Correspondence: Partha Saha, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata 700064, India. Tel.: +91 33 2337 0379; fax: +91 33 2337 4637; e-mail: email@example.com
Histone acetyl transferases (HATs) are important histone modifiers that affect critical cellular processes like transcription, DNA replication and repairs through highly dynamic chromatin remodelling. Our earlier studies recognized LdHAT1 as a substrate of the S-phase cell cycle kinase LdCyc1-CRK3 from Leishmania donovani. Here, we confirm through site-directed mutagenesis that RXL-like cyclin-binding (Cy) motif dependent interaction of LdHAT1 with LdCyc1 is essential for its phosphorylation at a canonical Cdk target site by the kinase complex. LdHAT1 acetylates K10 residue of a peptide derived from L. donovani histone H4 N-terminal tail. Interestingly, phosphorylation of LdHAT1 by the S-phase kinase inhibits its H4K10 acetylation activity, implicating an important mechanism of periodic regulation of histone acetylation during cell cycle progression.
Chromatin remodelling through various post-translational modifications such as acetylation, methylation, phosphorylation and ubiquitinylation of protruding histone tails of nucleosomal octamer controls access of the factors affecting transcription, replication and DNA repair (Ehrenhofer-Murray, 2004; Osley, 2004; Peterson & Laniel, 2004; An, 2007). The modifications also provide recognition sites for the plethora of protein factors facilitating DNA repair and regulated flow of genetic information. By and large, histone acetylation on lysine residues is important to disrupt the tight packing of chromatins essential for the initiation of processes like transcription. Expectedly, higher proportions of the acetylated histones are associated with promoter region of active genes compared to coding regions and silent portions of genomes. Moreover, several recent studies demonstrate the role of histone modifications in regulation of initiation of DNA replication. Studies in Drosophila (Aggarwal & Calvi, 2004) and Xenopus (Danis et al., 2004) have established the positive regulation of replication through histone acetylation. Direct involvement of the MYST family histone acetylase HBO1 in regulation of replication licensing through the formation of pre-replication complex has been shown (Miotto & Struhl, 2008). The preference of open chromatin structures with enriched histone H3 methylation and acetylation at metazoan origin has also been established recently (Rampakakis et al., 2009; Karnani et al., 2010). On the contrary, histone deacetylase Sir2 has been shown to interfere with pre-replicative complex (pre-RC) assembly in budding yeast regulating replication in a negative manner (Fox & Weinreich, 2008).
The MYST family is composed of a group of widely distributed but related histone acetyl transferases (HATs) that are involved in diverse cellular activities including activation of transcription, DNA repair, pre-replication complex formation and transcription silencing (Sapountzi & Cote, 2011). The MYST (derived from human MOZ, yeast Ybf2 or Sas2 and Sas3 and mammalian TIP60) family members contain a characteristic MYST domain including the canonical acetyl-CoA binding motif (A-motif) as well as a C2HC Zn-finger. The MYST HATs also contain other conserved domains like chromodomain and plant homeodomain for specific functions. One notable member of the family TIP60, a tumour suppressor, has been shown to be recruited at the DNA double-strand break site through the interaction of its chromodomain with histone H3 trimethylated on lysine 9 (H3K9me3) resulting in the activation of ATM kinase and initiation of repair (Sun et al., 2009). The HAT activity of the TIP60 has also been shown to be regulated through phosphorylation by cyclin B2/cdc2 (Lemercier et al., 2003), although its significance in cellular processes is not known. Hbo1, another important MYST family HAT, has been demonstrated to be essential for Cdt1-assisted loading of minichromosome maintenance (MCM) proteins to form pre-RC at eukaryotic replication origin (Miotto & Struhl, 2010).
