Histone H1 expression varies during the Leishmania major life cycle

Histone H1 expression varies during the Leishmania major life cycle

Molecular and Biochemical Parasitology 84 (1997) 215 – 227 Histone H1 expression varies during the Leishmania major life cycle Tanja M. Noll, Chantal...

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Molecular and Biochemical Parasitology 84 (1997) 215 – 227

Histone H1 expression varies during the Leishmania major life cycle Tanja M. Noll, Chantal Desponds, Sabina I. Belli, Theresa A. Glaser, Nicolas J. Fasel* Biochemistry Institute, Uni6ersity of Lausanne, Ch. des Bo6eresses 155, 1066 Epalinges, Switzerland Received 16 April 1996; accepted 14 November 1996

Abstract The deduced amino acid sequence of Leishmania major sw3 cDNA reveals the presence of characteristic histone H1 amino acid motifs. However, the open reading frame is of an unusually small size for histone H1 (105 amino acids) because it lacks the coding potential for the central hydrophobic globular domain of linker histones present in other eukaryotes. Here, we provide biochemical evidence that the SW3 protein is indeed a L. major nuclear histone H1, and that it is differentially expressed during the life cycle of the parasite. Due to its high lysine content, the SW3 protein can be purified to a high degree from L. major nuclear lysates with 5% perchloric acid, a histone H1 preparative method. Using an anti-SW3 antibody, this protein is detected as a 17 kDa or as a 17/19 kDa doublet in the nuclear subfraction in different L. major strains. The nuclear localization of the SW3 protein is further supported by immunofluorescence studies. During in vitro promastigote growth, both the sw3 cytoplasmic mRNA and its protein progressively accumulate within parasites from early log phase to stationary phase. Within amastigotes, the high level of H1 expression is maintained but decreases when amastigotes differentiate into promastigotes. Together, these observations suggest that the different levels of this histone H1 protein could influence the varying degrees of chromatin condensation during the life-cycle of the parasite, and provide us with tools to study this mechanism. © 1997 Elsevier Science B.V. Keywords: Protozoa; Leishmania; Histone H1; Differentiation

1. Introduction

Abbre6iations: FITC, fluoresceine isothiocyanate. * Corresponding author. Tel.: + 41 21 6925732; fax: + 41 21 6925705; e-mail: [email protected]

In higher eukaryotes, DNA and histones are organized into nucleosomes to form chromatin. Nucleosomes consist of an octamer of four core histones H2A, H2B, H3 and H4 around which the

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DNA is wrapped [1]. A fifth histone, the linker H1, binds proximate and distal nucleosomes. The length of histone H1 molecules of higher eukaryotes ranges in size from 190 to 220 amino acids. The highly charged carboxy-terminal sequences rich in proline, lysine and alanine residues, bind to and interact with both the phosphate backbone of the spacer DNA and the N-terminal tails of the core histones [2,3]. The central globular domain of about 80 amino acids makes contact with the spacer DNA sequence outside the nucleosome core, closing two turns of DNA coiled around the nucleosome core particle and bridging juxtaposed nucleosomes to form the 30 nm chromatin fiber [1,4]. Reversible phosphorylation of threonine residues at specific sites in both the N- and C-terminal arms of histone H1, regulates its interaction with DNA and the nucleosome core, although this is still under debate [5 – 7]. It is generally thought that linker histones may play a critical role in maintaining the 30 nm chromatin fiber. Recent reports of H1 knockouts in the protozoan Tetrahymena thermophila have shown that these proteins are not essential for cell survival but are involved in chromatin packaging [8]. Among the histones, the linker histone H1 is the least conserved and genes encoding multiple variants and subtypes whose functions are not yet known have been identified in higher eukaryotes. Some H1 subtypes have been shown to be expressed in stage- and tissue-specific manners, and in some organisms, a differential intranuclear distribution of histone H1 variants has been demonstrated [9 – 11]. The large number of different patterns of expression of these H1 subtypes suggests functional differences [12] which could be in part responsible for the variations in chromatin structure that exist within the genome and during development [11,13,14]. In Trypanosomatidae, it is conceivable that the differences in chromatin condensation observed in the various developmental stages could be correlated with structural peculiarities of histone H1 or H1-like proteins. Therefore, it is possible that trypanosomatids have developed stage specific histone variants which greatly differ in their

