Molecular cloning and analysis of expression of the Leishmania infantum histone H4 genes

Molecular and Biochemical Parasitology 90 (1997) 439–447

Molecular cloning and analysis of expression of the Leishmaniainfantum histone H4 genes1

Manuel Soto, Luis Quijada, Carlos Alonso *, Jose M. Requena

Centro de Biologıa Molecular Se6ero Ochoa (CSIC-UAM), Uni6ersidad Autonoma de Madrid, 28049 Madrid, Spain

Received 21 July 1997; received in revised form 30 September 1997; accepted 3 October 1997

Abstract

In the present work, we describe the sequence, organization and expression of histone H4 genes in the protozoanparasite Leishmania infantum. The predicted L. infantum histone H4 is a polypeptide of 100 amino acids with amolecular mass of 11.5 kDa. Comparison of the amino acid sequence of Leishmania histone H4 with the rest ofhistone H4 sequences indicates that this is the most divergent sequence reported to date. The genomic distributionanalysis of histone H4 genes indicates that there must be up to seven gene copies. A single size-class histone H4mRNA of 0.6 kb was detected, whose level dramatically decreases from logarithmic to stationary phase. However, theLeishmania histone H4 mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis,suggesting a regulation by a replication-independent mechanism. © 1997 Elsevier Science B.V.

Keywords: Leishmania infantum ; Histone H4; Expression; Aphidicolin; Hydroxyurea; DNA synthesis

1. Introduction

Protozoan parasites of the genus Leishmaniaare etiological agents of a spectrum of severe

diseases known as leishmaniasis. These parasitespossess a digenetic life cycle with two discretemorphological phases: the promastigote, whichdevelops extracellularly within the gut of the in-sect vector, and the amastigote that is specializedto survive within the macrophage phagolysosomeof vertebrate host. Leishmania species, and otherrelated kinetoplastid protozoa, are placed in themost primitive branch of the eukaryote evolution[1]. Related to this phylogenetic location, theseorganisms possess very peculiar features of geneexpression and organization. Among them, the

Abbre6iations: EST, expression site tags; UTR, untranslatedregion.

* Corresponding author. Tel.: +34 1 3974863; fax: +34 13974799; e-mail: [email protected]

1 Note: Nucleotide sequence data reported in this paper havebeen submitted to the EMBL/GenBank/DDBJ databases withthe accession numbers Y13915 (cDNA LiH4-1) and Y13916(cDNA LiH4-2).

0166-6851/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.

PII S 0166 -6851 (97 )00178 -3

M. Soto et al. / Molecular and Biochemical Parasitology 90 (1997) 439–447440

organization of the nuclear genome differs fromthat of higher eukaryotes. The nuclear envelopepersists during cell division and chromosomes arenot visualized at any phase of the cell cycle.Although chromatin is organized in nucleosomes,higher-order structures as 30 nm fibers are notobserved [2,3]. The biochemical characterizationof histones from several species of kinetoplastidsshowed that they have altered electrophoretic mo-bility when compared to the histones from verte-brates [4–8]. The differences in mobility of thepurified histones suggest changes in hydrophobic-ity, charge and size, and, therefore, may influencethe histone-histone and histone-DNA interac-tions. In fact, protein-DNA interactions are lessstable in trypanosome chromatin than in chro-matin of higher eukaryotes [2,6]. Also, it has beensuggested that these different biochemical proper-ties of trypanosome histones could be related tothe lack of chromatin condensation [2]. In recentyears, several histone genes from kinetoplastidshave been isolated and characterized. Thus, thesequences of the genes coding for histone H2Afrom both T. cruzi [9] and L. infantum [10], thehistone H2B from T. cruzi [11] and L. enriettii[12], the histone H3 from T. cruzi [13] and L.infantum [14], and the histone H1 from L. major[15] and T. cruzi [16] have been described. Al-though amino acid sequences of histone H4 frag-ments from T. cruzi [17] and T. brucei [18] havebeen reported, to date a complete sequence of thishistone in kinetoplastids was not available.

In this study, we describe the isolation andcharacterization of the L. infantum histone H4coding genes. The divergent primary structure ofthis histone may be a key to understanding thechromatin structure of trypanosomes. Further-more, we have analyzed some regulatory aspectsoperating on the histone H4 gene expression.

