Gene 538 (2014) 8–11
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Identification of the centromere-specific histone H3 variant in Lotus japonicus Ahmet L. Tek ⁎,1, Kazunari Kashihara 1, Minoru Murata 1, Kiyotaka Nagaki 1 Institute of Plant Science and Resources, Okayama University, Kurashiki 710–0046, Japan
a r t i c l e
i n f o
Article history: Accepted 11 January 2014 Available online 22 January 2014 Keywords: Centromere CenH3/CENP-A Green fluorescent protein Leguminosae Fabaceae
a b s t r a c t The centromere is a structurally and functionally specialized region present on every eukaryotic chromosome. Lotus japonicus is a model legume species for which there is very limited information on the centromere structure. Here we cloned and characterized the L. japonicus homolog of the centromere-specific histone H3 gene (LjCenH3) encoding a 159-amino acid protein. Using an Agrobacterium-based transformation system, LjCenH3 tagged with a green fluorescent protein was transferred into L. japonicus cells. The centromeric position of LjCENH3 protein was revealed on L. japonicus metaphase chromosomes by an immunofluorescence assay. The identification of LjCenH3 as a critical centromere landmark could pave the way for a better understanding of centromere structure in this model and other agriculturally important legume species. Published by Elsevier B.V.
1. Introduction The centromere is a structurally and functionally specialized chromosome region that forms the kinetochore, a multiprotein complex. Many kinetochore proteins are conserved among eukaryotes and perform processes essential to chromosome function, including attachment, generation of motive force, and cell cycle regulation, among others. In contrast to the functional conservation at centromeres, centromere DNA sequences are highly variable (Henikoff et al., 2001). Histone proteins, including histone H3, are ubiquitous components of DNA–protein complexes. Only at eukaryotic centromeres wrapping the centromeric DNA, there is a specialized variant of histone H3 protein, called centromere-specific histone H3, CENH3 (Henikoff et al., 2001). CENH3 aids in kinetochore–DNA association, and indeed functional centromeres are defined by the presence of CENH3 (Henikoff et al., 2001). CENH3 homologs have been identified and functionally characterized in many plant species, such as the model plant Arabidopsis, Arabidopsis thaliana (Talbert et al., 2002); soybean, Glycine max (Tek et al., 2010); Chinese milk vetch, Astragalus sinicus (Tek et al., 2011); common bean, Phaseolus vulgaris (Iwata et al., 2013). Structural features of CENH3 provide important insights into the establishment and function of centromeres. CENH3 is distinguished from the canonical
Abbreviations: CenH3, centromere-specific histone H3; LjCenH3, L. japonicus centromere-specific histone H3; HFD, histone fold domain; GFP, green fluorescent protein; Mbp, megabase pair; FISH, fluorescence in situ hybridization; cDNA, complementary DNA; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; UTR, untranslated region; DAPI, 4′,6-diamidino-2-phenylindole. ⁎ Corresponding author at: Department of Agronomy, Faculty of Agriculture, Harran University, Şanlıurfa, 63300, Turkey. Tel.: +90 414 318 3694; fax: +90 414 318 3682. E-mail addresses: [email protected] (A.L. Tek), [email protected] (K. Nagaki). 1 Tel.: +81 86 434 1207; fax: +81 86 434 1208. 0378-1119/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.gene.2014.01.034
histone H3 by a highly variable and divergent N terminal tail and a slightly longer loop 1 region (Henikoff et al., 2001). In addition, the histone fold domain (HFD) of the terminal part of CENH3 is highly conserved in plants, while its N-terminal tail shows adaptive evolution (Talbert et al., 2002). CENH3 proteins tagged by green fluorescent protein (GFP) have provided considerable insights into centromere organization (Jin et al., 2008) and function (Ravi et al., 2010) (Dubin et al., 2010). GFP-tagged CENH3 was used to demonstrate a cell type-dependent chromatin organization of centromeres in transgenic A. thaliana lines (Fang and Spector, 2005). In a pioneering study with direct implications on plant breeding, the A. thaliana CENH3 homolog linked with GFP was applied to eliminate one of the parental chromosome complements, thereby leading to the production of haploid progenies (Ravi and Chan, 2010). For a detailed analysis of the centromere structure, the availability of an efficient transformation system is crucial especially in model organisms. Lotus japonicus is an autogamus diploid (2n = 2× = 12) model species for nitrogen-fixing legumes because of its small genome (470 Mbp), growth characteristics and an effective Agrobacterium transformation system (Handberg and Stougaard, 1992). A draft sequence of the L. japonicus genome with a coverage of 67% was analyzed to determine genic and genomic features including repetitive elements (Sato et al., 2008; Young et al., 2005). The availability of genome sequence positions L. japonicus well in the genomic era (Cannon et al., 2009). However, the centromere research for L. japonicus lagged behind in comparison to the other models such as Arabidopsis (Copenhaver et al., 1999). So far, three types of DNA sequences have been implicated in the centromeric regions of L. japonicus. A telomere repeat-related sequence and a bacterial artificial clone derived repetitive element, Ljcen1, were localized at L. japonicus centromeric primary constrictions based on fluorescence in situ hybridization (FISH) (Pedrosa et al., 2002). In addition, a centromere-associated retroelement, LjRE2, was
A.L. Tek et al. / Gene 538 (2014) 8–11
used as a FISH probe to detect L. japonicus centromeres (Ohmido et al., 2010; Sato et al., 2008). However, the importance of these three DNA sequences for functional centromeres of L. japonicus remains to be elucidated. In an effort to identify the functional components of the legume centromeres, we chose the model species L. japonicus and concentrate on the CenH3 that could ultimately help resolve the issues associated with centromeres mentioned above. Therefore, in this study, we cloned the L. japonicus CenH3 cDNA and determined its DNA and protein sequence. The LjCenH3 cDNA was fused in frame with GFP-encoding sequences in a Gateway-based DNA construct. Subsequently, this construct was transformed to L. japonicus cells to determine the localization of LjCENH3 on L. japonicus chromosomes in situ. 2. Materials and methods 2.1. Plant material Plant material was obtained from Legume Base at the National BioResource Project (NBRP), University of Miyazaki, Japan. The plants were grown under regular greenhouse conditions. The young plants with fresh leaves were clonally propagated in several pots. During the experiments, several rounds of plant propagation were performed to keep fresh plant material available. The plants were watered as needed. The L. japonicus LjA cell line used for transformation was obtained from RIKEN Bio-Resource Center, Japan.
