting replication and chromosome condensation. In trypanosomatids, histone H1 lacks the globular domain and is sompared with the histone of other eukaryotes. We have previously shown that inTrypanosoma cruzi, the agent of Chagas’ disease, hist1 is phosphorylated and this increases its dissociation from chromatin. Here, we demonstrate using mass spectrometry analysisT. cruziistone H1 is only phosphorylated at the serine 12 in the sequence SPKK, a typical cyclin-dependent kinase site. We also found aetween the phosphorylation state of histone H1 and the cell cycle. Hydroxyurea and lactacystin, which, respectively, arrest para1/S and G2/M stages of the cell cycle, increased the level of histone H1 phosphorylation. Cyclin-dependent kinase-related enzymend less intensely the TzCRK1 were able to phosphorylate histone H1 in vitro. Histone H1 dephosphorylation was prevented byarasites with okadaic acid but not with calyculin A. These findings suggest thatT. cruzihistone H1 phosphorylation is promoted by cycependent kinases, present during S through G2 phase of the cell cycle, and its dephosphorylation is promoted by specific phosp2004 Published by Elsevier B.V.

eywords:Histone H1; Phosphorylation; Cell cycle;Trypanosoma cruzi; Phosphatase; CDK

Abbreviations: a.m.u., atomic mass unit; ESI-TOF-MS, electrosprayonization-time of flight-mass spectrometry; ESI-IT-MS, electrosprayonization-ion trap-mass spectrometry;m/z, mass to charge ratio; CDKs, cy-lin dependent kinases; HU, hydroxyurea; OA, okadaic acid; AUT-PAGE,olyacrylamyde gel electrophoresis containing acetic acid; urea and Triton-F116; TzCRK1,T. cruzicyclin related kinase 1; TzCRK3,T. cruzicyclin

elated kinase 3.∗ Corresponding author. Tel.: +55 115 751 996; fax: +55 115 571 5877.E-mail address:sergio@ecb.epm.br (S. Schenkman).

1 Present address: Laboratorio de Parasitologia, Instituto Butantan, Saoaulo, SP, 05503-900, Brazil.

1. Introduction

Histone H1, also known as linker histone, consistsconserved central globular domain flanked by a relatishort amino- and a long carboxy-terminal tail. The globdomain seems to interact with linker DNA outside the nuosome core, and the tails with the linker DNA and withamino-terminal tails of core histones[1]. Histone H1 affectmany features of chromatin structure and function. It slizes the high-order structure of chromatin[2,3] and affectsnucleosome position and spacing[4,5]. It is involved in chromatin assembly during replication[6], chromatin remodelin[7] and condensation[8], gene transcription[9] and cell apop

166-6851/$ – see front matter © 2004 Published by Elsevier B.V.oi:10.1016/j.molbiopara.2004.12.007

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2 J.P.C. da Cunha et al. / Molecular & Biochemical Parasitology xxx (2005) xxx–xxx

tosis [10]. However, the exact mechanism of interaction of39

this protein with the nucleosome remains unknown[11,12].40

In addition, the essentiality of histone H1 is also a matter of41

controversy, as histone H1 knockouts are viable[13].42

Different variants of the protein and post-translational43

modifications, mainly phosphorylation, seem to be involved44

in these histone H1 functions. It is well known that histone45

H1 phosphorylation increases as the cell progresses into the46

cell cycle, with specific phosphorylation events occurring at47

interphase, before mitosis. These phosphorylation events are48

catalyzed by cyclin-dependent protein kinases (CDKs)[1],49

mainly CDC2/cdc28 (yeasts CDK1), which are active from50

the S to M phase of the cell cycle[14–18], and are important51

for chromosome condensation. Many histone H1 phospho-52

rylation events occur at S/TPXK sites along the tails, but,53

as reviewed earlier, there is no obvious correlation between54

the sites and a particular stage of the cell cycle[19]. More re-55

cently, molecular modeling studies have shown that the phos-56

phorylation of histone H1 at CDC2 sites of the tail domains57

modifies the interaction of the protein with the DNA[20].58

Indeed, replacement of threonines and serines by alanine in59

these phosphorylation sites in mammalian histone H1 de-60

creases the protein mobility in the nucleus[18]. The phos-61

phorylation at these sites seems to be required to promote62

chromatin replication in vitro[6]. As histone H1 becomes63

hyperphosphorylated before mitosis, when the chromosomes64

c ain65

i at66

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c68

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related to the cell cycle control in these organisms. These find-95

ings and the fact that trypanosomatids are disease-promoting96

agents, prompted us to study in more detail the localization97

of the phosphorylation sites ofT. cruzihistone H1, the cell 98

cycle regulation of this event, and the enzymes involved in99

the phosphorylation and dephosphorylation steps. We found100

that histone H1 is phosphorylated when cells progress from101

the S to M phase of the cell cycle at a typical CDK consensus102

site in an organism that does not condense chromosomes in103

mitosis and does not have transcriptional control. 104

2. Material and methods 105

2.1. Parasites, cell cycle synchronization and FACs 106

analysis 107

T. cruzi (Y strain) epimastigote forms were cultured in108

liver infusion-tryptose medium, supplemented with 10% FBS109

at 28◦C [41]. Tissue culture derived trypomastigotes were110

obtained from infected LLCMK2 cells as described[42]. 111

The medium containing parasites was collected and cen-112

trifuged at 1000×g, the pellets were washed with phosphate-113

buffered saline (PBS) and immediately used, or stored at114

−70◦C. Hydroxyurea treatment was done as described[43]. 115

For G2 phase blockage, 1.5× 108 parasites at the exponen-116

t 117

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ondense[8], and the localization of histone H1 is uncertn the chromosome structure[21], it has been proposed thistone H1 phosphorylation might allow the binding ofessory factors to promote chromosome condensation[22].