Genome sequencing has revealed that four MYST family HATs are encoded by genomes of Leishmania major and Trypanosoma cruzi and three by that of Trypanosoma brucei (Ivens et al., 2005). The early branching trypanosomatid parasites including T. brucei, T. cruzi and Leishmania spp. cause potentially fatal diseases like sleeping sickness, Chagas disease and leishmaniases, respectively, affecting millions of people worldwide (Chatelain & Ioset, 2011). These parasites have many unique features in their biphasic life cycle such as concerted replication of nuclear genome and kinetoplastid DNA in a single copy of mitochondria, polycistronic message formation and nearly complete dependence on the post-transcriptional mechanism for differential gene expression (Gull, 2001; Hammarton et al., 2003). In these organisms, the tails of core histones have divergent sequences compare to other eukaryotes (Alsford & Horn, 2004), and unusual modifications of the histones are also observed in several experiments (Janzen et al., 2006; Mandava et al., 2007). One of the MYST HATs TbHAT3 acetylates histone H4K4, although it is dispensable for growth (Siegel et al., 2008). Among the other MYST HATs, TbHAT1 is essential for telomeric silencing, and its involvement in DNA replication has also been implicated. TbHAT2, the other MYST HAT, is required for H4K10 acetylation and growth (Kawahara et al., 2008).
Recently, we have identified a putative HAT from Leishmania donovani, which is highly homologous to TbHAT1, during a search for potential substrates of a previously characterized S-phase cell cycle kinase LdCyc1-CRK3 (Banerjee et al., 2003, 2006; Maity et al., 2011). We term the protein as LdHAT1 and show by site-directed mutagenesis that it directly interacts with LdCyc1 through an RXL-like Cy-motif (Chen et al., 1996). LdHAT1 gets phosphorylated by the kinase on a specific threonine residue, and its acetyl transferase activity is modulated by such phosphorylation, suggesting a possible mechanism of regulation of chromatin remodelling by the S-phase cell cycle kinase.
Materials and methods
Leishmania donovani promastigotes strain AG83 (MHOM/IN/83/AG83) was grown in M199 medium (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen) and penicillin–streptomycin mixture at 22 °C with slow shaking.
Preparation of GST-LdCyc1-CRK3 active kinase complex
To prepare GST-LdCyc1-CRK3 kinase complex, bacterially expressed GST-LdCyc1 was first bound to glutathione–sepharose beads (GE Healthcare Lifesciences), and the bead-bound GST-LdCyc1 was then incubated with an extract of Sf9 cells expressing LdCRK3 in the binding buffer (50 mM Tris-HCl, pH 8.0 containing 50 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 0.1 mM EDTA, 0.1% Triton X-100, 10% glycerol, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl flouride (PMSF) and protease inhibitors) on a rotating wheel at 4 °C for overnight. The beads were washed three times with the same binding buffer, and the kinase complex was eluted with 50 mM Tris-HCl, pH 8.0, containing 10% glycerol, 10 mM reduced glutathione and PMSF.
Cloning and expression of LdHAT1
The complete ORF of LdHAT1 was cloned into pET21b vector, and the C-terminal 6His-tagged chimera was expressed in pG-JKE8 (TAKARA)-transformed Escherichia coli strain BL21 cells (expressing GroEL and GroES chaperons for greater solubility of the over-expressed protein) by induction with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37 °C for 3 h. Two mutants of LdHAT1, viz., LdHAT1ΔCy and LdHAT1-T394A were also cloned into pET21b vector and expressed as mentioned above. All 6His-tagged proteins were purified over Ni-NTA agarose beads.
Protein interaction assay
To perform interaction assay between LdCyc1 and LdHAT1, 5 μg of bacterially purified LdHAT1 protein was incubated on a rotating wheel at 4 °C for 1 h with glutathione beads bound to 0.2 μg of either GST or GST-LdCyc1 proteins in 50 mM Na-phosphate (pH 8.0) containing 250 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, 2 mM DTT and protease inhibitors. Subsequently, the beads were washed six times with the same buffer, and the bound proteins were analysed by immunoblot analysis with appropriate antibodies. Similar experiments were carried out with the mutant LdHAT1 proteins.