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codon at the C-terminus was fused to a 35S-promoter-driven GFP construct. Once the integrity of destination clones was verified by sequencing as described above, the construct was transferred into the Agrobacterium tumefaciens C58Ci strain by electroporation (Bio-Rad Gene pulser Xcell), and L. japonicus cultured cells were transformed (Mathur et al., 1998). 2.4. Immunofluorescence localization of GFP-LjCENH3 Immunofluorescence localization of GFP-tagged LjCENH3 was carried out as described previously (Nagaki et al., 2009a, 2009b; Tek et al., 2010). Briefly, the transformed tissue was fixed in 3% (w/v) paraformaldehyde for 20 min at 4 °C and digested with 1% (w/v) Cellulase Onozuka RS (Yakult Honsha Co., Minato-ku, Tokyo, Japan) and 0.5% (w/v) Pectolyase Y23 (Kikkoman, Noda, Chiba, Japan) for 1 h at 37 °C. The digested tissue was squashed in phosphate-buffered saline (PBS) on poly-L-lysine-coated slides (Matsunami, Kishiwada, Osaka, Japan). Slides were incubated with a 1:100 dilution of the mouse anti-GFP antibody (Acris Antibodies GmbH, Germany) in TNB buffer (0.1 M Tris– HCl pH 7.5, 0.15 M NaCl, and 0.5% (w/v) Roche Blocking Reagent) at 4 °C overnight. The antibody was detected with a 1:100 dilution of Alexa Fluor 546-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR, USA). Chromosomes counterstained with 4′,6diamidino-2-phenylindole (DAPI) were visualized under a Carl Zeiss Axioskop 2 Plus fluorescence microscope with a chilled charge-coupled device camera (Carl Zeiss, Axiocam). 3. Results and discussion
2.2. Cloning of L. japonicus centromere-specific histone H3 cDNA 3.1. Cloning of the L. japonicus histone H3 variant cDNA Total RNA was extracted from L. japonicus leaves using an RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA). First-strand cDNA synthesis and rapid amplification of cDNA ends (RACE) PCR reactions were performed by employing the SMARTer RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions. Based on the G. max CenH3 gene (Tek et al., 2010), a reverse primer, LjCenH3r, (5′-TCACCAAGGCCTTCCTATTCCTC-3′) was designed (see below for further details). First, a 5′-RACE-PCR amplification was performed to obtain the 5′ coding and untranslated regions (UTR). Second, a 3′-RACE-PCR amplification was conducted to recover the 3′ UTR using a coding region-specific forward primer, LjCenH3f, (5′-GGAACT GTGGCGCTTCGTGAGAT-3′). Amplified fragments were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) and sequenced using cycle sequencing reactions (BigDye Terminator v.1.1) and the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster, CA, USA). Sequence analyses were performed on Geneious Pro Bioinformatics Software (Biomatters, Auckland, New Zealand). The sequences obtained from the 5'- and 3'-RACE-PCR clones were assembled at the overlapping regions to recover the full length cDNA sequences. Multiple sequence alignment was carried out with the MUSCLE program (Edgar, 2004). 2.3. Creating a Gateway construct for tagging LjCENH3 and transformation LjCenH3 was fused with GFP by employing the Gateway cloning strategy (Invitrogen) (Nagaki et al., 2010). Gateway sequences shown in italics were added to 5′ and 3′ ends of LjCenH3 cDNA using the following pair of primers (GLjH3f: 5′-G GGG ACA AGT TTG TAC AAA AAA GCA GGC TTC ATG GCG AGA ATC AAG CAC-3′ and GLjH3r: 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TCA CCA AGG CCT TCC TAT T-3′). PCR was performed under the following conditions: 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min for 30 cycles. To construct the entry clone, the amplified PCR products were introduced into pDONR221 vector mediated by BP clonase (Invitrogen) in a BP recombination reaction. Entry clones were then recombined with pK7WGF2 binary vectors (Karimi et al., 2002) for the destination vector mediated by LR clonase II (Invitrogen) in an LR reaction. LjCenH3 with a stop
To identify a CenH3 homolog in L. japonicus, we searched the public L. japonicus sequence databases for proteins sharing sequence similarity with the G. max CenH3 gene (Tek et al., 2010). As no L. japonicus sequences were obtained with significant matches to GmCenH3, we used the sequence information available from GmCenH3 to clone the L. japonicus homolog based on a polymerase chain reaction (PCR). To obtain the full length transcript of CenH3 homolog from a L. japonicus cDNA pool, we used a rapid amplification of cDNA ends (RACE)-PCR strategy in which a 5′ gene specific primer designed from GmCenH3 was used to amplify transcript fragments (Fig. 1). DNA sequencing of the 5′ RACE product provided information with which to design a 3′-RACE primer to amplify the