functional aspects with respect to histones of higher eukaryotes. Similar to higher eukaryotes, the chromatin of trypanosomatids is organized into nucleosomes. However further condensation into chromosomes during cell division does not occur [15], and compact heterochromatin is present during interphase. The inability of trypanosomatid chromatin to form solenoids (30 nm fibers), and subsequently to generate visible chromosomes during cell division suggests marked differences in the structural organization of DNA [16–19]. Initially it was thought that the low level of chromatin condensation in kinetoplastids reflected the absence of H1. However, H1-like or histone H1 proteins have been recently identified in Crithidia fasciculata [17], Trypanosoma cruzi [19] and Trypanosoma brucei [20,21]. The deduced amino acid sequence of H1 histones of trypanosomatids arising from cDNA and genomic clones has revealed that these proteins lack the central globular domain [20]. Thus it is possible that the structural difference of protozoan histone H1 as compared to that of higher eukaryotes accounts for the differences in chromatin condensation seen in these organisms and/ or might be involved in controlling the transcriptional activity of individual genes as observed in other systems [22–25]. The sw3 gene identified in Leishmania major has an increased level of steady state mRNA in amastigotes as compared to promastigotes [26]. To date detailed studies on the expression of this gene at the protein level have not been made. The deduced amino acid sequence of SW3 (105 aa) reveals that despite significant homology to histone H1 of higher eukaryotes, it completely lacks the central hydrophobic globular domain. Here we provide biochemical evidence that the sw3 gene encodes a histone H1 protein localized to the nucleus of L. major parasites. Furthermore, the level of expression of this H1 histone varies during the life cycle of the parasite, suggesting a possible role in chromatin condensation, development, and/or a role in the regulation of gene expression during transition from promastigotes to amastigotes.

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2. Materials and methods

2.1. Parasites Leishmania major promastigotes (strains MRHO/SU/59/P and MRHO/IR/75/ER designated as LV39 and IR75 respectively) were grown at 26°C in Dulbecco’s modified Eagles medium (DMEM; Gibco-BRL) on solid rabbit blood agar [27], supplemented with 10% fetal calf serum (FCS) (Seromed) and 10 mg ml − 1 gentamicin. Amastigotes were maintained in vivo in BALB/c mice injected subcutaneously in the hind footpad with 2–5×107 stationary phase promastigotes ml − 1. Alternatively, amastigotes were maintained in Swiss nude mice as intramuscular back lesions.

2.2. Parasite protein analysis and immunoblotting Promastigotes were collected from in vitro cultures by centrifugation (10 min, 270 × g, 4°C), washed three times in phosphate-buffered saline (PBS) (8 mM Na2HPO4, 1.75 mM KH2PO4, 0.25 mM KCl, 137 mM NaCl) and resuspended in gel sample buffer (2% SDS, 10% glycerol, 2.5% 2mercaptoethanol, 80 mM Tris – HCl pH 6.8, 125 mg ml − 1 bromophenol blue) for analysis by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) [28]. Amastigotes were isolated according to previously published methods [29], and then prepared for SDS-PAGE analysis as described above. Routinely, proteins from 3 ×107 cells were heated to 100°C for 5 min in sample buffer, and then separated on 15% SDS-PAGE gels. Proteins were then electrotransferred to nitrocellulose (Schleicher and Schuell) using the semi-dry Sammy blotter (Schleicher and Schuell). Immunoblotting of membranes was carried out as described by [30]. Briefly, membranes were blocked with 5% dried milk powder, 0.05% Tween 20 in TBS (10 mM Tris–HCl, 150 mM NaCl, pH 7.4). Strips were cut from the same nitrocellulose filter and incubated separately either with preimmune sera or with the a415 antibody. Rabbit a415 anti-serum in blocking solution (1:1000 in 1% dried milk) was added to the membranes and incubated overnight at 4°C. The membranes were washed four times in 0.05% Tween 20 in TBS. Membranes were then

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incubated with horse radish peroxidase conjugated protein A (SIGMA) in blocking solution (1% dried milk) for 1 h at room temperature. They were then washed four times in 0.05% Tween 20 in TBS. Membranes strips were aligned on a filter paper next to each other and were developed by chemiluminescence (Amersham).