Cell cycle analysis
To synchronize L. donovani promastigotes, exponentially growing cells were blocked with 10 mM hydroxyurea (HU) for 36 h followed by releasing the arrest by re-suspending the cells in equal volume of growth medium, and cells were collected at different intervals. The synchronicity of cell cycle progression was confirmed by analysis in a flow cytometer (Supporting Information, Fig. S3). The population of cells from each time point was also examined by analysing the fluorescence and differential interference contrast (DIC) images of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells captured by a Zeiss Axio-observer Z1 inverted microscope (Fig. S3). Cells from different time intervals were lysed in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 0.2% Triton X-100, 1 mM EDTA and protease inhibitors, and the soluble extracts were analysed by immunoblotting with antibodies against LdHAT1 (mouse polyclonal) and actin.
The kinase assays were carried out with the increasing amount of GST-LdCyc1-CRK3 complex in 20 mM HEPES-KOH, pH 7.5, containing 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 5 mM NaF, 2 mM DTT, 50 μM [γ32P]ATP (2.7 μCi/nmole) and 1.0 μg of LdHAT1 in a total volume of 15 μL at 30 °C for 30 min. For the assays with the mutated proteins, LdHAT1ΔCy and LdHAT1-T394A were incubated with 0.2 μg of kinase GST-LdCyc1-CRK3 in the reaction buffer. The reaction products were analysed by SDS-PAGE followed by phosphorimager scanning in Typhoon scanner (GE Healthcare Lifesciences).
Generation of antibodies against acetylated peptides derived from L. donovani histone H4
Three peptides derived from N-terminus of L. donovani histone H4–containing specific acetylated lysine (LdH4K4Ac: AKGKAcRSADAC; LdH4K10Ac: SADAKAcGSQKC; LdH4K14Ac: KGSQKAcRQKKC) were synthesized and conjugated to carrier protein keyhole limpet haemocyanin. For each peptide, two rabbits were immunized, and the progress of immunization was monitored by ELISA assay. Specific antibodies were purified from the anti-sera having higher titre values through affinity column chromatography in a two-step process – first over a column containing a control non-acetylated peptide (AKGKRSADAKGSQKRQKKC) followed by a column containing the respective acetylated peptide. The specificities of the purified antibodies were checked by ELISA assay. The entire process was carried out by IMGENEX India, Bhubaneswar, India, on contract basis. The specificities of the antibodies were further verified by dot blot analysis in our laboratory.
HAT assay was performed with 1.6 μM of 6His-tagged LdHAT1 as enzyme in 50 mM HEPES-KOH, pH 8.0, containing 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 10 mM Na-butyrate, 0.1 mM Li3Acetyl-CoA and 50 μM of a peptide derived from L. donovani histone H4 N-terminus (AKGKRSADAKGSQKRQKKC) as substrate in a total volume of 20 μL. The reaction was carried out at 30 °C for 1 h, stopped by adding 5 μL of SDS-PAGE sample buffer, and the products were subjected to a modified Tris-Tricine SDS-PAGE for better resolution of smaller peptides (Schagger & von Jagow, 1987). Briefly, 18% polyacrylamide (18%T, 5%C) in 0.75 M Tris-HCl, pH 8.45, containing 30% ethylene glycol and 0.1% SDS was used as resolving gel with 0.1 M Tris containing 0.1 M Tricine and 0.1% SDS as electrophoresis buffer. Finally, the acetylated peptide was detected by immunoblotting with the antibodies raised against the peptides containing specific acetylated lysine residues as described above. The antibodies obtained after purification over non-acetylated peptides or Coomassie blue staining of the gel were used for checking the presence of equal amount of substrate peptide in different reactions.