Histone H1 of early protests, such as the Entamoeinetoplastids, Ciliates and Dinoflagellates, lacks the gl

ar domain, presenting just the regions corresponding t-terminal domains of most eukaryotic histones[1]. Accord-

ngly, there is no formation of the typical 30 nm fibers ao condensation of chromosomes during mitosis. In Kinlastids, a group containing several protozoan parasitesrypanosoma cruzi,Trypanosoma bruceiand several specif Leishmania), the presence of histone H1 has been exively described[23–31], but the function of this proteinoorly understood. Histone H1 mRNA is mainly expres

n replicating forms during the S phase, but is also founon-dividing cells[32–34]. We have recently shown that h

one H1 ofT. cruzi, the parasite that causes Chagas’ dises differentially phosphorylated in proliferating versus nroliferating forms of the parasite[35]. Also, we providedvidence that phosphorylated histone H1 is releasedhromatin more easily than the non-phosphorylated fuch histone H1 phosphorylation may not be related to

egulation of transcriptional activity, as this group of orgsms show a primitive control of gene expression, with mf their genes being regulated at the post-transcriptional

36]. As proteins similar to CDKs are present in the S pharypanosomatid extracts that can phosphorylate mammistone H1 in vitro[37–40], it is possible that the histone Hhosphorylation could be promoted by these CDKs an

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ial growth phase were incubated with 10�M lactacystinCalbiochem) dissolved in dimethyl sulfoxide for 24 h8◦C. Controls were performed with an equivalent volumimethyl sulfoxide. For flow cytometry analysis, 5× 106 par-sites were washed twice in PBS and fixed with 1 ml of 5ethanol in PBS at 4◦C for 10 min. The fixed parasites weashed once with PBS and incubated for 20 min at 37◦C with0�g ml−1 of DNAse-free RNase A, (Roche-Diagnosticamples were washed in PBS and resuspended in 5�lf PBS containing 20�g ml−1 propidium iodide and ana

yzed with a flow cytometer using the CELLQuest softwBecton–Dickinson Excalibur). The data correspondin0,000 events was analyzed using the WinMid 2.8 softwhen indicated, parasites were washed in PBS and att

o glass slides coated with 0.1% poly-l-lysine in PBS. Atached parasites were fixed with 4%p-formaldehyde in PBor 20 min, washed, stained with 10�g DAPI/ml, and oberved with a 100×/1.4 Plan-Apochromatic lens in a Nik600 fluorescence microscope.

.2. Histones and histone H1 extraction and AUT gellectrophoresis

Frozen epimastigotes or trypomastigotes (5× 108 para-ites) were resuspended in 1 ml of 10 mM potassiumamate, 250 mM sucrose, 2.5 mM CaCl2 and lysed by thddition of 0.1% Triton X100. The lysate was centrifugashed once with the same buffer without Triton X100

wice with the buffer lacking sucrose. All solutions contai.1 mM phenyl–methyl–sulfonyl fluoride, 0.2 mM benza

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dine, 5 mM butyric acid and 1 mM sodium fluoride. Para-145

site lysates were acid-extracted with 0.3N HCl or 5% per-146

chloric acid, for 2 h at 4◦C with shaking, as described[35].147

The insoluble material was removed by centrifugation at148

12,000×g for 15 min at 4◦C; acid-soluble proteins were149

precipitated with eight volumes of acetone, washed three150

times with acetone:0.1 M HCl (10:1, v/v) and twice with151

pure acetone, and then vacuum dried. Alternatively, the acid-152

soluble proteins (1 ml) were dialyzed three times against 2153

l of 1 mM triethanolamine containing 0.2 mM EDTA, and154

twice against double-distilled water. Dialyzed extracts were155

stored at−20◦C. Extracts were fractionated by AUT-PAGE156

as previously described[35].157

2.3. Histone H1 purification158

Perchloric acid extracts were solubilized in 0.1 M159

NaH2PO4, pH 6.8, and the insoluble material was removed by160

centrifugation at 15,000×g for 10 min. Samples containing161

about 250�g of protein in the soluble extract were loaded162

onto a Mono S column equilibrated with 0.1 M NaH2PO4,163

pH 6.8, and eluted with a linear gradient to 1 M NaCl in the164

same buffer at 0.5 ml min−1. Column eluates were monitored165

by UV absorbance at 220 nm. Fractions were collected and166

extensively dialyzed against water to remove salt, and then167

stored frozen at−20◦C.168

2169

th170

p171

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C d as173

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2.5. Mass spectrometry analysis 197