2.3. Acid extraction of histones Histones are extractable from nuclei by treatment with HCl, and H1 histones are specifically extractable with perchloric acid. Parasites were collected from in vitro cultures, washed twice in PBS and lysed in 140 mM NaCl, 1.5 mM MgCl2 10 mM Tris–HCl (pH 8.6), 0.5% NP40. Nuclei were pelleted at 6000× g for 3 min, at 4°C (Kontron). They were then resuspended in 0.25 N HCl or 5% perchloric acid, vortexed for 30 s and mixed on a rotating wheel at room temperature for 1 h. Insoluble proteins were pelleted at 6000 × g for 5 min. HCl supernatants containing histones were precipitated with eight volumes of ice cold acetone, and perchloric acid supernatants containing H1 histones were precipitated with eight volumes of cold ethanol. Samples were then centrifuged at 8000×g for 15 min, and the pellets resuspended in sample buffer for analysis by SDSPAGE followed by immunoblotting.

2.4. Isolation of nuclei In vitro cultured promastigotes were collected by centrifugation (5 min, 270 × g, 4°C), the pellet resuspended in 10 ml ice cold PBS, and then recentrifuged as described earlier. The pellet was then resuspended in 2 ml lysis buffer (140 mM NaCl, 1.5 mM MgCl2, 10 mM Tris–HCl (pH 8.6), 0.5% NP-40) containing 40 ml 200 mM vanadyl ribonucleoside complex, an RNAse inhibitor [31], and vortexed for 10 s. The samples were then centrifuged (6000× g, 3 min, 4°C) giving rise to a pellet corresponding to the nuclear fraction, and 2 ml of supernatant. The supernatant was further processed to extract cytoplasmic RNA. The nuclear pellet was frozen in liquid nitrogen and stored at −70°C. Isolation of amastigote nuclei was performed using a similar protocol.

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2.5. Production and purification of the a415 antibody The peptide sequence (NH2-MSSNSAAAAVSAATTSPQKS-COOH) deduced from the sw3 cDNA and corresponding to the first 20 amino acids of the gene was synthesized. Peptide synthesis was performed according to the F-moc, t-butyl strategy for solid phase synthesis, as described by Merrifield et al. and Atherton et al. [32,33]. The peptide (415) was purified by gel filtration using Sephadex-G25, and its molecular mass confirmed by mass spectrometry on an LDI 1700 Mass Monitor (Linear Scientific, Reno, NV). It was shown to be more than 90% pure. The lyophilized peptide was dissolved in PBS at 2 mg ml − 1 and injected into rabbits to raise specific antibodies (Eurogentec SA, Belgium). The SW3 antiserum (a415) used in the immunofluorescence studies was affinity purified on a peptide 415 linked to CNBr-activated Sepharose beads (LKB Pharmacia) according to standard procedures.

2.7. Indirect immunofluorescence Parasites were spread on polylysine (SIGMA, Nr. P 8920) treated slides (Dynatech), and fixed with acetone-methanol (1:1) for 10 min. Slides were subsequently incubated with primary antibody (a415) for 1 h at room temperature in a moist incubation chamber, and washed three times with PBS. The second anti-rabbit Ig antibody conjugated with fluoresceine isothiocyanate (FITC) (1:200) (Silenus, Morwell Diagnostics, Cat. Nr. RDAF) was added for 1 h at room temperature in a moist incubation chamber. The slides were then washed four times in PBS. When Hoechst staining was performed slides were incubated with Hoechst dye (Pierce) dilution 1/1000 for 15% at room temperature in the moist incuba-

2.6. RNA isolation and analysis Cytoplasmic RNA of the different stages of Leishmania major was isolated as described previously [34]. Supernatants (2 ml) obtained in the preparation of nuclei was added to 2 ml of 2× digestion buffer (0.2 M Tris – HCl pH 7.5, 25 mM EDTA, 0.3 M NaCl, 2% SDS) and 50 ml of Proteinase K (20 mg ml − 1) added. Digestion was performed at 37°C for 30 min. RNA preparations were purified by phenol-chloroform extraction and precipitated at − 20°C with 0.3 M CH3COONa and two volumes of ethanol. RNA (15 mg) were fractionated on a 0.8% agarose gel and transferred (Vacublot) to Genescreen plus membrane (NEN research products). Radioactive probes were generated by in vitro transcription of sw3 cDNA inserted in the vector pGEM-1 (Promega) which contains the T7 and Sp6 RNA polymerases promoters. Hybridization and washing were carried out as described previously [26].