remaining 3′ portion of the gene, as shown in Fig. 1. The assembled 852-bp mRNA sequence appears to represent a full length cDNA containing a complete 477-bp open reading frame flanked by 5′ and 3′ UTR (Fig. 1). The deduced 159 amino-acid protein is highly similar to known CENH3 proteins. We named this gene LjCenH3 (L. japonicus centromere-specific histone H3) and its corresponding protein LjCENH3 and deposited the sequence to GenBank under the accession number of KF214777. The LjCENH3 protein contains several key features that most CENH3 proteins share and differ from canonical histone H3s (Henikoff et al., 2001). A multiple sequence alignment of LjCENH3 with soybean GmCENH3 and Arabidopsis HTR12 highlights these features (Fig. 2). The histone fold domain of LjCENH3 has ~63% sequence identity with that of canonical LjH3.1 (Fig. 2). The N terminus is 20-residues longer and highly divergent compared to that of canonical LjH3.1 (Fig. 2). Canonical LjH3.1 has seven residues in the loop 1 region, while the LjCENH3 has eight amino acids, making it a single residue longer for the same region (Fig. 2). Structurally important regions at the LjCENH3 histone fold domain share high amino acid identity with homologous soybean GmCENH3 and Arabidopsis HTR12 as shown in black in Fig. 2. In contrast, the N terminus of LjCENH3 is more divergent between that of GmCENH3 and HTR12, which is in line with previous findings (Henikoff et al., 2001). Collectively, these bioinformatic properties suggest that LjCENH3 is the functional centromeric histone H3 variant of L. japonicus.
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Fig. 1. Nucleotide and deduced amino acid sequences of L. japonicus CenH3 cDNA. The 5′ and 3′ RACE primers used in the amplification are shown above the sequence with black arrows. The primers with the Gateway cloning sequences are drawn as tailed-white arrows at the beginning and end of the protein. The stop codon is shown with an asterisk. A 159-residue protein as the longest ORF is indicated below the cDNA sequence.
3.2. Transformation and localization of GFP-LjCENH3 in L. japonicus cells Tagging centromeres using CENH3-GFP fusion proteins is a direct means to test the localization of putative CENH3 proteins and has been documented in many plant species (Fang and Spector, 2005; Lermontova et al., 2006; Nagaki et al., 2010). Detectable signals solely at L. japonicus centromeres throughout the cell cycle would indicate that the gene we cloned encodes a centromere-specific histone H3 variant. Given the high sequence identity between LjCENH3 and GmCENH3, we have initially tested our previously raised GmCenH3 antibody on L. japonicus root-tip cells. However, the GmCenH3 antibody did not produce any specific signals on L. japonicus (data not shown). Towards this end, we applied a Gateway-based cloning strategy to fuse the LjCenH3 cDNA with GFP. The construct encodes an N-terminal translation fusion of the GFP with LjCenH3. Agrobacterium was used to introduce the GFP-LjCenH3 fusion into the L. japonicus LjA cell line. Regenerated cells subsequent to callus
induction were used to analyze the deposition of constitutively expressed GFP-LjCENH3 recombinant protein at the L. japonicus centromeres. Therefore, we performed an immunofluorescence assay using an anti-GFP antibody on transformed cells and observed the accumulation of the recombinant GFP-LjCENH3 specifically at the L. japonicus centromeres on metaphase chromosomes (Fig. 3). In this particular example that is derived from a rapidly dividing cell culture, an endoreduplicated hexaploid L. japonicus cell shows centromeric GFP-LjCENH3 localization on 36 metaphase chromosomes (Fig. 3). In cell lines going through tissue culture, occurrence of higher ploidy levels is common (Lermontova et al., 2006; Nagaki et al., 2010). L. japonicus centromeres showed typically two-dots at the outer edge of the centromere of each metaphase chromosome. As indicated previously (Lermontova et al., 2006), an exclusive centromeric localization and distinctive double-dot signals could suggest that GFP-LjCenH3 expression was within physiological range without overexpression, allowing us to determine the native localization. Since GFP-alone transformation as a negative control produces
Fig. 2. A multiple sequence alignment of plant CENH3 proteins. LjCENH3 (Lotus japonicus, KF214777), GmCENH3 (Glycine max, FK014964), HTR12 (Arabidopsis thaliana, AY086361) are used in the sequence alignment. LjH3.1, a canonical histone H3 from L. japonicus BI418913, is included for comparison. Note the important structural domains shown below the amino acids used for bioinformatic analysis. Conserved amino acids are shown in black.