For the determination of intact protein molecular mass,198

250 pmol of protein obtained from the Mono S fractions199

were dialyzed against water, lyophilized and resuspended200

in 100�l of 0.05% formic acid in 50% of acetonitrile. 201

Twenty microliters of these fractions were loaded onto a Q-202

TOF UltimaTM electrospray-time of flight-mass spectrometer203

(ESI-TOF-MS) (Waters, Micromass Ltd., Manchester, UK)204

at a flow rate of 5�l min−1 with an infusion pump (Harvard 205

Apparatus, Cambridge, MA). Mass spectra were acquired in206

the positive-ion mode. The source voltage was 5 KV and the207

capillary voltage was 100 V. The capillary temperature was208

150◦C. The spectra were acquired at the 50–4000m/z range. 209

Raw data were processed and MS1 spectra were deconvo-210

luted using the Mass Lynx 3.5 software (Waters, Micromass211

Ltd.). 212

To determine the phosphorylation site, 250 pmol of each213

dialyzed Mono S fraction were digested overnight with214

0.02�g trypsin (modified, sequencing-grade, Roche) in215

10 mM ammonium bicarbonate, pH 8.0, at 37◦C. The di- 216

gested samples were lyophilized and resuspended in 100�l 217

of water. This procedure was repeated three times to remove218

salt and the samples were dissolved in 80�l of 0.05% formic 219

acid in 5% acetonitrile. One microliters was loaded onto a mi-220

crocapillary column (PepMap C18, 15 cm× 75�m, LC Pack- 221

i rap-222

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l in,226

4 of227

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a d in229

8 and230

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.4. Drug treatments and kinase assays

T. cruzi epimastigote forms in the exponential growhase were treated for 24 h at 28◦C with the indicatedmounts of calyculin A (Calbiochem) or okadaic acid (Oalbiochem), previously dissolved in DMSO and storetock solutions at−70◦C. The number of parasites in tultures was measured and 1× 108 parasites were harvesty centrifugation and the histones extracted as descbove. Controls were performed with an equivalent volf DMSO. For the kinase assays, 100�g of parasite solubl

ractions were pre-cleared with protein A-agarose (Inven) and incubated with purified IgGs from anti-TzCRK3nti-TzCRK1 sera[44]. The protein A-agarose precipitat

mmunocomplexes were washed four times with phosphuffered saline and incubated with the corresponding kissay mixture (50 mM Tris–HCl, pH 7.5, 10 mM MgC2,mM DTT, 2.5 mM EGTA, 5 mM MnCl2, 0.5 mM sodiumuoride, 0.4 mM sodium orthovanadate, 5�Ci [�-32P]-ATP,0�M ATP and 0.1 mg ml−1 of histone H1 (Calbiochem. cruzihistone H1 or the recombinantT. cruziH1 protein)eactions were performed at 30◦C for 30 min in a total volme of 40�l and stopped with 5× Laemmli’s buffer. Sampleere analyzed by 12% SDS-PAGE, stained with Coomalue R-250 or electrotransferred to Hybond C membra

Amersham Biosciences) and exposed to X-ray films.uantification of the labeled H1 the gel was scanned whosphorimager Storm 820 (Amersham Biosciences) anorporation was determined using the Image-Quant softw

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ngs, USA) coupled to an electrospray ionization-ion tass spectrometer (ESI-IT-MS) (LCQ-Duo, ThermoFian, San Jose, CA) equipped with a nanospray source (oFinnigan). Peptides were eluted from the column w

inear gradient from 0 to 40% mobile phase B within 25 m0–100% mobile phase B within 10 min, at a flow rate00 ml min−1. Mobile phase A was 0.05% formic acid in 5cetonitrile and mobile phase B was 0.04% formic aci0% acetonitrile. The source voltage was set at 1.9 kV

he capillary temperature was set at 180◦C. The spectra weollected in the triple-play data-dependent mode. Pepere monitored at 300–2000m/z range, and the most abuant ions were submitted to zoom scan followed by MScan (isolation width of 2 a.m.u., and normalized collisnergy of 35%) for five times and then dynamically exclu

or 2.5 min. The collected MS/MS spectra were correlasing the TurboSequest software (Thermo Finnigan) w. cruzidatabase (containing translated ORFs of more0 amino acids from genomic and EST sequences), avat the web sitehttp://www.tcruzidb.org/.

. Results

.1. A single phosphorylation event occurs in T. cruziistone H1

We have previously found that the phosphorylated his1 migrates slower than the unphosphorylated form in pcrylamyde gel electrophoresis containing acetic acid,

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Fig. 1. Histone H1 purification: elution profile of phosphorylated and non-phosphorylated forms of histone H1 from the Mono S column. The perchlo-ric acid extract of parasites containing 250�g of protein were solubilized in0.1 M NaH2PO4, and loaded onto the column (Mono S 5/5). The fractions(250�l) were eluted with a linear NaCl gradient of 20 ml from 0 to 1 M at0.5 ml min−1. The inset depicts the Coomassie-stained AUT-PAGE of thecorresponding fractions from 17.5 to 19 ml. The slow-migrating band corre-sponds to the phosphorylated histone H1 (H1P) and the fast-migrating bandto the non-phosphorylated form (H1). Twelve microliters of each fractionwere loaded onto the gel. No protein was detected in the AUT-PAGE for thepeak indicated by the asterisk and therefore it does not correspond to histoneH1.

