Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

A Full Suite of Histone and Histone Modifying Genes Are Transcribed in the Dinoflagellate Lingulodinium

  • Sougata Roy,

    Affiliation Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, Montréal, Québec, Canada

  • David Morse

    david.morse@umontreal.ca

    Affiliation Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, Montréal, Québec, Canada

A Full Suite of Histone and Histone Modifying Genes Are Transcribed in the Dinoflagellate Lingulodinium

  • Sougata Roy, 
  • David Morse
PLOS
x

Abstract

Background

Dinoflagellates typically lack histones and nucleosomes are not observed in DNA spreads. However, recent studies have shown the presence of core histone mRNA sequences scattered among different dinoflagellate species. To date, the presence of all components required for manufacturing and modifying nucleosomes in a single dinoflagellate species has not been confirmed.

Methodology and Results

Analysis of a Lingulodinium transcriptome obtained by Illumina sequencing of mRNA shows several different copies of each of the four core histones as well as a suite of histone modifying enzymes and histone chaperone proteins. Phylogenetic analysis shows one of each Lingulodinium histone copies belongs to the dinoflagellate clade while the second is more divergent and does not share a common ancestor. All histone mRNAs are in low abundance (roughly 25 times lower than higher plants) and transcript levels do not vary over the cell cycle. We also tested Lingulodinium extracts for histone proteins using immunoblotting and LC-MS/MS, but were unable to confirm histone expression at the protein level.

Conclusion

We show that all core histone sequences are present in the Lingulodinium transcriptome. The conservation of these sequences, even though histone protein accumulation remains below currently detectable levels, strongly suggests dinoflagellates possess histones.

Introduction

Unlike typical eukaryotes, dinoflagellate chromatin is permanently organized into a cholesteric liquid crystal structure [1], [2], similar to structures observed in bacteria grown under stress conditions [3] or in sperm cell nuclei [4]. In the dinoflagellates, a combination of several factors may contribute to this structure, including a high concentration of divalent cations [5], a low ratio (1∶10) of basic protein to DNA [6], and amounts of DNA that can range from 1.5 pg/cell (half that in a haploid human cell) in Symbiodinium [7] to roughly 200 pg/cell in Lingulodinium [8]. The unique chromatin structure in dinoflagellates is presumably a derived characteristic since nuclei in Perkinsus, a genus thought to be ancestral to the dinoflagellates [9], have a typical eukaryotic appearance [10].

An additional factor that is also likely to contribute to the unique structure of the dinoflagellate chromatin is the apparent lack of histones. This view is supported by biochemical evidence showing that protein extracts after gel electrophoresis lack the typical and distinctive pattern of histones [11], [12] as well as by microscopic observations showing that nucleosomes are not visible in DNA spreads [13], [14]. Instead of histones, dinoflagellates use histone-like proteins (HLPs) [15], [16]. HLPs of different dinoflagellates are similar but not identical [17], and have been shown to bind DNA and can be modified post-translationally [18], [19].

In general, DNA synthesis is coupled to histone protein synthesis for efficient assembly into nucleosomes. In plants and lower eukaryotes such as yeasts and ciliates, replication dependent histone mRNAs rely mainly on transcriptional regulation to affect histone accumulation in the S phase [20], [21], [22]. The N-terminal region of the histone proteins generally contains a nuclear localization signal (NLS) [23], [24] that binds to the nuclear import family of karyopherins with the help of Nucleosome Assembly Protein (NAP) [23], [24], [25]. Once inside the nucleus, the histones and DNA are assembled into nucleosomes by the help of NAP and other histone chaperone proteins [26], [27]. Certain residues in histone N-terminal end undergo specific post-translational modifications such as acetylation, methylation, phosphorylation, ADP-ribosylation, ubiquitination, sumoylation and biotinylation [28]. Histone modification causes chromatin to reorganize and can result in epigenetic regulation of gene expression as well as affecting other DNA processes such as recombination, repair and replication [29].

A parsimonious explanation for the lack of nucleosomes and histones in dinoflagellate chromatin is that these organisms no longer contain or express histone genes. However, reports of histones H3 and H2A.X mRNA sequences in Pyrocystis and Alexandrium [30], [31] as well as by retrieval of all core histones and transcripts for two histone-modifying enzymes and a NAP from an environmental sample of dinoflagellates [32] cast considerable doubt on this idea. The environmental sample contains only dinoflagellate sequences, as their amplification exploited a splice leader (SL) sequence specifically trans-spliced to the 5′ end of all nuclear encoded dinoflagellate mRNAs [33]. However, this study could not determine if any one species of dinoflagellate contained the complete set of histones or if the core histones were scattered among many different species and thus unlikely to be functional.

We undertook the present study because a transcriptome profile from the dinoflagellate Lingulodinium polyedrum has allowed an in depth analysis of histone and histone modifying genes in a single species. We report here that this species expresses a full set of core histone genes as well as a variety of histone modifying enzymes and histone chaperone proteins at the RNA level. Despite the fact we have not been able to detect histone proteins in Lingulodinium extracts the presence and highly conserved sequence of these genes indicates that, in contrast to what has been previously thought, dinoflagellates do indeed have histones.

Materials and Methods

Cell Culture

Lingulodinium polyedrum cultures (formerly Gonyaulax polyedra; strain CCMP1936) were obtained from the Provasoli-Guillard Culture Center for Marine Phytoplankton (Boothbay Harbor, Maine) and grown in a modified seawater medium (f/2) [34]at constant temperature (19±1°C) in 12-h light/12-h dark cycles using cool white fluorescent light at an intensity of 50 µmol photons m−2·s−1. The beginning of light period is defined as LD 0, and the beginning of the dark period as LD 12. Cultures were grown to a cell density of 12–14,000 cells/mL. The samples were collected from the middle of the dark phase (LD 18) by filtering on Whatman 541 paper supported by a Buchner funnel, and either used immediately or frozen in liquid nitrogen and stored at −80°C until further use.

Acid Extraction of proteins

Histone proteins were obtained by trichloroacetic acid (TCA) precipitation of the acid soluble protein fraction as described previously [35], [36] with some minor modifications. After washing with 10 volumes of fresh f/2 medium the cells were suspended in ice-cold acid extraction buffer containing 10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 1.5 mM Phenyl methyl sulfonyl fluoride supplemented with 1 X EDTA-free Protease Inhibitor (from Roche) and HCl at a final concentration of 0.25 M. Cells were broken by three one-minute treatments in a bead beater with Zirconium beads at 4°C. The lysate was then incubated on a rotator for 1 hour at 4°C. Insoluble cell debris was removed by two sequential centrifugations at 11,000× g for 10 and 5 minutes, each at 4°C, and the supernatant retained. To this acid soluble fraction, 100% TCA was added drop by drop with simultaneous mixing by inverting the tubes several times until a final concentration of 33% (v/v) TCA was reached. The solution was then incubated overnight at 4°C and the acid soluble proteins were obtained by centrifugation at 16,000× g for 10 minutes at 4°C. To remove the acid, the pellet was carefully washed three times with ice-cold acetone using centrifugation at 16,000× g for 5 minutes at 4°C after each wash. The final pellet was air dried and dissolved in appropriate amount of ddH2O.