Fig. 1. Identification of the sw3 gene product in Leishmania major (LV39) parasites by immunoblotting. Cell lysates of stationary phase L. major promastigotes (lanes a – c) and amastigotes (lanes d – f) were prepared, and equivalent amounts of proteins separated on a 12.5% SDS-polyacrylamide gel. Proteins were transferred to nitro-cellulose and immunodetection was performed using the a415 antibody (lanes b and e), a rabbit pre-immune serum (lanes a and d) and the a415 in the presence of 1 mg ml − 1 of the immunizing peptide (lanes c and f). The molecular mass markers 14.4, 21.5 and 31.5 kDa (Bio-Rad, low range) are indicated.

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Fig. 2. Acid extraction of the SW3 protein from nuclei. Total histones were extracted from L. major LV39 (panel A) or IR75 (panel B) promastigote nuclei with 0.25 N HCl (lanes c and d) and histones H1 with 5% perchloric acid (lanes e and f). Total nuclear lysates are represented in lanes a and b. Proteins were separated on 12.5% SDS-PAGE gel transferred to nitro-cellulose and analyzed using preimmune serum (lane a, c and e) or a415 antibody (lanes b, d and f). A 21.5 kDa molecular mass marker is indicated.

tion chamber and washed four times in PBS. Otherwise, for confocal immunofluorescence, the cells were stained for DNA by treating fixed parasites with 0.5 mg ml − 1 RNAse (Boehringer) in PBS for 30 min at room temperature, and washing four times in PBS. The slides were then mounted in Mowiol solution with ethidium bromide at a concentration of 1 mg ml − 1.

(lanes d–f) cell lysates. To confirm the specificity of the antibody for the 17 and 19 kDa doublet, we competed the binding of the rabbit antiserum with the synthetic 415 peptide. Fig. 1 (lanes c and f) shows that the binding of the antibody to the 17 and 19 kDa doublet was specifically competed out with 1 mg ml − 1 415 peptide. Non-specific signals are observed in lanes a and d when immunoblots were probed with rabbit pre-immune serum.

3. Results

3.2. HCl and perchloric acid extraction of the sw3 gene product from L. major cell lysates

3.1. Identification of the sw3 gene product in L. major by immunoblotting A rabbit antiserum directed against a peptide (415) corresponding to the amino-terminus of the deduced amino acid sequence of sw3 (see Section 2) was generated. This anti-SW3 antibody (a415) specifically detected a 17 and 19 kDa protein doublet (Fig. 1, lanes b and e) in L. major (LV39 strain) promastigote (lanes a – c) and amastigote

The histone H1 nature of the SW3 protein was investigated using histone purification methods [35]. Due to its high lysine content, histone H1, similar to high mobility group non-histone chromosomal proteins, could be enriched specifically from other histones with 5% perchloric acid. Proteins were extracted from L. major promastigotes of the strains LV39 (Fig. 2A) and IR75 (Fig. 2B) with 0.25 N HCl (lanes c and d) or 5%

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still detectable in IR75 but at a very low level. Histone H1 size polymorphisms have also been detected in the non-virulent strain of Leishmania major LV119 (C. Desponds and N. Fasel, unpublished results). The presence of these different molecular weight species could be due to the expression of different H1 copies. The SW3 protein was not detected in either strains when blots were probed with preimmune serum (lanes a, c and e).

3.3. Nuclear localization of the SW 3 protein

Fig. 3. Biochemical nuclear localization of the sw3 gene product. Cytoplasmic (lanes a and b) and nuclear (lanes c and d) fractions of stationary phase Leishmania major promastigotes (LV39) were separated on a 12.5% SDS-polyacrylamide gel, followed by immunoblotting using an a415 antibody (lanes b and d), or rabbit pre-immune serum (lanes a– c). A 215 kDa molecular mass marker is indicated.

perchloric acid (lanes e and f). Extracted proteins, as well as total nuclear lysates (lanes a and b), were separated by SDS-PAGE and proteins detected by immunoblotting using the a415 antibody (lanes b, d and f) or as a control, rabbit preimmune serum (lanes a, c and e). The a415 antibody recognises a 17 and 19 kDa doublet in all fractions of the strain LV39 (Fig. 2A, lanes, b, d and f). In the IR75 strain, the 17 kDa protein is more abundant than the 19 kDa band (Fig. 2B, lanes b, d and f). However, the 19 kDa species is