and Triton-DF116 (AUT-PAGE), and is less abundant than248

the latter in exponentially growing cells[35]. Although the249

slow migrating band was converted to the fast migrating band250

by phosphatase treatment, we had no indication of which and251

how many phosphorylation sites or other post-translational252

modifications were present. To further characterize the hi-253

stone H1 forms inT. cruzi, the protein was purified from254

perchloric acid extracts followed by cation-exchange chro-255

matography, which allowed the separation of the two forms256

of histone H1 (Fig. 1). The slow-migrating band eluted first,257

followed by the fast-migrating band. Other peaks seen in the258

chromatogram did not corresponded to histone H1 as fol-259

lowed by AUT-PAGE (not shown). Fractions a and f (Fig. 1,260

inset) were then submitted to ESI-TOF-MS analysis. Both261

samples showed a profile with multiply charged ion species,262

each composed of several ions, representing possible post-263

translational modifications and/or different protein isoforms264

of histone H1 (not shown). Deconvolution analysis of the265

species present from 6000 to 9000 Da revealed a major group266

of ions around 8000 Da. Other minor molecular species with267

sizes around 6000, 6500, 7000, 7500, 8400 and 8700 were268

also detected. Detailed analysis of the major species shows269

a predominant molecular species of 7992 Da for the non-270

phosphorylated form (Fig. 2C). Six other isoforms at 7950, 271

7964.5, 7977.75, 8006.25, 8020.25 and 8033.75 Da, differing272

from each other by 14 Da, were also observed in the spectrum.273

These forms might correspond to different methylated forms,274

or expression of different genes of histone H1 expressed si-275

multaneously. For example glycine to alanine substitution,276

found in the histone H1 genes, could also lead to a 14 Da277

increase. A small amount of the monophosphorylated form278

(8072 Da) was also detected, probably a contamination of the279

preparation with the slow-migrating band. A major 8072 Da280

species was detected in the analysis of the purified slow-281

migrating phosphorylated form, most likely corresponding282

to the addition of a single phosphate group (80 Da) to the283

7992 Da form (Fig. 2D). No other peak with additional 80 Da284

was observed, indicating that the slow-migrating band is in-285

deed monophosphorylated. The same isoforms differing by286

14 Da were also detected in the monophosphorylated pro-287

tein preparation. These results strongly suggest that a major288

form of histone H1 with a single phosphorylation event is289

e 290

iated291

t ed at292

t and293

p 294

a es295

t ono-296

p fact297

t hory-298

l f the299

p 300

3 301

the302

p with303

Table 1Peptide sequences of histone H1 digested with trypsin and identified by tand

Peptide Ion species (m/z) Charge state

1 709.34 +22 477.63 +23 421.70 +24 825.40 +15 356.98 +26 514.34 +17 443.30 +18

leculactively.

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a Monoisotopic molecular mass.b Difference between predicted and experimental monoisotopic moc Sac and Sp, acetylated and phosphorylated serine residues, respe

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xpressed inT. cruzi.The same isoforms are mainly expressed in different

rypomastigote forms and in epimastigotes forms arresthe beginning of the S phase with hydroxyurea (HU),reviously shown to be phosphorylated[35]. ESI-TOF-MSnalysis, as shown inFig. 3, revealed that in both cas

he major histone H1 species is represented by the mhosphorylated form of 8072 Da, compatible with the

hat a similar histone H1 gene is expressed and phospated at different cell cycle and developmental stages oarasite.

.2. Identification of the histone H1 phosphorylation site

In order to identify the phosphorylated residue in H1,urified phosphorylated form of histone H1 was treated

em ESI-IT-MS

Mmia �Mb Identified peptide sequencec

1416.7 0.0 (−)2SacDAAVPPKKASpPK14

953.3 0.3 (−)2SacDAAVPPKK10

841.4 0.1 (K)26TAKKPAVK 33

825.4 0.0 (−)2SacDAAVPPK9

713.0 0.5 (K)45KKPAAAK 51

514.3 0.0 (K)20,34KPAAK24,38

443.3 0.0 (K)65,73KAPK68,76

426.2 0.0 (R)61HAAK 64

r masses.

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Fig. 2. ESI-TOF-MS profile of distinct native forms of histone H1. Deconvoluted mass spectra of the non-phosphorylated (A) and phosphorylated (B) formsof histone H1. Panel (C) shows the deconvoluted mass spectrum from 7500 to 8500 Da of the same sample as shown in (A) of the non-phosphorylated (7992.5Da) histone H1 and possible multiply methylated forms (7977.75, 7964.5, 7950.00, 8006.25, 8020.25 and 8033.75 Da) of histone H1. Panel (D) depicts thedeconvoluted mass spectrum of the same sample shown in (B) of the phosphorylated (8072.75 Da) and possible multiply methylated forms (8044.5, 8057.5and 8087.00 Da) of histone H1.