As a positive control, Saccharomyces cerevisiae (budding yeast) was cultured in 100 ml of 2X YPAD medium at 30°C to mid-log phase (A260 = 0.6). Cells were then harvested by centrifugation at 4°C for 5 min at 2,000× g and washed once with 10 volumes of ice-cold sterile Phosphate buffered saline (pH 7.2). All the procedures after this were the same as described above for Lingulodinium cells. All protein concentrations were measured using the Bradford assay (Bio-Rad).

SDS-PAGE and Immunoblotting

Lingulodinium and Saccharomyces acid soluble proteins along with molecular weight markers (Low Range-BIORAD) were resolved by SDS-15% Polyacrylamide gel electrophoresis (PAGE) as previously described [36]. To compare the protein profiles after electrophoresis, some gels were stained with Coomassie Blue, while others were used for western blotting. Western blotting was performed using commercial rabbit polyclonal antibodies for histones H3 (ab 1791, Abcam, USA) and H2B (sc-10808, Santa Cruz Biotechnology, USA). For Immunoblotting, the proteins from gels were transferred to the Hybond-P PVDF membranes (Amersham Biosciences) using the Transblot SD Semi-Dry Electrophoretic transfer cell (Bio-Rad) following the manufacturer's protocol. After blocking the membranes with 5% Non-fat dry milk in Tris-buffered saline buffer supplemented with 0.05% Tween-20, immuno-reaction was performed with H3 (1∶5000) and H2B (1∶1000) antibodies in the same buffer. After secondary antibody reaction and subsequent washings, the blots were developed with Chemiluminescent substrate (Millipore) and were exposed to the ImageQuant LAS 4000 (GE Healthcare) to capture the chemiluminescence.

In order to test the commercial H3 antibody for cross-reaction with the Lingulodinium protein, a tagged version of our H3 was expressed in bacteria. The H3 sequence was cloned by PCR using primers based on the transcriptome sequence (forward primer 5′-CATTACGCCTGACGCTGTCTACGTGC-3′ and reverse primer 5′- GTTAGCGTCTGCTGCTGACGGCTTC-3′) from a 1st strand cDNA sample prepared from Trizol (Invitrogen) extracted RNA using a reverse transcription reaction catalyzed by MMLV RT (Clontech) and the 5′ CDS primer A of the SMARTer RACE cDNA Amplification kit (Clontech). A second PCR, performed on the first PCR product using the forward primer 5′-TCAGTCggatccATGGCCCGCACGAAGCAG-3′ (containing a BamH1 site indicated by small letters) was used to allow directional cloning into the BamH1 and Sma1 restriction sites of the bacterial expression vector pQE30 (Qiagen). The cloned H3 was sequenced to confirm the correct reading frame and used to transform electrocompetent XL1 blue host cells. A single colony grown on LB-agar containing tetracycline and ampicillin was inoculated into 5 mL of the same medium and left to grow overnight at 37°C. One mL of the overnight culture was used to inoculate Twenty ml of fresh prewarmed (37°C) LB medium with antibiotics was inoculated with one mL overnight culture and grown with vigorous shaking at 37°C until OD600 of 0.5. H3 expression was induced by adding IPTG to a final concentration of 1 mM and the culture was grown for another 4 hours with shaking at 37°C. One ml of this culture was centrifuged at 5000× g for 3 minutes at 4°C and the cell pellet resuspended directly in 50 µl SDS-PAGE sample buffer and heated at 95°C for 5 min. The samples were centrifuged to remove debris and 30 µl of sample was loaded onto a 15% polyacrylamide gel. XL1 blue cells containing an empty vector were used as a control. Electrophoresis, transfer and immunoblotting were carried out as above.

Mass Spectrometric analysis

The total acid soluble protein pellet in acetone was also used for mass spectrometric analysis. Also, after fractionating the yeast and Lingulodinium acid soluble proteins in SDS 15% PAGE, the gels were stained with Coomassie Blue and several regions were excised from the gel, both from the Lingulodinium and yeast samples. The excised bands were destained and sent to the proteomic facility of l'Institut de recherche en immunologie et en cancérologie (IRIC) in Montreal, Canada. The tryptic digestion and LC-MS/MS sequencing for both the total acid extracted proteins and fractionated gel-excised bands were performed at the IRIC.

Bioinformatic and Phylogenetic Analysis

The sequences for histones and histone modifying enzymes reported here were retrieved from a Lingulodinium transcriptome assembled from roughly 300 million 76 bp Illumina paired end reads combined from several times under a LD cycle and conditions (manuscript in preparation, GenBank Accession numbers JO692619 through JO767447). The Illumina sequencing and assembly was performed at the Genome Quebec sequencing facility. The number of reads corresponding to each histone sequence was determined for RNA samples prepared over LD 6 and LD 18 cell cultures and reported as number of histone reads present per million. The number of reads for the histone sequences in the wild potato Solanum chacoense was retrieved from a similar project undertaken concurrently with the Lingulodinium samples.

Phylogenetic analysis was performed using an online tool obtained from the website www.phylogeny.fr [37]. Our workflow used the software MUSCLE to align the histone sequences, curation by GBlocks, phyML bootstrapping (100 times) to construct the tree and TreeDyn to visualize the tree. The same workflow was followed for all the phylogenetic analysis.