In order to confirm the nuclear localization of the SW3 protein shown earlier, immunoblotting of cellular subfractions as well as indirect immunofluorescence of L. major promastigotes and amastigotes were performed. Cell lysates for immunoblotting were prepared from L. major promastigotes as described in Section 2, and cytoplasmic (Fig. 3, lanes a and b) and nuclear (lanes c and d) fractions isolated by centrifugation. Fractions were separated by SDSPAGE, transferred to nitro-cellulose and analyzed by immunodetection. a415 antibodies detected a 17 and 19 kDa doublet only in the nuclear subfraction (lane d), and not in the cytoplasmic fraction (lane b). Pre-immune serum was used as a negative control in this experiment (lanes a and c). Indirect immunofluorescence using an a415 antibody was carried out to confirm the nuclear localization of the SW3 protein in L. major LV39 promastigotes (Fig. 4). An affinity purified a415 antibody (see Section 2), and a secondary FITC labeled anti-rabbit antibody were used. For each sample, the intracellular location of the kineto-

Fig. 4. The nuclear localization of the sw3 gene product by indirect immunofluorescence. Promastigotes from stationary phase in vitro cultures were spread onto polylysine treated slides, fixed with acetone-methanol (1:1) and stained as described in Materials and Methods. Phase contrast (panel A), nuclei and kinetoplast staining (Hoechst) (panel B) and immunofluorescence of promastigotes (strain LV39) stained with the a415 antibody (panel C) are represented. Analysis by confocal immunofluorescence is shown in Panels D to L. The promastigote histone H1 staining patterns are shown in Panels D to F. Panels G to I represent the competition of the a415 antibody binding to SW3 with 1 mg ml − 1 415 peptide in promastigotes, and Panels J to L represent the negative control where the first a415 antibody was omitted from the staining of promastigotes. The nucleus and kinetoplast were stained with 1 mg ml − 1 ethidium bromide mounting medium seen in red (Panels D, G and J) or white (Panels E, H and K). Detection of histone H1 by a second anti-rabbit Ig antibody conjugated with FITC is seen in green (Panel D) or white (Panels F). From left to right, the first column (Panels D, G and J) represents in color the double labelling with FITC (green) and ethidium bromide (red). The second and third column represents in black and white ethidium bromide staining only (Panels E, H and K) and FITC staining only (Panels F, I and L) respectively. Size bars are corresponding to 5 mm in panels A – F and 10 mm in panels G – L.

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plast and nucleus was established by ethidium bromide or Hoechst dye staining. As shown in the Fig. 4 (panel A– C), a comparison of the ethidium staining and of the immunofluorescence staining due to the presence of the SW3 polypeptide suggests a localization of the histone H1 in the nucleus. It can be pointed out that, although localized in the nucleus as suggested by analysis of the phase contrast image shown in Fig. 4A, both staining do not overlap perfectly suggesting a defined localization of histone H1 in the nucleus. To obtain a better representation of the SW3 localisation, we performed confocal microscopy. The SW3 protein localized to the nuclear periphery as evidenced by the green staining (representing histone H1) versus the red staining corresponding to ethidium bromide staining, and gave rise to a punctated type of immunofluorescence in promastigotes (panels C, D and F). In panels G–I, competition of the a415 antibody binding using synthetic 415 peptide, as well as labelling with the secondary antibody alone (Panels J to L) are shown as controls. It has been shown that condensed chromatin is in contact with the nuclear envelope during interphase and throughout mitosis in trypanosomes [36]. Thus, this type of immunofluorescence pattern could reflect the co-localization of the SW3 protein with condensed chromatin. No detectable differences in staining patterns were observed in amastigotes or in promastigotes of another Leishmania strain such as IR75 (data not shown). Thus, further evidence that the SW3 product is indeed a Leishmania H1 arises from its specific extraction from parasite cell lysates using immunoblotting as well as from immunofluorescence studies showing that the SW3 gene product was localized to the periphery of the nucleus, forming a pattern reminiscent of the distribution of heterochromatin in the nucleus of parasites [15,37].