trypsin and injected into a microcapillary column (PepMap304

C18) coupled to an ESI-IT-MS. After accurate molecular305

mass determination at MS1, major ion species were frag-306

mented, and several peptide sequences matching histone H1307

could be assigned (Table 1). The horizontal boxes mark308

identified sequences inT. cruzi histone H1 (Fig. 4). The309

dotted boxes represent repeated sequences and were there-310

fore uncertain. One of the peptides [M + 2H]2+ = 709.34;311

[M + H] + = 1417.68 matched the mass predicted for the N-312

terminus considering the addition of one acetyl (42 Da) and a313

phosphate group (80 Da), and the absence of the first methion-314

ine. This peptide has two possible phosphorylation sites-Ser315

2 and Ser 12. The first could be acetylated since it is well316

known that histone H1 lacks the initial methionine, and the317

first amino acid in this protein (Ser inT. cruzi) is acetylated in318

several organisms[45]. In fact, we found that the N-terminus319

is blocked inT. cruziby Edman sequencing (data not shown),320

as observed by Toro and co-workers[31]. When the doubly321

charged ion ([M + 2H]2+; m/z 709.2)m/z 1417.7 was frag-322

mented, it produced a spectrum with bothy- andb-ion series,323

showing that Ser 12 is phosphorylated and Ser 2 is acetylated324

(Fig. 5).The same serine is found in a conserved position of325

several histone H1 genes from CL Brener strain ofT. cruzi 326

and defines a typical CDK site (S/TPXK), quite similar to327

the sequence found in the C-terminus tail of the sea urchin328

histone H1 (Fig. 4). The corresponding phosphorylation site329

was also found in some, but not allT. bruceigenes. 330

3.3. The histone H1 phosphorylation is related to the 331

cell cycle 332

The finding that the phosphorylation site is a typical CDK333

site, and that parasites blocked at the G1/S transition with HU334

contain mostly the phosphorylated histone H1[35] led us to 335

investigate in more detail whether histone H1 phosphoryla-336

tion is related to the cell cycle inT. cruzi. The parasites were 337

treated with lactacystin, a proteasome inhibitor shown to ar-338

restT. bruceiin the G2 phase[46]. The drug stopsT. cruzi 339

growth and after 24 h most cells showed a doubled DNA340

(Fig. 2C) content as seen by flow cytometry analysis using341

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6 J.P.C. da Cunha et al. / Molecular & Biochemical Parasitology xxx (2005) xxx–xxx

Fig. 3. ESI-TOF-MS profile of histone H1 extracted from trypomastigote forms and epimastigotes treated with HU. Deconvoluted mass spectra of all fractionseluted from the Mono S column and containing histone H1 of trypomastigotes (A) or epimastigotes treated 20 h with HU (B). Panel (C) and (D) show thedeconvoluted mass spectrum from from 7300 to 8400 Da of the samples as shown in (A) and (B), respectively.

propidium iodide staining (Fig. 6A). The parasites remained342

fully motile, and one kinetoplast and one nucleus were ob-343

served in 95% of them (Fig. 6B). Sixty-five percent of the344

drug-treated parasites also showed two flagella (detected by345

staining the cells with antibodies against the flagellar Ca2+346

binding protein, not shown) in contrast to 5% of untreated347

cells, which we found to grow only when the cells reach the348

G2 phase of the cell cycle (manuscript in preparation). These349

findings indicate thatT. cruziarrests between G2 and M phase350

with lactacystin treatment as shown forT. brucei. The his- 351

Fig. 4. Alignment of histone H1 sequences and localization of the phosphorylation sites. ClustalX alignment of the histone H1 genes of the sea urchinStrongylocentrotus purpuratus(GenBank A32137),T. cruzistrains: Y (AAL02283) and CL Brener (C1 = TIGR database TSKTSC 8219 from 8241 to 8008 andC2 = TTSKTSC 8219 from 7580 to 7368),T. brucei1 (CAB76185) andT. brucei(CAB76189). The serine and threonine residues are marked with white lettersagainst a dark background. The boxes with solid lines indicate unique peptides and dotted lines boxes, repeated sequences identified by tandem ESI-IT-MS inT. cruzi(Y strain). The arrow shows the position of the phosphorylated serine in theT. cruzihistone H1.

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Fig. 5. Tandem ESI-IT-MS spectrum of a typical phosphorylated histone H1 peptide. The precursor ion ([M + 2H]2+) at m/z 709.22 of peptide2SacDAAVPPKKASpPK14 was fragmented, yielding detectableb-ion (b2–5, b9–11) and y-ion (y2, y4–11) series (B). Both series confirm that S12 is phos-phorylated. If unmodified, the difference betweenb10–11 andy2–3 is expected to be∼87 Da but, consistent with phosphorylation, is observed to be∼168 Da.The schematic fragmentation is shown in (A).