2. Materials and methods

2.1. Parasites

Promastigotes of Leishmania infantum (LEM75;zymodeme 1) were grown at 26°C in RPMI 1640medium (Gibco Paisley, UK) supplemented with

10% heat inactivated fetal calf serum (Flow Lab.,Irvine, UK). Experimental cultures were initiatedat 1×106 promastigotes ml−1 and subsequentlyharvested for study at different points during theirtransition from logarithmic (5–9×106 promastig-otes ml−1, days 2–3) to the stationary (4–6×107

promastigotes ml−1, days 6–7) phase of growth.

2.2. Library screening, subcloning and sequenceanalysis

A L. infantum poly (A)+ lgt11 cDNA library[10] was screened with the 32P-labeled nick trans-lated insert of a T. cruzi histone H4 EST-clone(kindly provided by Dr W. Degrave, DBBM-Fiocruz, Rio de Janeiro, Brazil) by in situ plaquehybridization [19]. Two hybridizing recombinantphages, called LiH4-1 and LiH4-2, were isolated.The cDNAs were subcloned into the EcoRI siteof the pUC18 plasmid for sequencing purposes.Both strands of the cDNAs were sequenced by thedideoxy chain termination method [20] using theSequenase Kit (United States Biochemical Corp.).The analysis of the DNA and amino acid se-quences was done using the University of Wiscon-sin Genetics Computer Group programs [21] andby accessing to the GenBank and EMBL databases of protein and DNA sequences.

2.3. Southern and Northern blot analysis

L. infantum DNA and RNA were isolated aspreviously described [22,23]. Promastigote totalDNA was digested with a variety of restrictionenzymes, subjected to electrophoresis in 0.8%agarose gels and transferred to nylon membranes(Hybond-N, Amersham) by standard procedures[19]. Total RNA was size separated on 1%agarose-formaldehyde gels [24] and electro-trans-ferred to nylon membranes using a LKB system(Pharmacia). Hybridizations, either for DNA orRNA analysis, were performed in 50% for-mamide, 6×SSC (1×SSC is 0.15 M NaCl, 0.015M Na citrate, pH 7.0), 0.1% SDS and 0.25 mgml−1 of herring sperm DNA at 42°C overnight.Final post-hybridization washes were performedin 0.1×SSC, 0.2% SDS at 50°C for 1 h. Forreuse, blots were treated with 0.1% SDS for 30

M. Soto et al. / Molecular and Biochemical Parasitology 90 (1997) 439–447 441

min at 95°C to remove the previously hybridizedprobe. Removal of the probe was verified byautoradiography.

2.4. Drug treatments and [methyl-3H]thymidineincorporation into DNA

The effects of DNA synthesis inhibitors wereanalyzed on logarithmic phase promastigote cul-tures (5×106 cells ml−1) after adding aphidicolinor hydroxyurea to a final concentrations of 10mM or 5 mM, respectively. Culture aliquots wereremoved at 0, 4, 6 and 8 h of incubation with thedrugs and processed for RNA isolation. Inhibi-tion of DNA synthesis was estimated by measur-ing [methyl-3H]thymidine incorporation intoDNA, in treated cultures relative to untreatedcultures. For this purpose, 100 m l of both drug-treated and -untreated promastigote cultures (5×106 parasites ml−1) were incubated with 10 mCi of[methyl-3H]thymidine (Amersham, 2.0 Cimmol−1) for 0, 4, 6 and 8 h. Thymidine incorpo-ration into DNA was determined using the Multi-Screen Assay System (Millipore) followingmanufacturer’s instructions.

3. Results

3.1. Isolation and characterization of histone H4cDNAs

In order to characterize the genes coding for theLeishmania histone H4, we screened a L. infantumcDNA library using as probe an EST-clonederived from the T. cruzi histone H4 gene. Twolgt11 clones, called LiH4-1 and LiH4-2, withinserts of 531 and 498 nucleotides, respectively,were isolated. Sequencing and database compari-son of these clones, revealed that both cDNAspossess an open reading frame of 300 nucleotidescoding for a protein sequence that could bealigned with that of histone H4. The nucleotideand deduced amino acid sequences of the twocDNAs are available from the EMBL/GenBank/DDBJ databases under accession numbersY13915 (clone LiH4-1) and Y13916 (clone LiH4-2). Differences in nucleotide sequence between

both cDNAs were evidenced, indicating that theyderived from the transcription of two different L.infantum histone H4 genes. Most of nucleotidechanges accumulated in the 5%-untranslated region(5%-UTR). Also, there were found 6 nucleotidechanges in the coding region, but only one pro-duced a variation in the encoded amino acid (aCys to Ser replacement at amino acid position83). Remarkably, the 3%-UTRs of both cDNAswere found to be the most conserved regions, thesolely existing difference is a T/C transition. Aremarkable feature, found in the 3%-UTRs, is thepresence of the inverted repeat GCTGTTTgct-gaAGGCAGT with potential to form a stem-loopstructure in the mRNA. Such structures havebeen involved in the regulation of the histone geneexpression of several organisms [25].