Results

All core histone and many histone modifying enzyme sequences are present in the Lingulodinium transcriptome

Analysis of a recent Illumina sequencing run (manuscript in preparation) identified the entire set of core histones, namely H2A, H2B, H3 and H4 from the dinoflagellate Lingulodinium polyedrum (Table 1). Partial splice leader sequence [33] was recovered from at least one of the histone sequences and all the sequences are GC-rich, a common characteristic of the dinoflagellate sequences [31]. In addition, L. polyedrum also expresses genes encoding enzymes that post-translationally modify histones, such as histone lysine methyltransferase (KMT), histone arginine methyltransferase (PRMT), histone acetyltransferase (KAT) and also histone deacetylases (from both HDAC and sirtuin 2 superfamilies) (Table 2). We also found histone chaperone proteins, which assists in nucleosome formation and chromatin remodelling (Table 2). Lingulodinium thus expresses a wide range of genes responsible for making and modifying nucleosomes.

thumbnail
Table 1. Description of histone sequences and their relative abundance in Lingulodinium.

https://doi.org/10.1371/journal.pone.0034340.t001

thumbnail
Table 2. Description of histone modifying enzymes and histone chaperones.

https://doi.org/10.1371/journal.pone.0034340.t002

Phylogenetic grouping identifies at least two major variants of all histone sequences within Lingulodinium

Surprisingly, the Lingulodinium transcriptome contains at least two variants of each histone sequence. We thus performed phylogenetic analyses to provide insight into the relationship between the different histone variants. Among the three H2A sequences retrieved, two belong to class H2A.X while the other groups with the eukaryotic H2A.Z proteins (Figure 1). This is the first report of a Z – like variant of histone H2A in any dinoflagellate. The two H2A.X sequences, JO760634 and JO759158, both contain a signature SQEF motif at the C-terminal end that is common to all dinoflagellate H2A.X sequences known so far [32] and as expected, all the dinoflagellate H2A.X variants cluster together. Interestingly, the two L. polyedrum H2B proteins belong to two different clades, one common to other dinoflagellate H2B (JO720817) and the other (JO694219) grouping within the superphylum Alveolata along with the ciliates and apicomplexans (Figure S1). Similarly, there are two well supported clades of H3 sequences, one phylogenetically indistinguishable from other eukaryotic H3 sequences and the other divergent (JO753891) form also found in Pyrocystis lunula (Figure S2). Unfortunately, there is insufficient phylogenetic resolution to determine the origin of the Lingulodinium H4 proteins (Figure S3). In general, however, it seems Lingulodinium contains not only a dinoflagellate specific histone but also an additional sequence with a more divergent origin.

thumbnail
Figure 1. Two variants of Histone H2A in Lingulodinium.

The cladogram of histone 2A.X and Z variants shows representatives from mammals, plants, fungus and members of the superphylum Alveolata. The representative sequences were obtained from Pubmed database and bear the first three letters from genus followed by two letters from species. The values in red at each node indicate the respective Bootstrap support value. Lingulodinium sequences are coloured in blue.

https://doi.org/10.1371/journal.pone.0034340.g001

Histone mRNAs abundance levels are uniform throughout

Replication –dependent histone sequences tend to accumulate during the S-phase of the cell cycle. In Lingulodinium, S-phase begins in the middle of the dark phase (LD18) for cells grown under a 12∶12 L∶D cycle [38]. We therefore compared the number of sequence reads in a sample from mid-day (LD6) with the LD18 sample. No significant variation in the mRNA abundance between the day and night is supported by the data (Table 1). In general, all the histone mRNAs seem to be of low abundance. By way of comparison, we found Lingulodinium histone mRNA abundance to be roughly 5 to 25-fold lower than in the plant Solanum chacoense.

Histone protein accumulation is below current detection limits

To reconcile the apparent lack of nucleosomes in dinoflagellates with the expression of all core histone transcripts in Lingulodinium, we evaluated the extent of histone protein accumulation using more sensitive techniques than those used previously. As shown previously [12], [15] acid extracted proteins from Lingulodinium do not have the typical pattern of histones such as found in yeast extracts using SDS PAGE followed by Coomassie blue staining (Figure 2). We used LC-MS/MS to analyze the Lingulodinium acid extracted proteins, and both the entire acid extracted protein fraction as well as acid extracted proteins that had been further fractionated by SDS-PAGE into the size range of yeast histones were tested. None of the histone core sequences from Lingulodinium were found in any of our samples although we were able to detect Lingulodinium histone-like protein, as expected (Table 3, Table S1). As a control, the same experiment was performed with an acid extracted fraction of a yeast extract, and histone sequences H2A and H2B were readily detected (Table 3).

thumbnail
Figure 2. The acid soluble protein profiles of Lingulodinium and Yeast differ.

A Coomassie blue stained gel containing roughly equivalent amount of acid extracted proteins from Lingulodinium and yeast in SDS-15% PAGE is shown here. The regions of the gel corresponding to yeast histones were excised and analysed by LC-MS/MS.

https://doi.org/10.1371/journal.pone.0034340.g002

thumbnail
Table 3. Proteins found by LC-MS/MS sequencing of total acid soluble proteins from Lingulodinium and yeast.

https://doi.org/10.1371/journal.pone.0034340.t003

In a separate approach, we tested Lingulodinium acid extracted proteins for a cross reaction with histone antibodies. We first tested a commercial anti-H3 directed against an epitope that shared 92% sequence identity with the Lingulodinium sequence. This antibody detected the yeast H3 with as little as 0.07 µg of total acid extracted protein, whereas as much as 20 µg of acid extracted protein from Lingulodinium did not show a reaction with any protein corresponding in size to the yeast H3 band (Figure 3). The high protein load of Lingulodinium polyedrum extracts show cross-reacting proteins with a significantly different mobility from the yeast H3, but the identity of these proteins is unknown. We also tested an antibody raised against the full length H2B sequence of mammalian origin, and again the antibody was unable to detect any band corresponding in size to that of yeast H2B (Figure S4). Again, at high concentrations of protein the antibody showed a cross reaction with a band with reduced mobility (∼30 kD) whose identity is also unknown. As a caveat, however, the H2B used to generate this antibody is only 63% similar to the predicted protein produced by the Lingulodinium polyedrum H2B.

thumbnail
Figure 3. Histone H3 protein levels in Lingulodinium are below current immunodetection limits.

(A) Acid extracted proteins electrophoresed on SDS-15% PAGE were subjected to Western blot analysis using a commercial H3 antibody. For the yeast and Lingulodinium samples, the value above each lane indicates the amount of protein loaded in micrograms, and the samples were run and treated with antibodies concurrently. No signal is detected in the Lingulodinium sample at a position corresponding to the yeast H3. (B) Western blots, performed using the same anti-H3 and an H3-expressing E. coli strain or an E. coli strain containing only the empty vector, demonstrate cross-reaction of the antibody with the Lingulodinium H3.

https://doi.org/10.1371/journal.pone.0034340.g003

Discussion

Nucleosomes are the basic structural and functional unit of chromatin in most eukaryotes, and are formed when roughly 150 bp of DNA wrap around a histone octamer (two each of H2A, H2B, H3 and H4). Dinoflagellates different from other eukaryotes in that DNA spreads do not show nucleosomes [39], [40], [41], 150 bp DNA fragments of DNA are not protected from microccocal nuclease digestion [14], [42] and gels of basic proteins do not show the typical histone protein pattern [15]. This general rule for dinoflagellates has only two known exceptions, the binucleate dinoflagellates such as Peridinium balticum (which have both typical eukaryotic and dinoflagellate nuclei) [43], [44] and members of the endoparasitic Perkinsus whose nuclei resemble those in a typical eukaryotic cell [10]. Perkinsus marinus is considered to be the ancestor of the dinoflagellate lineage [9], and not only contains all the core histone sequences [45] but lacks the HLPs found in other dinoflagellates.