tones were then extracted with perchloric acid and analyzed352

by AUT-PAGE. As shown inFig. 6C, histone H1 phospho-353

rylation increased in lactacystin-treated parasites, indicating354

that the histone H1 is phosphorylated when the parasites are355

arrested at G2/M phase.356

3.4. Characterization of kinases and phosphatases357

involved in histone H1 phosphorylation358

To investigate the nature of the enzymes involved in the359

histone H1 modifications, we examined whether histone H1360

phosphorylation could be catalyzed by either one of the two361

known cyclin-dependent kinases described inT. cruzi[47].T.362

cruzicyclin related kinase 1 (TzCRK1) is localized in the cy-363

toplasm and in discrete regions of the nucleus, and is highly364

concentrated in mitochondrial DNA (kinetoplast), suggest-365

ing a putative control function in this organelle[44]. T. cruzi366

cyclin related kinase 3 (TzCRK3) corresponds to the Cdc2367

protein, and several lines of evidence indicate that it is in-368

volved in the control of the cell cycle in trypanosomatids369

[38,39]. TzCRK3 is able to phosphorylate mammalian hi-370

stone H1 in vitro and its activity has been shown to in-371

crease in the S phase[39]. Therefore, nuclear extracts of372

epimastigotes were immunoprecipitated with immobilized373

anti-TzCRK1 or anti-TzCRK3 antibodies, and the adsorbed374

material incubated with the non-phosphorylated form of hi-375

s376

c and377

less by TzCRK1 immunoprecipitates. No significant phos-378

phorylation was seen using control antibodies, with the same379

amount of histone H1 protein as substrate as shown by the380

Ponceau staining. When mammalian histone H1 was used,381

both kinases were effective to the same extent, suggesting382

that TzCRK3 would be more specific for the parasite his-383

tone than TzCRK1. These results suggest that TzCRK3-like384

kinases could mediate in vivo the cell cycle-dependent H1385

phosphorylation inT. cruzi. 386

We next studied the effect of phosphatase inhibitors on hi-387

stone H1 phosphorylation in vivo. We found that okadaic acid388

(OA) at concentrations above 250 nM induced accumulation389

of the phosphorylated histone H1 (Fig. 8A and B, left panel) 390

and promoted growth arrest. The parasites were fully motile391

at 250 nM OA, but showed a 20-fold increase in the number392

of parasites with two nuclei (Fig. 8D), and eventually two 393

kinetoplasts, rarely seen in the control cells (Fig. 8C). The 394

same concentration of OA was shown to block the differen-395

tiation of parasites inhibiting a protein phosphatase 2A[48]. 396

In contrast, calyculin A at a concentration that blocked the397

growth ofT. cruzidid not induce an increase in the level of398

H1 phosphorylation (Fig. 8A, right panel). We also found 399

that calyculin A at 2.5 nM also increased the number of400

cells containing two nuclei as found for OA, confirming re-401

sults previously obtained by Orr et al.[49]. Therefore, both 402

phosphatase inhibitors seem to arrest cell at the citokinesis,403

b one404

H 405

UN

CO

tone H1 isolated from the parasite. As seen inFig. 7, T.ruzi histone H1 was phosphorylated by anti-TzCRK3

MOLBIO 9602 1–12

ut only OA increased the phosphorylation level of hist1.

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Fig. 6. Lactacystin blocksT. cruziin G2 and promotes histone H1 phospho-rylation. (A) Epimastigotes in the exponential growth phase were treatedwith DMSO (control) or 10 (M lactacystin to block cells in the G2 phase ofthe cell cycle. After 24 h, samples of each culture were submitted to flowcytometry after propidium iodide staining. Events of 10,000 were collectfor each sample. Single (1C) and double (2C) DNA content of cells areindicated. (B) Drug treated parasites were observed in a fluorescence micro-scope after DAPI staining (left figure) or under phase contrast (right figure).The position of the nucleus (N) kinetoplast (k), and flagella (f) are indi-cated. (C) Shows the AUT-PAGE of the acid-extracted histone H1 stainedwith Coomassie with the position of phosphorylated histone H1 (H1P) andnon-phosphorylated histone H1 (H1) indicated by arrows.

4. Discussion406

In the present study, we have characterized the histone H1407

phosphorylation site and the relation of this phosphorylation408

event with the cell cycle ofT. cruzi. In the Y strain ofT. cruzi,409

the histone H1 is expressed as major isoforms. These proteins410

are monophosphorylated at the serine 12, which corresponds411

to a typical cyclin-dependent kinase site defined by the con-412

sensus S/TPXK sequence. The histone H1 phosphorylation is413

increased when parasites are arrested in G1/S or G2/M stages414

of the cell cycle, and the candidate enzymes involved in the415

phosphorylation reactions are cdc2-related kinases, includ-416

ing the TzCRK3 and TzCRK1[39]. The dephosphorylation 417

reaction seems to occur directly or indirectly related to a418

protein phosphatase 2A, inhibited by OA[48]. Therefore, in 419

spite of the fact thatT. cruzi is an early diverging eukaryote 420

that neither condenses chromosomes during mitosis nor has421

a typical eukaryotic transcriptional control, it has a pattern422

of histone H1 phosphorylation according to the cell cycle423

similar to higher eukaryotes. 424

We found several 14 Da increments inT. cruzihistone H1. 425

These mass differences may represent methylation events,426

which have also been found in the histone H1 of other proto-427

zoa such asEuglena[28] andPhysarium[50] or to the expres- 428

sion of various members of the histone H1 gene family[23]. 429

However, our analysis point to a reduced heterogeneity of the430

histone H1 expressed genes. The most predominant form de-431

tected in the ESI-TOF-MS analysis (7992.5 Da) correspond432

to the mass based on the histone H1 cDNA identified in epi-433

mastigote forms of the Y strain used in this study (GenBank434

AAL02283). This cDNA predicts a protein of 8079.9 Da.435

Considering that the first methionine is removed from the N-436

terminus and the second serine is acetylated as found here; the437

corresponding mature protein would have 7991.8 Da. Other438

relatively less abundant species were also detected by ESI-439

TOF-MS and might correspond to isoforms of histone H1440

expressed in the parasite, including the genomic sequence441

found in the Y-strain (Accession number AAL02282). The442

s nd in443

t neity444

i t of445

g 446

nt in447

t H1448

g L-449

B 450

t sider-451

i ser-452

i teins453

o ddi-454

t hole455

g - 456

b d 457

g find-458

i tero-459

g entify460

t also461

w yla-462

t 463

12.464

T ro-465

t train466

i codes467

f site468

( tyla-469

t t of470

s of the471

h erine472

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MOLBIO 9602 1–12

ame pattern of expressed histone H1 isoforms was fourypomastigote forms, arguing that a reduced heteroges maintained during the life cycle, and that a major seenes is effectively expressed in the parasite.