3.4. H1 mRNA accumulation during parasite differentiation To investigate the variation in the expression of the sw3 gene during parasite differentiation, we quantified the steady state sw3 RNA by Northern blot analysis. It has been shown that during in

vitro growth of promastigotes from logarithmic to stationary phase, a sequential transformation from uninfectious procyclic forms to metacyclic infectious forms occurs [38]. This observation led us to investigate H1 expression at the RNA level during the transformation of amastigotes into promastigotes (Fig. 5A), as well as during the differentiation of promastigotes in liquid culture (Fig. 5B). LV39 amastigotes were isolated from infected BALB/c mice, and were left to transform into promastigotes in liquid culture. Cytoplasmic RNA was extracted from isolated amastigotes (Fig. 5A, lane a) and differentiating amastigotes after 5 (lane b), 8 (lane c), 12 (lane d), 24 (lane e), 48 (lane f) and 72 (lane g) h in culture. Samples were separated on a 0.8% agarose gel, transferred to a Gene Screen Plus™ membrane (NEN® Research Products) and probed using 32P-labelled sw3 RNA probes. Membranes were also probed with an a-tubulin riboprobe as a control for stable mRNA levels [38,39]. Hybridizing mRNA was detected as a broad band which could be due to the presence of two mRNAs species. As shown in the Fig. 5A, the level of steady state mRNA decreased when amastigotes were left to transform into promastigotes. No other significant variation in the sw3 RNA level could be observed during this differentiation process. Cytoplasmic RNA was also extracted from promastigotes grown at different cell densities, 5 × 106 cells ml − 1 (early logarithmic) (Fig. 5B lane a), 1.6 × 106 cells ml − 1 (logarithmic) (lane b), 4.7× 107 cells ml − 1 (early stationary) (lane c) and 7.3×107 cells ml − 1 (stationary) (lane d), and analyzed as described above. Sw3 mRNA accumulated during promastigote growth in vitro from logarithmic to stationary phases of growth (Fig. 5B). Quantitative analysis of Northern blots was carried out by densitometric scanning (measurements were performed under conditions where a linear correlation exists between the amount of the various mRNAs and the intensity of the bands on the autoradiograms) to quantitate the variation in the level of sw3 transcripts during promastigote differentiation. A two to three fold increase in sw3 mRNA level was observed during promastigote differentiation (Fig. 5C). In amastigotes transforming into promastigotes, the level of sw3

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Fig. 5. Northern blot analysis of the differential expression of sw3. (A) Cytoplasmic RNA (15 mg) from amastigotes isolated from lesions (lane a) and amastigotes left to differentiate in liquid culture for 5 (lane b), 8 (lane c), 12 (lane d), 24 (lane e), 48 (lane f) and 72 h (lane g), as well as cytoplasmic RNA isolated from promastigotes (B) grown to early logarithmic (5× 106 cells ml − 1, lane a), logarithmic (1.6× 106 cells ml − 1, lane b), early stationary (4.7 × 107 cells ml − 1, lane c) and stationary phase (7.3 ×107 cells ml − 1, lane d) were separated on a 0.8% agarose gel, transferred to Genescreen plus and hybridized to a 32P-labelled sw3 or an a-tubulin anti-sense riboprobe. Densitometric scans (C) of the Northern blot of differentiating promastigotes: expression of SW3 mRNA is standardized to the expression of the a-tubulin gene.

mRNA gradually decreased to reach levels observed in promastigotes cultured at low density.

3.5. The differential expression of the Sw3 protein Nuclear fractions of differentiating promastigotes, as well as amastigotes from L. major strains LV39 and IR75 were analyzed by SDS-PAGE and immunoblotting using the a415 antibody in order to correlate the differential expression of the sw3 gene at the RNA (Fig. 5) and protein level. LV39 promastigote nuclear lysates were prepared from parasites grown in vitro from logarithmic to stationary phase (Fig. 6A). Equivalent amounts of protein were transferred to nitrocellulose as determined by Ponceau S staining of membranes after protein transfer (data not shown). Fig. 6A shows

that the level of H1 expression gradually increases when promastigotes grow from low (6.0× 106 cells ml − 1) to high density (7.3 ×107 cells ml − 1) (lanes a–d). Nuclear lysates were also prepared from IR75 amastigotes isolated from BALB/c mice lesions (Fig. 6B, lane a) and from amastigotes left to transform to promastigotes in liquid culture for 24, 48 and 72 h in lane b, c and d respectively, after their isolation from lesions. The promastigotes were then grown at low density and used to obtain parasites at logarithmic (lane e) and stationary phases (lane f). In contrast to Fig. 6A, the analysis was performed with fixed numbers of cells loaded per lane. This figure shows that the expression decreases dramatically when amastigotes differentiate back to promastigotes (Fig. 6B, lanes a–d) but there is an increase in expression