Recently, high throughput sequencing has revealed that environmental samples of dinoflagellates transcripts contain not only the four core histones, but also two histone modification proteins and a NAP [32]. However, while these sequences are clearly dinoflagellate in origin, based on the distinguishing SL sequence at the 5′ end [33], it is not clear if they are all expressed in the same species. We show here that a single species of dinoflagellate expresses all the core histone (Figures S5, S6, S7, S8) as well as a wide range of histone modifying enzymes and histone chaperone proteins (Table 1 and 2). Furthermore, the gene profile is surprisingly complex, with at least two different variants of predicted histone sequence, one relatively close to other eukaryotic histones and the other more divergent (Figure 1 and Figures S1, S2, S3).

Among the core histones, histone H2A has several subtypes including H2A.1 and 2, H2A.X and H2A.Z. These subtypes each contain signature sequence elements that have been conserved throughout evolution and allow them to be readily identified [46], [47]. In mammals, all the major variants of H2A are present in varying proportions, whereas lower eukaryotes often replace the more common H2A.1 and 2 subfamily with H2A.X [48]. Lingulodinium also contains the H2A.X variant and in addition, an H2A.Z-like subtype previously unreported in dinoflagellates (Figure 1). These subtypes are thought to have specific functions, with H2A.X directly involved in DNA repair and genome integrity, which requires the phosphorylation of the C-terminal serine (S) of the SQ(D/E)(M/Y/F) motif [49], and H2A.Z involved in chromosome segregation, cell cycle progression and regulation of expression of cyclin genes, which is mediated by the H2A.Z localized in the promoter regions of these genes [50]. For the H2B and H3 histones, Lingulodinium maintains a general eukaryote form in addition to a divergent form common to other dinoflagellates (Figures S1, S2). Interestingly, two of the three H3 sequences in Lingulodinium conserve the key post-translational modification sites K4, K9, K27, K36 and K79 [51], while the other divergent forms lack the K27/K36, as in Pyrocystis H3 and K79 as in Karlodinium H3. For H4, we found two sequences (Figure S3), all with a conserved K20 site, which has been linked to transcription repression upon methylation [51]. Thus, the presence of all core histones, the conservation of sites typically modified, as well as the presence of histone modifying enzymes in the transcriptome (Table 2), all suggest that Lingulodinium should accumulate histone proteins.

We had originally anticipated that the amount of histone proteins expected for Lingulodinium could be estimated by assuming that the amount of protein produced from a transcript will be proportional to the amount of message independent from the organism in which the transcript is found. We therefore compared the amount of histone transcripts in Lingulodinium with those of the plant Solanum chacoense, as RNA samples from both were prepared, sequenced and analysed concurrently. In general, the abundance of histone messages in Lingulodinium is roughly 30 times less than that in S. chacoense (Table 1) and roughly 60-fold less than that reported for yeast [52]. However, immunoblotting was unable to detect H3 in Lingulodinium, even when the amount of Lingulodinium protein was 300 times greater than yeast. Furthermore, histone proteins were not detected by mass spectrometry (Table 3), either in total or gel fractionated acid soluble extracts, even though other proteins detected in the extracts had similar transcript levels as the Lingulodinium histones (Table 4). Thus, it seems histone abundance may be lower than would be predicted. It might also be of interest to test different extraction procedures for histones to see if this aids detection.

thumbnail
Table 4. mRNA abundance of expressed proteins detected by LC-MS/MS in an acid-extracted protein fraction.

https://doi.org/10.1371/journal.pone.0034340.t004

Histone modification has been linked to several functions such as chromatin remodelling and epigenetic regulation [53], and thus the finding that the Lingulodinium transcriptome also contains histone acetyltransferase and deacetylase enzymes as well as methyltransferases (Table 2) supports a role for histones in regulating gene expression. However, it must be noted that while histone deacetylases have a strong link to gene repression and heterochromatin formation [28], [54], [55], they can also target non-histone proteins and regulate DNA binding affinity, protein stability and protein-protein interaction, as well as modulate enzyme activity [56]. Sirtuin family proteins, deacetylases overrepresented in our transcriptome, were also reported in prokaryotes and archeae [57] where they function to regulate metabolism through important enzymes like acetyl-CoA synthetase [58]. Similarly, the SET domain K-methyltransferase that methylates histones can also methylate diverse proteins such as cytochrome c and the large subunit of Rubisco [59], [60]. A SET domain histone methyltransferase (NUE) has been reported in the pathogenic bacteria Chlamydia trachomatis [61]. Thus, it is possible the histone modifying enzymes in Lingulodinium might modify proteins other than the core histones. One prospective substrate could be the Lingulodinium HLPs, which have been reported to be acetylated [62]. Similarly, histone chaperone proteins also have important alternative roles other than those related to nucleosome assembly. NAP family proteins specifically interact with B-type cyclin [63], [64] and play a role in regulating cell cycle [65]. It would be of interest to determine if any of the histone modifying enzymes are, unlike the histones themselves, detectable immunologically.

The abundance of histone mRNA in Lingulodinium is between 5- and 25-fold lower than in the higher plant Solanum chacoense depending on the histone (Table 1). In eukaryotes, histones are found in both replication-dependent and replication-independent classes [66], with the mRNA abundance of replication-dependent histones coupled to the cell cycle as expected [67]. Transcriptional and posttranscriptional regulation can result in a 15- to 30-fold increase in mRNA accumulation with a peak during mid S phase [68], [69]. A comparison of histone mRNA levels at LD 6 and LD 18 (Table 1) does not show preferential abundance during the LD 18, the peak of S-phase in Lingulodinium [38], [70]. Thus, histone transcript accumulation is independent from the cell cycle in Lingulodinium.

Our results with Lingulodinium show that all core histone transcripts are present in a single species. Although histone protein levels remain below our current limit of detection, the presence of all four core histone proteins, the conservation of their sequence, and the presence of a large number of histone modifying enzymes all support the hypothesis that dinoflagellates have histones.

Supporting Information

Figure S1.