The open reading frame of clone LiH4-1 trans-lated into a polypeptide of 100 amino acids with adeduced molecular mass of 11.5 kDa and anisoelectric point of 11.3. The analysis of the aminoacid sequence revealed a high prevalence ofarginine residues (16%), a feature common to thehistones H4. The sequence of L. infantum histoneH4 was easily aligned with the homologous his-tones from other organisms (Fig. 1A), showing a56% of sequence identity (72% similarity) with theconsensus sequence for histone H4 predicted byWells [26]. However, these values of sequenceidentity are rather low taking into account theextreme conservation of histone H4 sequences[27]. Thus, within the groups of animals, fungi,and plants that overall sequence divergence is lessthan 8% for histone H4 [28]. Strong sequencedivergence is also observed when L. infantum H4sequence is compared with the Entamoeba his-tolytica histone H4 sequence, the most divergenthistone described to date [29]. Most of the aminoacids changes are located at the amino terminalend of the protein, and the alignment of thisregion with the rest of H4 sequences is question-able (Fig. 1A). However the N-terminal region ofL. infantum histone H4 was found very similar tothe chemically sequenced N-terminal fragments ofhistones H4 from T. cruzi (Fig. 1B) [17] and T.brucei [18]. Thus, it can be concluded that thisN-terminal sequence seems to be a specific featureof histone H4 from kinetoplastids.


Fig. 1. Comparison of histones H4 amino acid sequences. (A) Maximal alignment of Leishmania histone H4 (Li), with the consensussequence predicted by Wells [26] (Cons), and the H4 histone of Entamoeba histolytica [29]. Identical residues to the consensussequence are boxed. One gap (dot) was included to maximize the alignment; (B) amino acid sequence alignment of L. infantum andT. cruzi H4 amino terminal domains. The T. cruzi sequence was obtained by direct amino acid sequencing of the protein [17].Identical residues are boxed.

3.2. Genomic organization of Leishmania histoneH4 genes

When Southern blots of genomic DNA, di-gested with a variety of restriction enzymes, were

probed with the cDNA LiH4-1, a complex patternof hybridization bands was revealed (Fig. 2). Thepresence of multiple bands in all lanes pointed tothe existence of several histone H4 genes in the L.infantum genome. Thus, the presence of seven


hybridization bands in lanes containing DNA di-gested with ClaI, SalI or ApaLI, endonucleasesthat do not cut the LiH4 cDNAs, suggested theexistence of at least seven copies for histone H4gene. Furthermore, the existence of multiplebands in all lanes was taken as an indication thatthe histone H4 genes are not clustered in a singlelocus of the L. infantum genome. The existence ofmultiple genes dispersed along the L. infantumgenome has been reported for other histone genes[30–32].

3.3. Expression of the Leishmania histone H4genes

A Northern blot containing total RNA,poly(A)+ RNA and poly(A)− RNA was probedwith the cDNA LiH4-1. As shown in Fig. 3A, asingle hybridization band of approximately 0.6 kb

was seen in the total and poly(A)+ lanes, but notin the poly(A)− fraction. Thus, it can be con-cluded that either the expression of LeishmaniaH4 histone genes yields transcripts of similar sizesor only one of the histone H4 genes is activelytranscribed. The presence of a poly(A)+ tail inthe 3%-end of histone transcripts seems to be acommon feature in all the kinetoplastid histonemRNAs studied to date.

3.4. Regulation of histone mRNA le6els bygrowth phase

In order to determine the relationship betweenthe steady-state level of the H4 RNA and theparasite growth phase, a Northern blot containingequal amounts of RNA from logarithmic andstationary growth phase parasites, was probedwith the radiolabeled cDNA LiH4-1 (Fig. 3B,panel LiH4). It was observed that the steady statelevel of the H4 mRNAs was strongly down-regu-lated when parasites enter the stationary phase ofgrowth. Quantitative analysis of Northern blotswas carried out by densitometric scanning of thecorresponding autoradiograms. A ten to twelvefold decrease in histone H4 mRNA levels wasobserved in parasite cultures from logarithmicphase to stationary phase. These differences in themRNA levels, must be controlled by a specificmechanism connected with the proliferation stageof parasites, since in these conditions the levels ofthe a-tubulin transcripts only decreased 2-foldand the rRNAs did not change (Fig. 3B). Asimilar decrease in RNA levels, associated withthe growth phase, has been observed for other L.infantum histones [10,31].