Results and discussion
LdHAT1, a MYST family HAT, expresses at a constant level during cell cycle
One of the identified substrates of the S-phase cell cycle kinase LdCyc1-CRK3 from L. donovani was shown to contain a MYST (human Moz, Yeast Ybf2 and Sas2, and human TIP60) domain of HATs (Maity et al., 2011), although further characterization of activity of the protein and its regulation by phosphorylation were not carried out. The HAT protein from L. donovani is 97% identical to LmHAT1, which was grouped with the HAT1 from T. brucei and T. cruzi in a phylogenetic analysis (Kawahara et al., 2008). Therefore, we designate the 525 amino acid–containing protein as LdHAT1, which is also highly homologous to other MYST family HATs from diverse organisms (Fig. 1a and Fig. S1). Maximum homology is present along the C-terminal canonical MYST domain (amino acid 254–456 of LdHAT1), which contains the characteristic acetyl-CoA binding R/Qx2GxG/A-motif (A-motif). Like other family members, on the N-terminus of the MYST domain, the conserved C2HC (Cx2Cx12Hx3–5C) Zn-finger motif is also present in LdHAT1. As previously described (Maity et al., 2011), the cyclin-binding RXL-type Cy-motif (Chen et al., 1996) is located within the MYST domain in LdHAT1, although such a typical motif is absent in HsTIP60, DmMof and HsHbo1. However, a canonical Cdk target phosphorylation site (TPEK) is well-conserved within the MYST domain of LdHAT1 and in the other MYST family members (Fig. S1). In addition to the canonical Cdk phosphorylation site, five minimal sites (T/S-P) are also present in the molecule in a scattered manner. Interestingly, catalytically critical Glu residue, corresponding to Glu338 in the prototype yeast Esa1 (Berndsen et al., 2007), is located within the canonical Cdk target site (TPEK), implicating an interesting regulatory mechanism if the Thr residue is actually phosphorylated by cell cycle kinases. Moreover, similar to HsTIP60 and DmMof, a Chromatin Organization Modifier (chromo) domain is located towards the N-terminus of LdHAT1. Chromodomain of heterochromatin protein HP1 was shown to interact with methylated lysine9 residue of histone H3 to recruit the regulator at appropriate location (Jacobs & Khorasanizadeh, 2002; Nielsen et al., 2002). Chromodomain can also function as RNA-interacting module to target the regulators to the specific chromosome site as was observed in case of DmMof (Akhtar et al., 2000). The presence of chromodomain in LdHAT1, therefore, raises its possible role in crosstalk between methylation and acetylation histones and/or RNA-mediated chromatin remodelling in the parasites.
As phosphorylation of LdHAT1 by S-phase Cdk raised the possibility of its involvement in cell cycle–related periodic activities, its expression profile was analysed during cell cycle progression of L. donovani promastigotes. Polyclonal anti-sera against the purified LdHAT1 were raised in mice, and one of them was shown to detect a specific band of expected size in immunoblot analysis with the extract of L. donovani promastigotes (Fig. S2). The same anti-serum was used subsequently to analyse extracts from the synchronized cells. Uniform changes in morphology of the cell population with the progress of cell cycle along with corroborating flow cytometer analysis profiles ascertained the synchronicity of the promastigote culture (Fig. S3). Analysis of protein extracts from such synchronously growing cells showed the presence of LdHAT1 protein at equal amounts in different cell cycle phases of L. donovani promastigotes (Fig. 1b). As the level of LdHAT1 found to be invariable during cell cycle, it would be interesting to study the effect of phosphorylation by the S-phase kinase on its activity.
LdHAT1 directly interacts with LdCyc1 through its Cy-motif
LdHAT1 was shown previously to interact with L. donovani S-phase cyclin LdCyc1 in a RXL-like Cy-motif-dependent manner by peptide competition assay (Maity et al., 2011). To further confirm the contribution of Cy-motif in the interaction, the putative Cy-motif of LdHAT1 was altered (290RRLVV→RDDVV, LdHAT1ΔCy), and the mutated protein was used in the interaction assay. As shown in Fig. 2a, the wild-type protein was found to interact with GST-LdCyc1, whereas the interaction with LdHAT1ΔCy was almost completely abolished, proving the involvement of Cy-motif during direct interaction between the proteins. The observation also confirmed the identity of an active Cy-motif in the molecule. The mutation at the putative Cdk phosphorylation site (394TPEK→APEK, LdHAT1-T394A) of the protein did not affect the interaction (Fig. 2a), confirming further the specific involvement of Cy-motif in the binding.