Cladogram of histone H2B. The cladogram of histone sequences shows representatives from mammals, plants, fungus and members of the superphylum Alveolata. The representative sequences were obtained from Pubmed database and bear the first three letters from genus followed by two letters from species. The values in red at each node indicate the respective Bootstrap support value. Lingulodinium sequences are coloured in blue.

https://doi.org/10.1371/journal.pone.0034340.s001

(JPG)

Figure S2.

Cladogram of histone H3. The cladogram of histone sequences shows representatives from mammals, plants, fungus and members of the superphylum Alveolata. The representative sequences were obtained from Pubmed database and bear the first three letters from genus followed by two letters from species. The values in red at each node indicate the respective Bootstrap support value. Lingulodinium sequences are coloured in blue.

https://doi.org/10.1371/journal.pone.0034340.s002

(JPG)

Figure S3.

Cladogram of histone H4. The cladogram of histone sequences shows representatives from mammals, plants, fungus and members of the superphylum Alveolata. The representative sequences were obtained from Pubmed database and bear the first three letters from genus followed by two letters from species. The values in red at each node indicate the respective Bootstrap support value. Lingulodinium sequences are coloured in blue.

https://doi.org/10.1371/journal.pone.0034340.s003

(JPG)

Figure S4.

Histone H2B protein is not detected in Lingulodinium (TIFF). Western blotting with H2B antibody is shown here. The amount of protein (in micrograms) per lane is written above each lane.

https://doi.org/10.1371/journal.pone.0034340.s004

(PDF)

Figure S5.

Alignment of H2A sequences. Multiple sequence alignment of histone H2A from yeast, human and Lingulodinium is shown.

https://doi.org/10.1371/journal.pone.0034340.s005

(JPG)

Figure S6.

Alignment of H2B sequences. Multiple sequence alignment of histone H2B from yeast, human and Lingulodinium is shown.

https://doi.org/10.1371/journal.pone.0034340.s006

(JPG)

Figure S7.

Alignment of H3 sequences. Multiple sequence alignment of histone H3 from yeast, human and Lingulodinium is shown.

https://doi.org/10.1371/journal.pone.0034340.s007

(JPG)

Figure S8.

Alignment of H4 sequences. Multiple sequence alignment of histone H4 from yeast, human and Lingulodinium is shown.

https://doi.org/10.1371/journal.pone.0034340.s008

(JPG)

Table S1.

LC-MS/MS identification of acid soluble proteins from Lingulodinium extracts fractionated on SDS PAGE.

https://doi.org/10.1371/journal.pone.0034340.s009

(DOCX)

Author Contributions

Conceived and designed the experiments: SR DM. Performed the experiments: SR. Analyzed the data: SR DM. Contributed reagents/materials/analysis tools: SR. Wrote the paper: SR DM.

