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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
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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
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th170
p171
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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
m nni-223
g Ther-224
m ith a225
l in,226
4 of227
2 %228
a d in229
8 and230
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d /MS234
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tone245
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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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J.P.C. da Cunha et al. / Molecular & Biochemical Parasitology xxx (2005) xxx–xxx 5
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
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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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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
PR
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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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