Cy-motif of LdHAT1 is required for its phosphorylation by S-phase cell division kinase
LdHAT1 was demonstrated to be phosphorylated in vitro by LdCyc1-CRK3 complex (Fig. 2b) (Maity et al., 2011). As the substrate docking on the cyclin moiety was shown to be important for phosphorylation, to investigate the effect of Cy-motif of LdHAT1 on its phosphorylation, LdHAT1ΔCy was used as substrate in a kinase assay of LdCyc1-CRK3 complex. As observed, LdHAT1ΔCy was not efficiently phosphorylated by the kinase complex compared to the wild-type protein (Fig. 2c, lanes 4 and 5). As the mutation in Cy-motif of LdHAT1 was shown to disrupt its interaction with LdCyc1 (Fig. 2a), the inhibition of the phosphorylation established the requirement of its docking through the Cy-motif on MRAIL-motif on LdCyc1 (Banerjee et al., 2003) for the phosphorylation on the target serine/threonine residue. LdHAT1 was also shown to contain a putative Cdk phosphorylation site on its C-terminal end. To confirm whether Thr-394 in the motif TPEK was phosphorylated by the kinase complex, the threonine residue was changed to alanine, and the mutant LdHAT1-T394A was used as substrate. As shown in Fig. 2c, the phosphorylation was completely abolished because of the mutation (lane 6), suggesting that the S-phase kinase LdCyc1-CRK3 targets Thr-394 for phosphorylation. It is interesting to note that Thr-394 is located very close to conserved catalytically critical Glu residue raising the possibility of regulation of HAT activity because of the incorporation of a phosphate group. Therefore, it is important to study the effect on the activity of LdHAT1 by phosphorylation of the Thr residue by the cell kinase.
Phosphorylation of LdHAT1 by S-phase kinase down-regulates its L. donovani histone H4-K10 acetylation activity
It was previously implicated that HAT1 from T. brucei could acetylate histone H4 from the parasite (Kawahara et al., 2008). Therefore, to characterize the histone acetylation activity of LdHAT1, in vitro assays were carried out using a peptide substrate derived from the N-terminus of L. donovani histone H4. To identify the lysine residue that was specifically acetylated by LdHAT1, three antibodies were raised against L. donovani histone H4–derived peptides acetylated on K4, K10 or K14 residue, respectively. Specificities of the antibodies were ensured by dot blot analysis, which showed no cross-reactivity (Fig. 3a). Once the specificities were confirmed, the antibodies were used to identify the lysine residue on the peptide derived from N-terminus of L. donovani histone H4 acetylated by LdHAT1. As shown in Fig. 3b, the peptide acetylated by LdHAT1 could be detected only by anti-H4K10Ac antibody, but not with other two antibodies (data not shown), suggesting that the acetyltransferase from L. donovani specifically acetylates H4K10 residue. As LdHAT1 was shown to be phosphorylated by S-phase kinase LdCyc1-CRK3, it would be interesting to investigate any possible effect of such phosphorylation on its histone acetylation activity. To explore such a possibility, H4K10 acetylation activity of non-phosphorylated LdHAT1 was compared with that of LdHAT1 phosphorylated by LdCyc1-CRK3. As depicted in Fig. 3c, the phosphorylated form of LdHAT1 did not show any H4K10 acetylation activity, suggesting the regulation of histone H4 acetylation by S-phase cell cycle kinase. Intriguingly, LdHAT1ΔCy and LdHAT1-T394A mutants also did not show any acetylation activity (Fig. 3d) implicating the contribution of the mutated residues in the enzymatic activity.
Previous studies demonstrated the acetylation of K4, K10 and K14 residues of the N-terminal tail of histone H4 from T. brucei and T. cruzi (da Cunha et al., 2006; Janzen et al., 2006; Mandava et al., 2007). Moreover, cell cycle-dependent post-S-phase enhancement of K4, K10 and K14 acetylation of histone H4 was observed in T. cruzi (Nardelli et al., 2009). Therefore, the observed inhibition of histone H4K10 acetylation by LdHAT1 owing to its phosphorylation by S-phase kinase in the present studies could correlate such cell cycle–specific periodic acetylation. It will be interesting to study the effect of inhibition of the HAT activity on the S-phase events.
The work was partially supported by the project grant [37(1044)/00/EMR-II] from Council of Scientific and Industrial Research (CSIR), India and intramural grant from Department of Atomic Energy, Government of India.
Conflict of interest
The authors have no conflict of interest to declare.