References

  1. 1. Rill RL, Livolant F, Aldrich HC, Davidson MW (1989) Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes. Chromosoma 98: 280–286.RL RillF. LivolantHC AldrichMW Davidson1989Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes.Chromosoma98280286
  2. 2. Chow MH, Yan KT, Bennett MJ, Wong JT (2010) Birefringence and DNA condensation of liquid crystalline chromosomes. Eukaryot Cell 9: 1577–1587.MH ChowKT YanMJ BennettJT Wong2010Birefringence and DNA condensation of liquid crystalline chromosomes.Eukaryot Cell915771587
  3. 3. Frenkiel-Krispin D, Levin-Zaidman S, Shimoni E, Wolf SG, Wachtel EJ, et al. (2001) Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection. EMBO J 20: 1184–1191.D. Frenkiel-KrispinS. Levin-ZaidmanE. ShimoniSG WolfEJ Wachtel2001Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection.EMBO J2011841191
  4. 4. Livolant F (1984) Cholesteric organization of DNA in the stallion sperm head. Tissue Cell 16: 535–555.F. Livolant1984Cholesteric organization of DNA in the stallion sperm head.Tissue Cell16535555
  5. 5. Levi-Setti R, Gavrilov KL, Rizzo PJ (2008) Divalent cation distribution in dinoflagellate chromosomes imaged by high-resolution ion probe mass spectrometry. Eur J Cell Biol 87: 963–976.R. Levi-SettiKL GavrilovPJ Rizzo2008Divalent cation distribution in dinoflagellate chromosomes imaged by high-resolution ion probe mass spectrometry.Eur J Cell Biol87963976
  6. 6. Herzog M, Soyer MO (1981) Distinctive features of dinoflagellate chromatin. Absence of nucleosomes in a primitive species Prorocentrum micans E. Eur J Cell Biol 23: 295–302.M. HerzogMO Soyer1981Distinctive features of dinoflagellate chromatin. Absence of nucleosomes in a primitive species Prorocentrum micans E.Eur J Cell Biol23295302
  7. 7. Lajeunesse TC (2005) “Species” radiations of symbiotic dinoflagellates in the Atlantic and Indo-Pacific since the Miocene-Pliocene transition. Mol Biol Evol 22: 570–581.TC Lajeunesse2005“Species” radiations of symbiotic dinoflagellates in the Atlantic and Indo-Pacific since the Miocene-Pliocene transition.Mol Biol Evol22570581
  8. 8. Holm-Hansen O (1969) Algae: amounts of DNA and organic carbon in single cells. Science 163: 87–88.O. Holm-Hansen1969Algae: amounts of DNA and organic carbon in single cells.Science1638788
  9. 9. Saldarriaga JF, McEan ML, Fast NM, Taylor FJR, Keeling PJ (2003) Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage. Int J Syst Evol Microbiol 53: 355–365.JF SaldarriagaML McEanNM FastFJR TaylorPJ Keeling2003Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage.Int J Syst Evol Microbiol53355365
  10. 10. Azevedo C (1989) Fine structure of Perkinsus atlanticus n. sp. (Apicomplexa, Perkinsea) parasite of the clam Ruditapes decussatus from Portugal. J Parasitol 75: 627–635.C. Azevedo1989Fine structure of Perkinsus atlanticus n. sp. (Apicomplexa, Perkinsea) parasite of the clam Ruditapes decussatus from Portugal.J Parasitol75627635
  11. 11. Rizzo PJ (1981) Comparative aspects of basic chromatin proteins in dinoflagellates. Biosystems 14: 433–443.PJ Rizzo1981Comparative aspects of basic chromatin proteins in dinoflagellates.Biosystems14433443
  12. 12. Vernet G, Sala-Rovira M, Maeder M, Jacques F, Herzog M (1990) Basic nuclear proteins of the histone-less eukaryote Crypthecodinium cohnii (Pyrrhophyta): two-dimensional electrophoresis and DNA-binding properties. Biochim Biophys Acta 1048: 281–289.G. VernetM. Sala-RoviraM. MaederF. JacquesM. Herzog1990Basic nuclear proteins of the histone-less eukaryote Crypthecodinium cohnii (Pyrrhophyta): two-dimensional electrophoresis and DNA-binding properties.Biochim Biophys Acta1048281289
  13. 13. Herzog M, Soyer M-O (1981) Distinctive features of dinoflagellate chromatin. Absense of nucleosomes in a primitive species Prorocentrum micans. Eur J Cell Biol 23: 295–302.M. HerzogM-O Soyer1981Distinctive features of dinoflagellate chromatin. Absense of nucleosomes in a primitive species Prorocentrum micans.Eur J Cell Biol23295302
  14. 14. Bodansky S, Mintz LB, Holmes DS (1979) The mesokaryote Gyrodinium cohnii lacks nucleosomes. Biochem Biophys Res Commun 88: 1329–1336.S. BodanskyLB MintzDS Holmes1979The mesokaryote Gyrodinium cohnii lacks nucleosomes.Biochem Biophys Res Commun8813291336
  15. 15. Rizzo PJ, Nooden LD (1972) Chromosomal proteins in the dinoflagellate alga Gyrodinium cohnii. Science 176: 796–797.PJ RizzoLD Nooden1972Chromosomal proteins in the dinoflagellate alga Gyrodinium cohnii.Science176796797
  16. 16. Rizzo PJ, Burghardt RC (1982) Histone-like protein and chromatin structure in the wall-less dinoflagellate Gymnodinium nelsoni. Biosystems 15: 27–34.PJ RizzoRC Burghardt1982Histone-like protein and chromatin structure in the wall-less dinoflagellate Gymnodinium nelsoni.Biosystems152734
  17. 17. Rizzo PJ (1981) Comparative aspects of basic chromatin proteins in dinoflagellates. Biosystems 14: 433–443.PJ Rizzo1981Comparative aspects of basic chromatin proteins in dinoflagellates.Biosystems14433443
  18. 18. Chudnovsky Y, Li JF, Rizzo PJ, Hastings JW, Fagan TF (2008) Cloning, expression and characterization of a histone-like protein from the marine dinoflagellate Lingulodinium polyedrum (Dinophyceae). J Phycol 38: 543–550.Y. ChudnovskyJF LiPJ RizzoJW HastingsTF Fagan2008Cloning, expression and characterization of a histone-like protein from the marine dinoflagellate Lingulodinium polyedrum (Dinophyceae).J Phycol38543550
  19. 19. Chan YH, Wong JT (2007) Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3. Nucleic Acids Res 35: 2573–2583.YH ChanJT Wong2007Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3.Nucleic Acids Res3525732583
  20. 20. Kapros T, Robertson AJ, Waterborg JH (1995) Histone H3 transcript stability in alfalfa. Plant Mol Biol 28: 901–914.T. KaprosAJ RobertsonJH Waterborg1995Histone H3 transcript stability in alfalfa.Plant Mol Biol28901914
  21. 21. Tanimoto EY, Rost TL, Comai L (1993) DNA Replication-Dependent Histone H2A mRNA Expression in Pea Root Tips. Plant Physiol 103: 1291–1297.EY TanimotoTL RostL. Comai1993DNA Replication-Dependent Histone H2A mRNA Expression in Pea Root Tips.Plant Physiol10312911297
  22. 22. Taoka K, Ohtsubo N, Fujimoto Y, Mikami K, Meshi T, et al. (1998) The modular structure and function of the wheat H1 promoter with S phase-specific activity. Plant Cell Physiol 39: 294–306.K. TaokaN. OhtsuboY. FujimotoK. MikamiT. Meshi1998The modular structure and function of the wheat H1 promoter with S phase-specific activity.Plant Cell Physiol39294306
  23. 23. Mosammaparast N, Guo Y, Shabanowitz J, Hunt DF, Pemberton LF (2002) Pathways mediating the nuclear import of histones H3 and H4 in yeast. J Biol Chem 277: 862–868.N. MosammaparastY. GuoJ. ShabanowitzDF HuntLF Pemberton2002Pathways mediating the nuclear import of histones H3 and H4 in yeast.J Biol Chem277862868