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Fig. 6. Histone H1 expression fluctuates during the differentiation of Leishmania major. (A) Histone H1 content in differentiating promastigotes. Nuclear lysates of promastigotes grown at DENSITY 1 (lane a), density 2 (lane b) etc. Promastigotes cultured in low density conditions were grown to logarithmic density (lane e) or stationary density lane f (B) Amastigotes isolated from a lesion (lane a) or from amastigotes differentiating in liquid culture for 24 (lane b), 48 (lane c) and 72 h (lane d) were fractionated on a 12.5% SDS-polyacrylamide gel, transferred to nitro-cellulose membrane and immunodetection was performed using a415 antibodies. (C and D) The accumulation of the sw3 gene product during promastigote differentiation occurs early in metacyclogenesis. Nuclear lysates (C) and total cell lysates (D) from promastigotes grown to logarithmic (6.3 × 106 cells ml − 1, lane a), early stationary (3.6 ×107 cells ml − 1 , lane b) and stationary phase (6.5 ×107 cells ml − 1, lane c) were separated on a 12.5% SDS-PAGE gels, transferred to nitro-cellulose, and immunodetection was performed using a415 antibody (A) or Ab336 antibody (B) respectively. Equivalent amounts of protein were loaded, and the molecular mass markers 14.4, 21.5, 31.0 and 45 kDa (Bio-Rad, low range) are indicated.

when procyclic promastigotes differentiate into metacyclic promastigotes (Fig. 6B, lanes e and f) correlating with the developmental expression of histone H1 at the mRNA level. It should also pointed out that there is no difference in the expression pattern between LV39 and IR75.

3.6. H1 accumulates early in metacyclogenesis The hydrophilic surface protein, gene B product, has been previously identified in L. major as metacyclic specific [40]. In order to define the timing of H1 induction in the succession of events during metacyclogenesis, we monitored the appearance of the infectious form of the parasites in promastigote liquid cultures using the Ab336 anti-

body (kindly provided by D.F. Smith) directed against the metacyclic specific gene B protein (Fig. 6D). Appearance of the metacyclic specific surface protein and SW3 protein were analyzed simultaneously in promastigotes growing in vitro (Fig. 6C and D). Nuclear extracts (Fig. 6C, lanes a–c) and total cell lysates (Fig. 6D, lanes a–c) of promastigotes grown at various densities were analyzed by gel electrophoresis and immunoblotting with a415 (Fig. 6C) or Ab336 (Fig. 6D) antibodies. Fig. 6 shows that in both instances, the level of expression of histone H1 as suggested by the amount of 17 and 19 kDa species is increasing during differentiation towards the metacyclic stage. Thus, the accumulation of the H1 protein follows an expression pattern very

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similar to the expression pattern of metacyclic gene B protein.

4. Discussion The sw3 cDNA clone has a deduced amino acid sequence which has a composition similar to H1 histones identified in other trypanosomatids. Although similar in amino acid composition to H1 histones of other eukaryotes, it lacks a characteristic central hydrophobic globular domain [26]. The biochemical and immunocytochemical characterization of histone H1 presented in this paper represents the first report of a nuclear histone H1 in Leishmania, and simultaneously the first account of histone H1 mRNA and protein variation throughout the life-cycle of a trypanosomatid parasite. We generated an antibody to the amino terminal peptide of SW3 (a415) which recognizes a 17 and 19 kDa protein doublet in both promastigotes and amastigotes of the L. major strain LV39. This doublet reflects the presence of two histone H1 polypeptides possessing similar epitopes, possibly arising from two mRNA transcripts. Although we can not definitively exclude that they arise as a result of a post-translational phosphorylation of SW3 as seen for higher eukaryote H1 histones, we tend to believe that the size difference between the 17 and 19 kDa proteins is more likely to be the result of expression from different genes than post-translational modification of one gene product. This assumption is supported by recent results confirming the presence of different copies of the sw3 gene which could code for the different polypeptides (S. Belli and N. Fasel, unpublished results). Size polymorphisms of the SW3 protein have been observed amongst different strains of L. major as shown in the results. Specifically, there is a difference in the ratio between the 17 and 19 kDa proteins in LV39 and IR75. IR75 expresses much less of the 19 kDa protein, and it is only seen on immunoblots after a long exposure after chemiluminescence. Even though the amount of the 19 kDa protein is lower than that seen in the LV39 strain, this protein is still developmentally regulated as seen for the 17 kDa protein. RNAse