When we searched the histone H1 genes presehe T. cruzi genome, we found nine different histoneenes in theT. cruzi database generated from the Crener strain (http://www.genedb.org/genedb/tcruzi). From

hese, three genes encode the 7992 Da protein conng that the first methionine is removed and the firstne is acetylated. The other six genes predict prof 7342, 7964, 8322, 8418, 8432 and 8434 Da. In a

ion, when searching the individual readings at the wenome database at TIGR (http://www.tigrblast.tigr.org/erlast/index.cgi?project=tca1), the most frequently founene was the one encoding the 7992 Da isoform. These

ngs suggest that the histone H1 gene family is not so heeneous. Nevertheless, further studies are required to id

he different forms of histone H1 being expressed, andhen, whether, and at which amino acid position meth

ions may occur.The phosphorylation site was identified as the serine

his is a typical CDK site and it is conserved in the peins encoded by eight different genes of CL-Brener sdentified in the genome database. Only one gene enor an 8400 Da protein that lacks the phosphorylationdiscounted the initial methionine and adding the aceion in the first serine). Analysis of the whole genome seequences at TIGR also revealed that less than 15%istone H1 sequences predict for proteins without the s

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MOLBIO 9602 1–12

J.P.C. da Cunha et al. / Molecular & Biochemical Parasitology xxx (2005) xxx–xxx 9

Fig. 7. H1 phosphorylation by TzCRK3 and TzCRK1. (A) Forty micrograms of soluble fractions ofT. cruziepimastigote nuclear extracts were preclarified withprotein A-agarose and incubated with anti-TzCRK1 (�-CRK1), anti-TzCRK3 (�-CRK3) or normal rabbit serum (NRS) as a control. The immunocomplexeswere incubated with 3�g of purified histone H1 from the parasite (T. cruziH1), or commercial mammalian H1 protein in the presence of [�32P]-ATP asdescribed in Experimental Procedures. After the incubation the samples were analyzed by SDS-PAGE, transferred to nitrocellulose membranes, stained withPonceau S, and exposed to X-ray film. The figure shows, in top, the phosphorimage and corresponding Ponceau stain of the gel. The arrows indicate the positionof T. cruziand mammalian histone H1 in the gel. In the bottom part of the figure, the histogram represents the phosphorylation of each protein expressed inarbitrary units determined from scanning the gel on a phosphorimager. The results are mean of four independent experiments.

phosphorylation site. Also in other strains the phosphory-473

lation site is conserved. For example, the histone H1 from474

Tulahuen strain contain one, two or three putative phospho-475

rylation sites in tandem and in this strain several histone H1476

bands are detected in AUT-PAGE[29]. If different histone H1477

genes without phosphorylated serine were expressed, a pro-478

tein with 8400 Da would be seen in our analysis, which was479

not the case. Therefore, a major type of histone H1 gene is480

expressed and it is differently phosphorylated at the different481

stages of the cell and life cycle. Differently fromT. cruzi, sev-482

eral histone H1 genes ofT. bruceilack the CDK consensus,483

while a few displayed a putative CDK phosphorylation site484

[51]. Interestingly, a threonine at position 11 instead of serine485

could be phosphorylated inT. brucei[25]. Whether all genes486

are expressed and whether the same type of phosphorylation487

mechanism occurs, it remains to be investigated.488

The fact that histone H1 phosphorylation is related to the489

cell cycle is supported by several independent sets of evi-490

dences. The first one was provided by the blockage in the491

G2/M stage by lactacystin treatment. Flow cytometry and492

morphological analysis indicate thatT. cruzi does not un- 493

dergo mitosis in the presence of this inhibitor, and under this494

condition, the histone is mostly present in the phosphorylated495

form. We cannot exclude that inhibition of the proteosome496

could indirectly affect a phosphatase or kinase activity. Sec-497

ond, our data showing that TzCRK3 phosphorylate more effi-498

ciently the parasite histone H1 in vitro compared to TzCRK1499

suggest that a cdc-2 like kinase might be involved in the500

phosphorylation in vivo. Considering that a limited number501

of serine and threonine residues (and CDK sites) are avail-502

able in the parasite histone, the efficiency of labeling of the503

parasite histone H1 with the�-TzCRK3 immunoprecipitates 504

was higher than the mammalian histone H1 per protein mass.505

Moreover, the weak labeling with TzCRK1, which was ac-506

tive against the mammalian histones supports the notion that507

CRK3 activity is more specific towards the parasite protein.508

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10 J.P.C. da Cunha et al. / Molecular & Biochemical Parasitology xxx (2005) xxx–xxx