  24. 24. Mosammaparast N, Jackson KR, Guo Y, Brame CJ, Shabanowitz J, et al. (2001) Nuclear import of histone H2A and H2B is mediated by a network of karyopherins. J Cell Biol 153: 251–262.N. MosammaparastKR JacksonY. GuoCJ BrameJ. Shabanowitz2001Nuclear import of histone H2A and H2B is mediated by a network of karyopherins.J Cell Biol153251262
  25. 25. Fries T, Betz C, Sohn K, Caesar S, Schlenstedt G, et al. (2007) A novel conserved nuclear localization signal is recognized by a group of yeast importins. J Biol Chem 282: 19292–19301.T. FriesC. BetzK. SohnS. CaesarG. Schlenstedt2007A novel conserved nuclear localization signal is recognized by a group of yeast importins.J Biol Chem2821929219301
  26. 26. Krude T (1999) Chromatin assembly during DNA replication in somatic cells. Eur J Biochem 263: 1–5.T. Krude1999Chromatin assembly during DNA replication in somatic cells.Eur J Biochem26315
  27. 27. Park YJ, Luger K (2006) The structure of nucleosome assembly protein 1. Proc Natl Acad Sci U S A 103: 1248–1253.YJ ParkK. Luger2006The structure of nucleosome assembly protein 1.Proc Natl Acad Sci U S A10312481253
  28. 28. Vaquero A, Loyola A, Reinberg D (2003) The constantly changing face of chromatin. Sci Aging Knowledge Environ 2003: RE4.A. VaqueroA. LoyolaD. Reinberg2003The constantly changing face of chromatin.Sci Aging Knowledge Environ2003RE4
  29. 29. Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21: 381–395.AJ BannisterT. Kouzarides2011Regulation of chromatin by histone modifications.Cell Res21381395
  30. 30. Okamoto OK, Hastings JW (2003) Genome-wide analysis of redox-regulated genes in a dinoflagellate. Gene 321: 73–81.OK OkamotoJW Hastings2003Genome-wide analysis of redox-regulated genes in a dinoflagellate.Gene3217381
  31. 31. Hackett JD, Scheetz TE, Yoon HS, Soares MB, Bonaldo MF, et al. (2005) Insights into a dinoflagellate genome through expressed sequence tag analysis. BMC Genomics 6: 80.JD HackettTE ScheetzHS YoonMB SoaresMF Bonaldo2005Insights into a dinoflagellate genome through expressed sequence tag analysis.BMC Genomics680
  32. 32. Lin S, Zhang H, Zhuang Y, Tran B, Gill J (2010) Spliced leader-based metatranscriptomic analyses lead to recognition of hidden genomic features in dinoflagellates. Proc Natl Acad Sci U S A 107: 20033–20038.S. LinH. ZhangY. ZhuangB. TranJ. Gill2010Spliced leader-based metatranscriptomic analyses lead to recognition of hidden genomic features in dinoflagellates.Proc Natl Acad Sci U S A1072003320038
  33. 33. Zhang H, Hou Y, Miranda L, Campbell DA, Sturm NR, et al. (2007) Spliced leader RNA trans-splicing in dinoflagellates. Proc Natl Acad Sci U S A 104: 4618–4623.H. ZhangY. HouL. MirandaDA CampbellNR Sturm2007Spliced leader RNA trans-splicing in dinoflagellates.Proc Natl Acad Sci U S A10446184623
  34. 34. Guillard RR, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can J Microbiol 8: 229–239.RR GuillardJH Ryther1962Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran.Can J Microbiol8229239
  35. 35. Chavez-Blanco A, Segura-Pacheco B, Perez-Cardenas E, Taja-Chayeb L, Cetina L, et al. (2005) Histone acetylation and histone deacetylase activity of magnesium valproate in tumor and peripheral blood of patients with cervical cancer. A phase I study. Mol Cancer 4: 22.A. Chavez-BlancoB. Segura-PachecoE. Perez-CardenasL. Taja-ChayebL. Cetina2005Histone acetylation and histone deacetylase activity of magnesium valproate in tumor and peripheral blood of patients with cervical cancer. A phase I study.Mol Cancer422
  36. 36. Shechter D, Dormann HL, Allis CD, Hake SB (2007) Extraction, purification and analysis of histones. Nat Protoc 2: 1445–1457.D. ShechterHL DormannCD AllisSB Hake2007Extraction, purification and analysis of histones.Nat Protoc214451457
  37. 37. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, et al. (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36: W465–469.A. DereeperV. GuignonG. BlancS. AudicS. Buffet2008Phylogeny.fr: robust phylogenetic analysis for the non-specialist.Nucleic Acids Res36W465469
  38. 38. Dagenais-Bellefeuille S, Bertomeu T, Morse D (2008) S-phase and M-phase timing are under independent circadian control in the dinoflagellate Lingulodinium. J Biol Rhythms 23: 400–408.S. Dagenais-BellefeuilleT. BertomeuD. Morse2008S-phase and M-phase timing are under independent circadian control in the dinoflagellate Lingulodinium.J Biol Rhythms23400408
  39. 39. Rizzo PJ (2003) Those amazing dinoflagellate chromosomes. Cell Res 13: 215–217.PJ Rizzo2003Those amazing dinoflagellate chromosomes.Cell Res13215217
  40. 40. Moreno Diaz de la Espina S, Alverca E, Cuadrado A, Franca S (2005) Organization of the genome and gene expression in a nuclear environment lacking histones and nucleosomes: the amazing dinoflagellates. Eur J Cell Biol 84: 137–149.S. Moreno Diaz de la EspinaE. AlvercaA. CuadradoS. Franca2005Organization of the genome and gene expression in a nuclear environment lacking histones and nucleosomes: the amazing dinoflagellates.Eur J Cell Biol84137149
  41. 41. Costas E, Goyanes V (2005) Architecture and evolution of dinoflagellate chromosomes: an enigmatic origin. Cytogenet Genome Res 109: 268–275.E. CostasV. Goyanes2005Architecture and evolution of dinoflagellate chromosomes: an enigmatic origin.Cytogenet Genome Res109268275
  42. 42. Shupe K, Rizzo PJ (1983) Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus, Peridinium balticum, and Crypthecodinium cohnii. Journal of Eukaryotic Microbiology 30: 599–606.K. ShupePJ Rizzo1983Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus, Peridinium balticum, and Crypthecodinium cohnii.Journal of Eukaryotic Microbiology30599606
  43. 43. Rizzo PJ, Cox ER (1977) Histone occurrence in chromatin from Peridinium balticum, a binucleate dinoflagellate. Science 198: 1258–1260.PJ RizzoER Cox1977Histone occurrence in chromatin from Peridinium balticum, a binucleate dinoflagellate.Science19812581260
  44. 44. Tomas RN, Cox ER, Steidinger KA (1973) PERIDINIUM BALTICUM (LEVANDER) LEMMERMANN, AN UNUSUAL DINOFLAGELLATE WITH A MESOCARYOTIC AND AN EUCARYOTIC NUCLEUS. Journal of Phycology 9: 91–98.RN TomasER CoxKA Steidinger1973PERIDINIUM BALTICUM (LEVANDER) LEMMERMANN, AN UNUSUAL DINOFLAGELLATE WITH A MESOCARYOTIC AND AN EUCARYOTIC NUCLEUS.Journal of Phycology99198
  45. 45. Postberg J, Forcob S, Chang WJ, Lipps HJ (2010) The evolutionary history of histone H3 suggests a deep eukaryotic root of chromatin modifying mechanisms. BMC Evol Biol 10: 259.J. PostbergS. ForcobWJ ChangHJ Lipps2010The evolutionary history of histone H3 suggests a deep eukaryotic root of chromatin modifying mechanisms.BMC Evol Biol10259
  46. 46. West MH, Bonner WM (1980) Histone 2A, a heteromorphous family of eight protein species. Biochemistry 19: 3238–3245.MH WestWM Bonner1980Histone 2A, a heteromorphous family of eight protein species.Biochemistry1932383245