A.L. Tek et al. / Gene 538 (2014) 8–11
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Conflict of interest The authors declare no conflict of interest.
Acknowledgments We thank to Dr. Douglas R. Cook of University of California, Davis, USA, Dr. Ingo Schubert of Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany and anonymous reviewers for valuable contributions and critical reading of the manuscript. This work was supported by the Fellowship Programs of the Japan Society for the Promotion of Science (JSPS), the Turkish Higher Education Council, and Harran University. Fig. 3. Immunofluorescence localization of the GFP-LjCENH3 fusion protein on a L. japonicus 6× hexaploid transgenic cell containing 36 metaphase chromosomes. (a) The chromosomes are stained with DAPI. (b) The localization of GFP-LjCENH3 is detected with mouse anti-GFP antibody and with Alexa Fluor 546-conjugated goat anti-mouse antibody and shown in red. (c) The merged image of (a) and (b). The scale bar represents 10 μm.
much weaker signals randomly distributed on nuclei and cytoplasm (Nagaki et al., 2010), possibility of centromeric localization due to the GFP tag alone is negligible. The functional characterization of LjCENH3 should serve an important centromere mark for L. japonicus chromosomes in genetic and genomic studies as discussed in detail below. This study establishes L. japonicus as a legume model for especially studying CENH3 in an efficient Agrobacterium transformation compatible system. In addition, since the sexual crossings using GFP-CENH3 plants produce progenies with chromosomal missegration (Ravi and Chan, 2010), it will be possible to test the production of haploid L. japonicus plants with a potential to use in legume crop improvement. 4. Conclusions We have demonstrated that LjCenH3 cDNA can be cloned using gene specific RACE-PCR primers derived from a conserved region of GmCENH3. The bioinformatic analysis showed that LjCENH3 has a conserved HFD similar to other plant CENH3 proteins. In addition, the N-terminal tail is much longer and divergent showing the features that are consistent with previous reports on CENH3. Furthermore, we provide evidence that the GFP-LjCENH3 recombinant protein localizes at the L. japonicus centromeres throughout the cell cycle following an Agrobacterium transformation. Research on model legumes could have dramatic effects on the agronomically important legume crops as illustrated previously (Cannon et al., 2009). Despite a model for legume species, there is only limited information regarding the centromere structure of L. japonicus (Ohmido et al., 2010; Pedrosa et al., 2002). The identification of LjCenH3 could serve as an initial step to help position the centromeres on cytogenetic and genetic maps (Ohmido et al., 2010; Sato et al., 2008). Such positioning will also improve mapping efforts in agronomically important legume crops (Cannon et al., 2009). In addition, it will be possible to extend the analysis to identify L. japonicus centromeric DNA sequences by chromatin affinity purification of HaloTag7-fused LjCENH3 protein (Nagaki et al., 2012) and other L. japonicus centromeric proteins such as CENP-C, mis12 (Nagaki et al., 2009b). Ultimately, given the suggestion that centromeres in different legumes are not syntenic and could possibly be evolutionarily new (Cannon et al., 2009), this study and others (Iwata et al., 2013; Tek et al., 2010; Tek et al., 2011) might provide crucial insights into a unified legume centromere evolution and its mechanisms.
References Cannon, S.B., May, G.D., Jackson, S.A., 2009. Three sequenced legume genomes and many crop species: rich opportunities for translational genomics. Plant Physiol. 151, 970–977. Copenhaver, G.P., 1999. Genetic definition and sequence analysis of Arabidopsis centromeres. Science 286, 2468–2474. Dubin, M., Fuchs, J., Gräf, R., Schubert, I., Nellen, W., 2010. Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis. Nucleic Acids Res. 38, 7526–7537. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Fang, Y., Spector, D.L., 2005. Centromere positioning and dynamics in living Arabidopsis plants. Mol. Biol. Cell 16, 5710–5718. Handberg, K., Stougaard, J., 1992. Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J. 2, 487–496. Henikoff, S., Ahmad, K., Malik, H.S., 2001. The centromere paradox: stable inheritance with rapidly evolving DNA. Science 293, 1098–1102. Iwata, A., et al., 2013. Identification and characterization of functional centromeres of the common bean. Plant J. 76, 47–60. Jin, W., Lamb, J.C., Zhang, W., Kolano, B., Birchler, J.A., Jiang, J., 2008. Histone modifications associated with both A and B chromosomes of maize. Chromosome Res. 16, 1203–1214. Karimi, M., Inzé, D., Depicker, A., 2002. Gateway vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7, 193–195. Lermontova, I., Schubert, V., Fuchs, J., Klatte, S., Macas, J., Schubert, I., 2006. Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain. Plant Cell 18, 2443–2451. Mathur, J., et al., 1998. Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension. Plant J. 13, 707–716. Nagaki, K., Kashihara, K., Murata, M., 2009a. A centromeric DNA sequence colocalized with a centromere-specific histone H3 in tobacco. Chromosoma 118, 249–257. Nagaki, K., Kashihara, K., Murata, M., 2009b. Characterization of the two centromeric proteins CENP-C and MIS12 in Nicotiana species. Chromosome Res. 17, 719–726. Nagaki, K., Terada, K., Wakimoto, M., Kashihara, K., Murata, M., 2010. Centromere targeting of alien CENH3s in Arabidopsis and tobacco cells. Chromosome Res. 18, 203–211. Nagaki, K., Shibata, F., Kanatani, A., Kashihara, K., Murata, M., 2012. Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco. Plant Cell Rep. 31, 771–779. Ohmido, N., Ishimaru, A., Kato, S., Sato, S., Tabata, S., Fukui, K., 2010. Integration of cytogenetic and genetic linkage maps of Lotus japonicus, a model plant for legumes. Chromosome Res. 18, 287–299. Pedrosa, A., Sandal, N., Stougaard, J., Schweizer, D., Bachmair, A., 2002. Chromosomal map of the model legume Lotus japonicus. Genetics 161, 1661–1672. Ravi, M., Chan, S.W., 2010. Haploid plants produced by centromere-mediated genome elimination. Nature 464, 615–618. Ravi, M., et al., 2010. The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana. Genetics 186, 461–471. Sato, S., et al., 2008. Genome structure of the legume, Lotus japonicus. DNA Res. 15, 227–239. Talbert, P.B., Masuelli, R., Tyagi, A.P., Comai, L., Henikoff, S., 2002. Centromeric localization and adaptive evolution of an Arabidopsis histone H3 variant. Plant Cell 14, 1053–1066. Tek, A.L., Kashihara, K., Murata, M., Nagaki, K., 2010. Functional centromeres in soybean include two distinct tandem repeats and a retrotransposon. Chromosome Res. 18, 337–347. Tek, A.L., Kashihara, K., Murata, M., Nagaki, K., 2011. Functional centromeres in Astragalus sinicus include a compact centromere-specific histone H3 and a 20-bp tandem repeat. Chromosome Res. 19, 969–978. Young, N.D., et al., 2005. Sequencing the genespaces of Medicago truncatula and Lotus japonicus. Plant Physiol. 137, 1174–1181.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Identification of the centromere-specific histone H3 variant in Lotus japonicus Ahmet L. Tek ⁎,1, Kazunari Kashihara 1, Minoru Murata 1, Kiyotaka Nagaki 1 Institute of Plant Science and Resources, Okayama University, Kurashiki 710–0046, Japan
a r t i c l e
i n f o
Article history: Accepted 11 January 2014 Available online 22 January 2014 Keywords: Centromere CenH3/CENP-A Green fluorescent protein Leguminosae Fabaceae
a b s t r a c t The centromere is a structurally and functionally specialized region present on every eukaryotic chromosome. Lotus japonicus is a model legume species for which there is very limited information on the centromere structure. Here we cloned and characterized the L. japonicus homolog of the centromere-specific histone H3 gene (LjCenH3) encoding a 159-amino acid protein. Using an Agrobacterium-based transformation system, LjCenH3 tagged with a green fluorescent protein was transferred into L. japonicus cells. The centromeric position of LjCENH3 protein was revealed on L. japonicus metaphase chromosomes by an immunofluorescence assay. The identification of LjCenH3 as a critical centromere landmark could pave the way for a better understanding of centromere structure in this model and other agriculturally important legume species. Published by Elsevier B.V.
1. Introduction The centromere is a structurally and functionally specialized chromosome region that forms the kinetochore, a multiprotein complex. Many kinetochore proteins are conserved among eukaryotes and perform processes essential to chromosome function, including attachment, generation of motive force, and cell cycle regulation, among others. In contrast to the functional conservation at centromeres, centromere DNA sequences are highly variable (Henikoff et al., 2001). Histone proteins, including histone H3, are ubiquitous components of DNA–protein complexes. Only at eukaryotic centromeres wrapping the centromeric DNA, there is a specialized variant of histone H3 protein, called centromere-specific histone H3, CENH3 (Henikoff et al., 2001). CENH3 aids in kinetochore–DNA association, and indeed functional centromeres are defined by the presence of CENH3 (Henikoff et al., 2001). CENH3 homologs have been identified and functionally characterized in many plant species, such as the model plant Arabidopsis, Arabidopsis thaliana (Talbert et al., 2002); soybean, Glycine max (Tek et al., 2010); Chinese milk vetch, Astragalus sinicus (Tek et al., 2011); common bean, Phaseolus vulgaris (Iwata et al., 2013). Structural features of CENH3 provide important insights into the establishment and function of centromeres. CENH3 is distinguished from the canonical
Abbreviations: CenH3, centromere-specific histone H3; LjCenH3, L. japonicus centromere-specific histone H3; HFD, histone fold domain; GFP, green fluorescent protein; Mbp, megabase pair; FISH, fluorescence in situ hybridization; cDNA, complementary DNA; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; UTR, untranslated region; DAPI, 4′,6-diamidino-2-phenylindole. ⁎ Corresponding author at: Department of Agronomy, Faculty of Agriculture, Harran University, Şanlıurfa, 63300, Turkey. Tel.: +90 414 318 3694; fax: +90 414 318 3682. E-mail addresses: [email protected] (A.L. Tek), [email protected] (K. Nagaki). 1 Tel.: +81 86 434 1207; fax: +81 86 434 1208. 0378-1119/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.gene.2014.01.034