3.5. Histone H4 mRNA le6els and DNA synthesis

The histone gene expression of lower andhigher eukaryotes is regulated in a replication-de-pendent manner. High levels of the transcriptsaccumulate during DNA synthesis (S phase) anddrop to much lower levels in the absence of DNAsynthesis [33]. In addition, inhibition of DNAsynthesis with drugs such as hydroxyurea andaphidicolin causes a rapid reduction in histonemRNA levels [34,35]. Thus, in the present work,

Fig. 2. Southern blot analysis of the genomic distribution ofhistone H4 genes. Approximately 2 mg of total DNA weredigested with the restriction enzymes BamHI (lane B), ClaI(lane C), SalI (lane S), SmaI (lane M), ApaLI (lane A), andelectrophoresed on an 0.8% agarose gel. Following blotting thefilter was hybridized with the radiollabeled LiH4-1 cDNA.Numbers at the left indicate the sizes (in kb) of the l DNAdigested with HindIII.


Fig. 3. Northern blot analysis of the histone H4 mRNAs. (A) Total RNA (5 mg; lane T), 2 mg of polyadenylated RNA (lane A+)and 10 mg of non-polyadenylated RNA (lane A−) from L. infantum were electrophoresed on 1%-agarose/formaldehyde gel. Afterblotting the filter was hybridized with the radiollabeled LiH4-1 cDNA; (B) 5 mg of total RNA from promastigotes at eitherlogarithmic growth phase (lane L) or stationary growth phase (lane S) were fractionated on 1% agarose/formaldehyde gels andtransferred to nylon membranes. The filter was hybridized with probe LiH4-1 (panel LiH4), and after autoradiographic exposure,the same filter was subsequently re-hybridized with a T. cruzi a-tubulin probe [44] (panel a-Tub) and a L. infantum 24S-a rDNAprobe (panel rRNA). The size of hybridization bands (in kb) are indicated.

we have investigated the effect of aphidicolin andhydroxyurea treatments on the steady state levelsof the Leishmania histone H4 transcripts. In orderto establish the amounts of drug to be assayed, wehave taken advantage of previous studies onDNA synthesis inhibition in Leishmania-relatedspecies of the Trypanosomatidae [36,37]. Asshown in Fig. 4B, L. infantum DNA synthesis wasinhibited by more than 90% in the presence of 5mM hydroxyurea (8 h incubation with drug) andby 80% when 10 mM aphidicolin was used. RNAsamples were prepared from parasites treated witheach drug for increasing intervals of up to 8 h andanalyzed by Northern blotting (Fig. 4A). Remark-ably, the L. infantum H4 mRNA levels did notdecrease during the time of the treatments withthese inhibitors of DNA synthesis. Instead, anevident accumulation of the mRNAs was ob-served after 6 h of drug-treatments. Therefore, thepresent data imply that H4 mRNA levels in L.infantum are not directly coupled to DNA synthe-sis.

4. Discussion

Parasites of the Leishmania genus, as well asother related kinetoplastids, have been located inthe most primitive branch of the evolutionary treeof eukaryotes [1]. As a reflection of their evolu-tionary distance with other eukaryotes is the factthat primary structures of kinetoplastid histonesare the most divergent described to date[27,28,38]. In this work, we report the first com-plete sequence for the histone H4 from a memberof the Trypanosomatidae. In contrast to the ex-treme conservation of the histone H4 sequencethroughout evolution, Leishmania H4 sequencehas been found to be strongly divergent. Thus, forexample, while the human and wheat histone H4sequences differ only in two amino acid positions[26], the human and L. infantum sequences differby 43 amino acid positions. Remarkably, most ofthe substitutions are located in the amino terminalregion. In fact, the alignment of the 18 N-terminalamino acids of the L. infantum H4 sequence to the