  47. 47. Thatcher TH, Gorovsky MA (1994) Phylogenetic analysis of the core histones H2A, H2B, H3, and H4. Nucleic Acid Research 22: 174–179.TH ThatcherMA Gorovsky1994Phylogenetic analysis of the core histones H2A, H2B, H3, and H4.Nucleic Acid Research22174179
  48. 48. Baxevanis AD, Landsman D (1996) Histone Sequence Database: a compilation of highly-conserved nucleoprotein sequences. Nucleic Acids Res 24: 245–247.AD BaxevanisD. Landsman1996Histone Sequence Database: a compilation of highly-conserved nucleoprotein sequences.Nucleic Acids Res24245247
  49. 49. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273: 5858–5868.EP RogakouDR PilchAH OrrVS IvanovaWM Bonner1998DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.J Biol Chem27358585868
  50. 50. Dhillon N, Oki M, Szyjka SJ, Aparicio OM, Kamakaka RT (2006) H2A.Z functions to regulate progression through the cell cycle. Mol Cell Biol 26: 489–501.N. DhillonM. OkiSJ SzyjkaOM AparicioRT Kamakaka2006H2A.Z functions to regulate progression through the cell cycle.Mol Cell Biol26489501
  51. 51. Robin P, Fritsch L, Philipot O, Svinarchuk F, Ait-Si-Ali S (2007) Post-translational modifications of histones H3 and H4 associated with the histone methyltransferases Suv39h1 and G9a. Genome Biol 8: R270.P. RobinL. FritschO. PhilipotF. SvinarchukS. Ait-Si-Ali2007Post-translational modifications of histones H3 and H4 associated with the histone methyltransferases Suv39h1 and G9a.Genome Biol8R270
  52. 52. Velculescu VE, Zhang L, Zhou W, Vogelstein J, Basrai MA, et al. (1997) Characterization of the yeast transcriptome. Cell 88: 243–251.VE VelculescuL. ZhangW. ZhouJ. VogelsteinMA Basrai1997Characterization of the yeast transcriptome.Cell88243251
  53. 53. Suganuma T, Workman JL (2010) Signals and Combinatorial Functions of Histone Modifications. Annu Rev Biochem. T. SuganumaJL Workman2010Signals and Combinatorial Functions of Histone Modifications.Annu Rev Biochem
  54. 54. Verdin E, Dequiedt F, Kasler HG (2003) Class II histone deacetylases: versatile regulators. Trends Genet 19: 286–293.E. VerdinF. DequiedtHG Kasler2003Class II histone deacetylases: versatile regulators.Trends Genet19286293
  55. 55. Margueron R, Trojer P, Reinberg D (2005) The key to development: interpreting the histone code? Curr Opin Genet Dev 15: 163–176.R. MargueronP. TrojerD. Reinberg2005The key to development: interpreting the histone code?Curr Opin Genet Dev15163176
  56. 56. Glozak MA, Sengupta N, Zhang X, Seto E (2005) Acetylation and deacetylation of non-histone proteins. Gene 363: 15–23.MA GlozakN. SenguptaX. ZhangE. Seto2005Acetylation and deacetylation of non-histone proteins.Gene3631523
  57. 57. Frye RA (2000) Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun 273: 793–798.RA Frye2000Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins.Biochem Biophys Res Commun273793798
  58. 58. Starai VJ, Celic I, Cole RN, Boeke JD, Escalante-Semerena JC (2002) Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 298: 2390–2392.VJ StaraiI. CelicRN ColeJD BoekeJC Escalante-Semerena2002Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine.Science29823902392
  59. 59. Martzen MR, McCraith SM, Spinelli SL, Torres FM, Fields S, et al. (1999) A biochemical genomics approach for identifying genes by the activity of their products. Science 286: 1153–1155.MR MartzenSM McCraithSL SpinelliFM TorresS. Fields1999A biochemical genomics approach for identifying genes by the activity of their products.Science28611531155
  60. 60. Trievel RC, Beach BM, Dirk LM, Houtz RL, Hurley JH (2002) Structure and catalytic mechanism of a SET domain protein methyltransferase. Cell 111: 91–103.RC TrievelBM BeachLM DirkRL HoutzJH Hurley2002Structure and catalytic mechanism of a SET domain protein methyltransferase.Cell11191103
  61. 61. Pennini ME, Perrinet S, Dautry-Varsat A, Subtil A (2010) Histone methylation by NUE, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis. PLoS Pathog 6: e1000995.ME PenniniS. PerrinetA. Dautry-VarsatA. Subtil2010Histone methylation by NUE, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis.PLoS Pathog6e1000995
  62. 62. Chudnovsky Y, Li JF, Rizzo PJ, Hastings JW, Fagan T (2002) CLONING, EXPRESSION, AND CHARACTERIZATION OF A HISTONE-LIKE PROTEIN FROM THE MARINE DINOFLAGELLATE LINGULODINIUM POLYEDRUM. Journal of Phycology 38: 543–550.Y. ChudnovskyJF LiPJ RizzoJW HastingsT. Fagan2002CLONING, EXPRESSION, AND CHARACTERIZATION OF A HISTONE-LIKE PROTEIN FROM THE MARINE DINOFLAGELLATE LINGULODINIUM POLYEDRUM.Journal of Phycology38543550
  63. 63. Canela N, Rodriguez-Vilarrupla A, Estanyol JM, Diaz C, Pujol MJ, et al. (2003) The SET protein regulates G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 activity. J Biol Chem 278: 1158–1164.N. CanelaA. Rodriguez-VilarruplaJM EstanyolC. DiazMJ Pujol2003The SET protein regulates G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 activity.J Biol Chem27811581164
  64. 64. Kellogg DR, Kikuchi A, Fujii-Nakata T, Turck CW, Murray AW (1995) Members of the NAP/SET family of proteins interact specifically with B-type cyclins. J Cell Biol 130: 661–673.DR KelloggA. KikuchiT. Fujii-NakataCW TurckAW Murray1995Members of the NAP/SET family of proteins interact specifically with B-type cyclins.J Cell Biol130661673
  65. 65. Kellogg DR, Murray AW (1995) NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast. J Cell Biol 130: 675–685.DR KelloggAW Murray1995NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast.J Cell Biol130675685
  66. 66. Wu RS, Bonner WM (1981) Separation of basal histone synthesis from S-phase histone synthesis in dividing cells. Cell 27: 321–330.RS WuWM Bonner1981Separation of basal histone synthesis from S-phase histone synthesis in dividing cells.Cell27321330
  67. 67. Robbins E, Borun TW (1967) The cytoplasmic synthesis of histones in hela cells and its temporal relationship to DNA replication. Proc Natl Acad Sci U S A 57: 409–416.E. RobbinsTW Borun1967The cytoplasmic synthesis of histones in hela cells and its temporal relationship to DNA replication.Proc Natl Acad Sci U S A57409416
  68. 68. Ehinger A, Denison SH, May GS (1990) Sequence, organization and expression of the core histone genes of Aspergillus nidulans. Mol Gen Genet 222: 416–424.A. EhingerSH DenisonGS May1990Sequence, organization and expression of the core histone genes of Aspergillus nidulans.Mol Gen Genet222416424
  69. 69. Heintz N, Sive HL, Roeder RG (1983) Regulation of human histone gene expression: kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle. Mol Cell Biol 3: 539–550.N. HeintzHL SiveRG Roeder1983Regulation of human histone gene expression: kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle.Mol Cell Biol3539550
  70. 70. Bertomeu T, Rivoal J, Morse D (2007) A dinoflagellate CDK5-like cyclin-dependent kinase. Biol Cell 99: 531–540.T. BertomeuJ. RivoalD. Morse2007A dinoflagellate CDK5-like cyclin-dependent kinase.Biol Cell99531540