histone H3 by a highly variable and divergent N terminal tail and a slightly longer loop 1 region (Henikoff et al., 2001). In addition, the histone fold domain (HFD) of the terminal part of CENH3 is highly conserved in plants, while its N-terminal tail shows adaptive evolution (Talbert et al., 2002). CENH3 proteins tagged by green fluorescent protein (GFP) have provided considerable insights into centromere organization (Jin et al., 2008) and function (Ravi et al., 2010) (Dubin et al., 2010). GFP-tagged CENH3 was used to demonstrate a cell type-dependent chromatin organization of centromeres in transgenic A. thaliana lines (Fang and Spector, 2005). In a pioneering study with direct implications on plant breeding, the A. thaliana CENH3 homolog linked with GFP was applied to eliminate one of the parental chromosome complements, thereby leading to the production of haploid progenies (Ravi and Chan, 2010). For a detailed analysis of the centromere structure, the availability of an efficient transformation system is crucial especially in model organisms. Lotus japonicus is an autogamus diploid (2n = 2× = 12) model species for nitrogen-fixing legumes because of its small genome (470 Mbp), growth characteristics and an effective Agrobacterium transformation system (Handberg and Stougaard, 1992). A draft sequence of the L. japonicus genome with a coverage of 67% was analyzed to determine genic and genomic features including repetitive elements (Sato et al., 2008; Young et al., 2005). The availability of genome sequence positions L. japonicus well in the genomic era (Cannon et al., 2009). However, the centromere research for L. japonicus lagged behind in comparison to the other models such as Arabidopsis (Copenhaver et al., 1999). So far, three types of DNA sequences have been implicated in the centromeric regions of L. japonicus. A telomere repeat-related sequence and a bacterial artificial clone derived repetitive element, Ljcen1, were localized at L. japonicus centromeric primary constrictions based on fluorescence in situ hybridization (FISH) (Pedrosa et al., 2002). In addition, a centromere-associated retroelement, LjRE2, was
A.L. Tek et al. / Gene 538 (2014) 8–11
used as a FISH probe to detect L. japonicus centromeres (Ohmido et al., 2010; Sato et al., 2008). However, the importance of these three DNA sequences for functional centromeres of L. japonicus remains to be elucidated. In an effort to identify the functional components of the legume centromeres, we chose the model species L. japonicus and concentrate on the CenH3 that could ultimately help resolve the issues associated with centromeres mentioned above. Therefore, in this study, we cloned the L. japonicus CenH3 cDNA and determined its DNA and protein sequence. The LjCenH3 cDNA was fused in frame with GFP-encoding sequences in a Gateway-based DNA construct. Subsequently, this construct was transformed to L. japonicus cells to determine the localization of LjCENH3 on L. japonicus chromosomes in situ. 2. Materials and methods 2.1. Plant material Plant material was obtained from Legume Base at the National BioResource Project (NBRP), University of Miyazaki, Japan. The plants were grown under regular greenhouse conditions. The young plants with fresh leaves were clonally propagated in several pots. During the experiments, several rounds of plant propagation were performed to keep fresh plant material available. The plants were watered as needed. The L. japonicus LjA cell line used for transformation was obtained from RIKEN Bio-Resource Center, Japan.
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codon at the C-terminus was fused to a 35S-promoter-driven GFP construct. Once the integrity of destination clones was verified by sequencing as described above, the construct was transferred into the Agrobacterium tumefaciens C58Ci strain by electroporation (Bio-Rad Gene pulser Xcell), and L. japonicus cultured cells were transformed (Mathur et al., 1998). 2.4. Immunofluorescence localization of GFP-LjCENH3 Immunofluorescence localization of GFP-tagged LjCENH3 was carried out as described previously (Nagaki et al., 2009a, 2009b; Tek et al., 2010). Briefly, the transformed tissue was fixed in 3% (w/v) paraformaldehyde for 20 min at 4 °C and digested with 1% (w/v) Cellulase Onozuka RS (Yakult Honsha Co., Minato-ku, Tokyo, Japan) and 0.5% (w/v) Pectolyase Y23 (Kikkoman, Noda, Chiba, Japan) for 1 h at 37 °C. The digested tissue was squashed in phosphate-buffered saline (PBS) on poly-L-lysine-coated slides (Matsunami, Kishiwada, Osaka, Japan). Slides were incubated with a 1:100 dilution of the mouse anti-GFP antibody (Acris Antibodies GmbH, Germany) in TNB buffer (0.1 M Tris– HCl pH 7.5, 0.15 M NaCl, and 0.5% (w/v) Roche Blocking Reagent) at 4 °C overnight. The antibody was detected with a 1:100 dilution of Alexa Fluor 546-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR, USA). Chromosomes counterstained with 4′,6diamidino-2-phenylindole (DAPI) were visualized under a Carl Zeiss Axioskop 2 Plus fluorescence microscope with a chilled charge-coupled device camera (Carl Zeiss, Axiocam). 3. Results and discussion