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protection assays and the characterization of the genomic copies of the sw3 gene show preliminary evidence to suggest that the genome of IR75 contains a sw3 gene copy which could code for a 19 kDa SW3 protein slightly different from the 19 kDa protein present in LV39, and which may not be efficiently recognised by our a415 antibody (T. Noll and N. Fasel, unpublished results). As yet, it is unclear as to why there is this size and probably sequence polymorphism and if it plays any functional role in the chromatin condensation pattern. The H1 protein that we have described is regulated throughout parasite differentiation. The accumulation of H1 mRNA and protein in non dividing metacyclic promastigotes and in amastigotes was observed. Moreover, despite an overall two to three fold increase in sw3 steady state RNA expression between procyclic promastigotes and amastigotes or metacyclic promastigotes, the level of expression of other H1 gene copies appears to be increased up to ten fold during promastigote maturation as measured by RNAse protection assays (T. Noll and N. Fasel unpublished results). This suggests that other copies of H1 are down-regulated to obtain a global change in expression of two to three fold. If this difference in sw3 mRNA steady state plays an essential role compared to translational control will be unravelled by characterization of the expression of the individual copies. The level of H1 histone is already abundant prior to complete promastigote differentiation into metacyclic forms, where the levels of the SW3 protein and stationary promastigote marker gene B product [40] were compared. Histone H1 proteins may be involved in the reversible interruption of parasite proliferation during metacyclogenesis. In T. brucei, histone H1 variants have been postulated to participate in the regulation of cell proliferation and differentiation [41] as has been postulated for higher eukaryotes [23]. The presence of specific H1 variants in stationary stages of the parasite’s life cycle is reminiscent of the accumulation of H1 subtypes during terminal differentiation in many eukaryotic systems [13,42,43]. Interpretation at the level of induction of expression of histone H1 is however rendered difficult by the fact that the total amount of

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protein per cell decreases in the metacyclic stages compared with procyclic forms [44]. Thus, it is possible that the amount of histone H1 remains constant at a single cell basis while the amount of other proteins decreases. In Fig. 6A, equivalent amounts of protein have been loaded, whereas in Fig. 6B, fixed numbers of cells per lane have been analysed. In both cases, an increase in histone H1 is detected suggesting that histone H1 is upregulated in expression in every cell, and its higher level of expression is only partially due to reduction of other proteins. It should also be pointed out that histone H1 expression could be linked to the cell cycle. Although it is not possible yet to analyse synchronous populations of Leishmania, analysis of promastigotes freshly derived from amastigotes can give preliminary indications since in this population, asynchrony in the cell cycle could be reduced. Our results tend to show that in this case the variation in expression of histone H1 is more dramatic. Thus, interruptions of cell cycle may play an important role in trypanosomes and other protozoan parasites, not only with respect to cellular proliferation, but also during progression through their developmental system controlling infectivity and survival in the host by transition to a non-dividing stage, preadapted for survival within the next host and preceding the next proliferative phase. It could also be the case in the transformation process from amastigote to promastigote in which no division occurs during the first 24 h [45]. Further investigation is now possible to define the role and importance of the described H1 and other variants in such developmental and cell cycle controls as adaptations of the parasite to the changing of environmental conditions.

Acknowledgements We thank Dr Paola Romagnoli, Thierry Laroche and Michael Schro¨ter for assistance with immunofluorescence experiments and confocal microscope imagery as well as M. Allegrini and P. Dubied for their photographic skills. We also thank Dr Deborah Smith (Imperial College, Lon-

don) for kindly providing us with the Ab336 antibody and Prof. Peter Overath for his advice in the immunolocalization studies. This work was supported by grants of the Swiss National Fund for Scientific research No 31-36343-92 for N. Fasel, No 31-040881.94 for C. Bron and N.F., and No 31-40712-94 for J. Maue¨l and T. Glaser, as well as by special grants of Sovarec S.A. and the foundation ‘Recherche et Sante´’ to N. Fasel.

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