Fig. 8. Okadaic acid, but not calyculin A, induces histone H1 phosphory-lation. Epimastigotes in the exponential growth phase were treated with theindicated concentration of OA and calyculin A. After 24 h of treatment, analiquot was taken, acid extracted, and the solubilized histones analyzed byAUT-PAGE (A). In (B), bars show the quantification ratio of the H1 andH1P bands, and the dots indicate the relative growth (in percentage) after24 h measured by counting the number of parasites. The doubling time ofT. cruziepimastigotes is between 22 and 24 h. Phase contrast images andDAPI staining of parasites untreated (C), treated with 250 nM OA (D), or2.5 nM calyculin A (E). The arrows indicate the presence and the positionof two nuclei in a single parasite. The numbers of parasites with two nucleiis indicated in the right of the figures, and are mean± standard deviation offour different experiments. Bars are 2�m.

Although, it is difficult to correlate in vitro with in vivo sub-509

strates, there are several reports showing that TzCRK3 is one510

of the cdc2 related kinases in trypanosomatids[52] and, in as-511

sociation with different cyclins, it seems to define the targets512

for the cell cycle control, including a histone H1 kinase activ-513

ity in trypanosomatids. Therefore, it is most likely involved514

in histone H1 phosphorylation.515

In T. cruzi, the TzCRK3 activity is present in the S phase516

and increases largely at the G2/M transition. When the par-517

asite undergoes mitosis it is inactivated[39]. The fact that518

histone H1 is phosphorylated in HU-arrested cells when519

TzCRK3 activity is low, suggests that other active kinase520

complexes in the S phase could also phosphorylate histone521

H1 in vivo. Indeed, we have found that the same serine 12522

is phosphorylated in HU treated cells (not shown). A pos-523

sible kinase involved in this phosphorylation could be the524

TzCRK1, which is predominant after HU arrest[39], but 525

other kinases induced by drug treatment could also be in-526

volved. 527

We have observed that OA, but not calyculin prevented hi-528

stone H1 dephosphorylation at the concentrations that arrest529

cell growth and promoted increase in the number of cells with530

two nuclei. These findings suggest that only an OA sensitive531

protein phosphatase is involved in histone H1 dephospho-532

rylation, as both drugs seem to inhibit arrest cell cycle at533

cytokinesis. Thus, the prevention of cytokinesis per se does534

not seem to cause histone H1 phosphorylation. Neverthe-535

less, further experiments are required to determine how they536

block cytokinesis and whether the inhibition of histone H1537

dephosphorylation is a direct consequence of a phosphatase538

inhibition or of the cell cycle arrest at different moments539

of cytokinesis. Interestingly, calyculin A, induce, while OA540

prevent the transformation of trypomastigotes in amastigotes541

[48,53], suggesting that specific protein phosphatases may542

have unique roles in cell cycle and differentiation inT. cruzi. 543

The exact role of histone H1 phosphorylation in most eu-544

karyotes remains an open question. The expression of consti-545

t ates546

t pro-547

t the548

D is-549

t PXK550

m leo-551

s uld552

m 553

r DNA554

r . In555

a ld556

b n it557

o ould558

i os-559

p ism560

u ce of561

t 562

A 563

ss564

t o 565

M ID,566

I nd567

F read-568

i in569

g was570

s o 571

E olvi-572

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MOLBIO 9602 1–12

utively phosphorylated proteins in several systems indichat phosphorylation of the N and C-terminal tails of theein might be involved in the release of the protein fromNA. Molecular modeling and structure predictions of h

one H1 bound to nucleosomes also show that the S/Totifs, targets of phosphorylation, interact with the nuc

omal DNA, and it is conceivable that phosphorylation coediate the release of histone H1 from chromatin[20]. The

elease, occurring in the S phase, may be required foreplication and folding of the new chromatin structurenalogy, inT. cruzithe phosphorylation of histone H1 coue required for chromatin replication, and for this reasoccurs at the beginning of the S phase. This finding w

ndicate an early evolutionary acquisition of histone phhorylation related to the cell cycle control in an organnable to form 30 nm chromatin fibers due to the absen

he globular domain of histone H1.

cknowledgments

We are grateful to Dr. Sirlei Daffre for providing acceo the LC-MS system at ICB-USP, Sao Paulo, Dr. Antoni. Camargo for the use of the Q-tof system at CAT-CEP

nstituto Butantan, Sao Paulo, Dr. Beatriz A. Castilho aernando M. Dossin for suggestions, comments and

ng the manuscript, and Evania Barbosa Azevedo for helprowing and maintaining the parasite cultures. This workupported by grants from Fundac¸ao de Amparoa Pesquisa dstado de Sao Paulo and Conselho Nacional de Desenv

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J.P.C. da Cunha et al. / Molecular & Biochemical Parasitology xxx (2005) xxx–xxx 11

mento Cientıfico e Tecnologico, Brazil. ICA and SS are the573

recipints of research fellowships from the Conselho Nacional574

para o Desenvolvimento Cientıfico e Tecnologico (CNPq),575

Brazil. MTTI work was supported by grants from the Na-576

tional Research Council (CONICET), the WHO (TDR) and577

the University of Buenos Aires (UBA).578

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