2.2. Cloning of L. japonicus centromere-specific histone H3 cDNA 3.1. Cloning of the L. japonicus histone H3 variant cDNA Total RNA was extracted from L. japonicus leaves using an RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA). First-strand cDNA synthesis and rapid amplification of cDNA ends (RACE) PCR reactions were performed by employing the SMARTer RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions. Based on the G. max CenH3 gene (Tek et al., 2010), a reverse primer, LjCenH3r, (5′-TCACCAAGGCCTTCCTATTCCTC-3′) was designed (see below for further details). First, a 5′-RACE-PCR amplification was performed to obtain the 5′ coding and untranslated regions (UTR). Second, a 3′-RACE-PCR amplification was conducted to recover the 3′ UTR using a coding region-specific forward primer, LjCenH3f, (5′-GGAACT GTGGCGCTTCGTGAGAT-3′). Amplified fragments were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) and sequenced using cycle sequencing reactions (BigDye Terminator v.1.1) and the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster, CA, USA). Sequence analyses were performed on Geneious Pro Bioinformatics Software (Biomatters, Auckland, New Zealand). The sequences obtained from the 5'- and 3'-RACE-PCR clones were assembled at the overlapping regions to recover the full length cDNA sequences. Multiple sequence alignment was carried out with the MUSCLE program (Edgar, 2004). 2.3. Creating a Gateway construct for tagging LjCENH3 and transformation LjCenH3 was fused with GFP by employing the Gateway cloning strategy (Invitrogen) (Nagaki et al., 2010). Gateway sequences shown in italics were added to 5′ and 3′ ends of LjCenH3 cDNA using the following pair of primers (GLjH3f: 5′-G GGG ACA AGT TTG TAC AAA AAA GCA GGC TTC ATG GCG AGA ATC AAG CAC-3′ and GLjH3r: 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TCA CCA AGG CCT TCC TAT T-3′). PCR was performed under the following conditions: 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min for 30 cycles. To construct the entry clone, the amplified PCR products were introduced into pDONR221 vector mediated by BP clonase (Invitrogen) in a BP recombination reaction. Entry clones were then recombined with pK7WGF2 binary vectors (Karimi et al., 2002) for the destination vector mediated by LR clonase II (Invitrogen) in an LR reaction. LjCenH3 with a stop
To identify a CenH3 homolog in L. japonicus, we searched the public L. japonicus sequence databases for proteins sharing sequence similarity with the G. max CenH3 gene (Tek et al., 2010). As no L. japonicus sequences were obtained with significant matches to GmCenH3, we used the sequence information available from GmCenH3 to clone the L. japonicus homolog based on a polymerase chain reaction (PCR). To obtain the full length transcript of CenH3 homolog from a L. japonicus cDNA pool, we used a rapid amplification of cDNA ends (RACE)-PCR strategy in which a 5′ gene specific primer designed from GmCenH3 was used to amplify transcript fragments (Fig. 1). DNA sequencing of the 5′ RACE product provided information with which to design a 3′-RACE primer to amplify the remaining 3′ portion of the gene, as shown in Fig. 1. The assembled 852-bp mRNA sequence appears to represent a full length cDNA containing a complete 477-bp open reading frame flanked by 5′ and 3′ UTR (Fig. 1). The deduced 159 amino-acid protein is highly similar to known CENH3 proteins. We named this gene LjCenH3 (L. japonicus centromere-specific histone H3) and its corresponding protein LjCENH3 and deposited the sequence to GenBank under the accession number of KF214777. The LjCENH3 protein contains several key features that most CENH3 proteins share and differ from canonical histone H3s (Henikoff et al., 2001). A multiple sequence alignment of LjCENH3 with soybean GmCENH3 and Arabidopsis HTR12 highlights these features (Fig. 2). The histone fold domain of LjCENH3 has ~63% sequence identity with that of canonical LjH3.1 (Fig. 2). The N terminus is 20-residues longer and highly divergent compared to that of canonical LjH3.1 (Fig. 2). Canonical LjH3.1 has seven residues in the loop 1 region, while the LjCENH3 has eight amino acids, making it a single residue longer for the same region (Fig. 2). Structurally important regions at the LjCENH3 histone fold domain share high amino acid identity with homologous soybean GmCENH3 and Arabidopsis HTR12 as shown in black in Fig. 2. In contrast, the N terminus of LjCENH3 is more divergent between that of GmCENH3 and HTR12, which is in line with previous findings (Henikoff et al., 2001). Collectively, these bioinformatic properties suggest that LjCENH3 is the functional centromeric histone H3 variant of L. japonicus.
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Fig. 1. Nucleotide and deduced amino acid sequences of L. japonicus CenH3 cDNA. The 5′ and 3′ RACE primers used in the amplification are shown above the sequence with black arrows. The primers with the Gateway cloning sequences are drawn as tailed-white arrows at the beginning and end of the protein. The stop codon is shown with an asterisk. A 159-residue protein as the longest ORF is indicated below the cDNA sequence.