Fig. 4. Effect of the DNA synthesis inhibition on the histone H4 RNA levels. (A) Logarithmic growth cultures of L. infantumpromastigotes were incubated with 10 mM aphidicolin or 5 mM hydroxyurea for 0, 4, 6 and 8 h. Five mg of total L. infantum RNAfrom each time point in both treatments were analyzed by Northern blotting and probed with LiH4-1 cDNA (panel LiH4). As acontrol, the filter was re-hybridized with a L. infantum 24S-a rDNA, showing that similar amounts of RNA per lane were loaded(panel rRNA). (B) Kinetics of DNA synthesis inhibition by aphidicolin (solid squares) and hydroxyurea (open circles). Thepercentage of inhibition of DNA synthesis was estimated by measuring [methyl-3H]thymidine incorporation into DNA indrug-treated cultures relative to untreated cultures. The data points shown are the mean (9SD) of triplicate samples.

rest of the H4 sequences remains questionable.Structural studies indicate that the amino terminaldomain of histones H4 is exposed outside of thenucleosome and not involved in the interactionwith the H3 histone for nucleosome formation[39]. Thus, it can be postulated that the aminoterminal domain of Leishmania histone H4 has alower evolutionary pressure than the rest of themolecule due to a more relaxed functional impli-cation of this region in nucleosome assembly.Also, the amino-terminal domain of L. infantumhistone H3 was found to be extremely divergent[14]. It is possible that the higher variability ofLeishmania histones, in the amino terminal do-mains of histones H3 and H4, is the basis for theobserved variability in chromatin structure ofkinetoplastids [2,3,40].

Another question addressed in the presentstudy has been the regulation of histone H4mRNA levels in Leishmania. Histone biosynthesisis tightly linked to cellular DNA replication inboth higher and lower eukaryotes. The histoneregulation appears to occur during the cell cycleitself and during transitions between proliferatingand quiescent cells [25,33,41]. The coupling ofhistone mRNA regulation to DNA replicationthat is observed during the cell cycle can be

studied by using DNA synthesis inhibitors [34,35].The present data indicate that inhibition of L.infantum DNA synthesis by either aphidicolin orhydroxyurea does not promote a decrease in thehistone H4 mRNA levels. Thus, it can be con-cluded that histone H4 mRNA levels in L. infan-tum are not directly coupled to DNA synthesis.According to previous results on the regulation ofL. infantum histone H3 [31] and L. enriettii his-tone H2B [12], it can be stated that histonemRNA levels in Leishmania are not directly cou-pled to DNA replication. However, our data indi-cate that the L. infantum histone H4 mRNA levelsare down-regulated when promastigotes enter sta-tionary phase of growth. Similar down-regulationby growth phase has been observed in L. infantumfor both histone H2A and histone H3 mRNAlevels [10,31]. Thus, it appears that histonemRNA levels are regulated by a mechanism cou-pled to cellular growth. This conclusion agreeswith the observation that the level of L. enriettiiH2B mRNA is several fold higher in promastig-otes than in amastigotes, considering that pro-mastigotes have a more rapid rate of division thanamastigotes [12]. However, it must be noticed thatthe regulation of L. major histone H1 seems to beclearly different to the Leishmania core histones.


Thus, during promastigote growth, histone H1mRNA progressively accumulates from early log-arithmic phase to stationary phase [42]. Further-more, amastigotes show high level of H1expression that decreases when amastigotes differ-entiate into promastigotes. It appears that,though in both cases histones mRNA levels areunder growth control, the requirement of histoneH1 during growth is quite different to the require-ments of core histones. Thus, it can be concludedthat histone H1 must play a different role thancore histones during both growth phase and cellcycle.

The fact that Leishmania histone mRNA levelsare not directly coupled to DNA synthesis butthat the histone mRNA levels are sensitive to thecellular division rate (growth control) can betaken as an indication of cell cycle regulation ofhistones. In this way, the recent work on T. bruceihistone mRNA regulation performed by Ersfeldet al. [43] should be mentioned. These authors,using fluorescence in situ hybridization, demon-strate that histone H2A, H2B and H4 RNA levelspeaks during S phase and are not detectable dur-ing all other stages of the cell cycle.

Acknowledgements

We thank Dr W. Degrave and A. Brandao forkindly providing us with the T. cruzi H4 ESTclone. This work was supported by grants I+D0020/94 from Comunidad Autonoma de Madrid,PTR94-0091 from Plan Nacional de InvestigacionCientıfica y Desarrollo, and BIO96-0405 fromPrograma Nacional de Biotecnologıa. An institu-tional grant from Fundacion Ramon Areces isalso acknowledged.

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Molecular cloning and analysis of expression of the Leishmania infantum histone H4 genes

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