3.2. Transformation and localization of GFP-LjCENH3 in L. japonicus cells Tagging centromeres using CENH3-GFP fusion proteins is a direct means to test the localization of putative CENH3 proteins and has been documented in many plant species (Fang and Spector, 2005; Lermontova et al., 2006; Nagaki et al., 2010). Detectable signals solely at L. japonicus centromeres throughout the cell cycle would indicate that the gene we cloned encodes a centromere-specific histone H3 variant. Given the high sequence identity between LjCENH3 and GmCENH3, we have initially tested our previously raised GmCenH3 antibody on L. japonicus root-tip cells. However, the GmCenH3 antibody did not produce any specific signals on L. japonicus (data not shown). Towards this end, we applied a Gateway-based cloning strategy to fuse the LjCenH3 cDNA with GFP. The construct encodes an N-terminal translation fusion of the GFP with LjCenH3. Agrobacterium was used to introduce the GFP-LjCenH3 fusion into the L. japonicus LjA cell line. Regenerated cells subsequent to callus
induction were used to analyze the deposition of constitutively expressed GFP-LjCENH3 recombinant protein at the L. japonicus centromeres. Therefore, we performed an immunofluorescence assay using an anti-GFP antibody on transformed cells and observed the accumulation of the recombinant GFP-LjCENH3 specifically at the L. japonicus centromeres on metaphase chromosomes (Fig. 3). In this particular example that is derived from a rapidly dividing cell culture, an endoreduplicated hexaploid L. japonicus cell shows centromeric GFP-LjCENH3 localization on 36 metaphase chromosomes (Fig. 3). In cell lines going through tissue culture, occurrence of higher ploidy levels is common (Lermontova et al., 2006; Nagaki et al., 2010). L. japonicus centromeres showed typically two-dots at the outer edge of the centromere of each metaphase chromosome. As indicated previously (Lermontova et al., 2006), an exclusive centromeric localization and distinctive double-dot signals could suggest that GFP-LjCenH3 expression was within physiological range without overexpression, allowing us to determine the native localization. Since GFP-alone transformation as a negative control produces
Fig. 2. A multiple sequence alignment of plant CENH3 proteins. LjCENH3 (Lotus japonicus, KF214777), GmCENH3 (Glycine max, FK014964), HTR12 (Arabidopsis thaliana, AY086361) are used in the sequence alignment. LjH3.1, a canonical histone H3 from L. japonicus BI418913, is included for comparison. Note the important structural domains shown below the amino acids used for bioinformatic analysis. Conserved amino acids are shown in black.
A.L. Tek et al. / Gene 538 (2014) 8–11
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Conflict of interest The authors declare no conflict of interest.
Acknowledgments We thank to Dr. Douglas R. Cook of University of California, Davis, USA, Dr. Ingo Schubert of Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany and anonymous reviewers for valuable contributions and critical reading of the manuscript. This work was supported by the Fellowship Programs of the Japan Society for the Promotion of Science (JSPS), the Turkish Higher Education Council, and Harran University. Fig. 3. Immunofluorescence localization of the GFP-LjCENH3 fusion protein on a L. japonicus 6× hexaploid transgenic cell containing 36 metaphase chromosomes. (a) The chromosomes are stained with DAPI. (b) The localization of GFP-LjCENH3 is detected with mouse anti-GFP antibody and with Alexa Fluor 546-conjugated goat anti-mouse antibody and shown in red. (c) The merged image of (a) and (b). The scale bar represents 10 μm.
much weaker signals randomly distributed on nuclei and cytoplasm (Nagaki et al., 2010), possibility of centromeric localization due to the GFP tag alone is negligible. The functional characterization of LjCENH3 should serve an important centromere mark for L. japonicus chromosomes in genetic and genomic studies as discussed in detail below. This study establishes L. japonicus as a legume model for especially studying CENH3 in an efficient Agrobacterium transformation compatible system. In addition, since the sexual crossings using GFP-CENH3 plants produce progenies with chromosomal missegration (Ravi and Chan, 2010), it will be possible to test the production of haploid L. japonicus plants with a potential to use in legume crop improvement. 4. Conclusions We have demonstrated that LjCenH3 cDNA can be cloned using gene specific RACE-PCR primers derived from a conserved region of GmCENH3. The bioinformatic analysis showed that LjCENH3 has a conserved HFD similar to other plant CENH3 proteins. In addition, the N-terminal tail is much longer and divergent showing the features that are consistent with previous reports on CENH3. Furthermore, we provide evidence that the GFP-LjCENH3 recombinant protein localizes at the L. japonicus centromeres throughout the cell cycle following an Agrobacterium transformation. Research on model legumes could have dramatic effects on the agronomically important legume crops as illustrated previously (Cannon et al., 2009). Despite a model for legume species, there is only limited information regarding the centromere structure of L. japonicus (Ohmido et al., 2010; Pedrosa et al., 2002). The identification of LjCenH3 could serve as an initial step to help position the centromeres on cytogenetic and genetic maps (Ohmido et al., 2010; Sato et al., 2008). Such positioning will also improve mapping efforts in agronomically important legume crops (Cannon et al., 2009). In addition, it will be possible to extend the analysis to identify L. japonicus centromeric DNA sequences by chromatin affinity purification of HaloTag7-fused LjCENH3 protein (Nagaki et al., 2012) and other L. japonicus centromeric proteins such as CENP-C, mis12 (Nagaki et al., 2009b). Ultimately, given the suggestion that centromeres in different legumes are not syntenic and could possibly be evolutionarily new (Cannon et al., 2009), this study and others (Iwata et al., 2013; Tek et al., 2010; Tek et al., 2011) might provide crucial insights into a unified legume centromere evolution and its mechanisms.
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