MARGEN-00216; No of Pages 5 Marine Genomics xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana"☆ Leïla Tirichine a,⁎, Xin Lin a,1, Yann Thomas a, Bérangère Lombard b, Damarys Loew b, Chris Bowler a,⁎ a b
Environmental and Evolutionary Genomics Section, Institut de Biologie de l'École Normale Supérieure (IBENS), CNRS UMR 8197 INSERM U1024, 46 rue d'Ulm 75005 Paris, France Institut Curie, Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, 26 rue d'Ulm 75248 Cedex 05 Paris, France
a r t i c l e
i n f o
Article history: Received 29 July 2013 Received in revised form 27 October 2013 Accepted 21 November 2013 Keywords: Phaeodactylum tricornutum Thalassiosira pseudonana Epigenetics Mass spectrometry Post-translational histone modifications
a b s t r a c t Post-translational modifications of histones affect many biological processes by influencing higher order chromatin structure that affects gene and genome regulation. It is therefore important to develop methods for extracting histones while maintaining their native post-translational modifications. While histone extraction protocols have been developed in multicellular and single celled organisms such as yeast and Arabidopsis, they are inefficient in diatoms that have a silica cell wall that is likely to hinder histone extraction. We report in this work a rapid and reliable method for extraction of large amounts of high quality histones from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. The protocol is an important enabling step permitting downstream applications such as western blotting and mass spectrometry. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Post-translational modifications (PTMs) on the N-terminal tails of histones play important roles in many biological processes, e.g., by regulating chromatin dynamics and transcription. Combinatorial PTMs of histones give rise to the histone code which defines active or repressed chromatin states which in turn regulate gene expression. Chromatin, the site where all these modifications take place and are regulated, is composed of nucleosomes in which 146 bp DNA is wrapped around an octamer composed of two copies of each of the four core histones, H2A, H2B, H3 and H4. Nucleosomes are linked to each other with the linker histone H1 which is important for nucleosome stability and higher order chromatin structures. The protruding tails of histones are subject to several covalent modifications that include methylation, acetylation, phosphorylation, ubiquitination, sumoylation, and adenosine-diphosphate ribosylation.
☆ The publisher would like to inform the readership that this article is a reprint of a previously published article. An error occurred on the publisher’s side which resulted in the publication of this article in a wrong issue. As a consequence, the publisher would like to make this reprint available for the reader's convenience and for the continuity of the papers involved in the Special Issue. For citation purposes, please use the original publication details; Marine Genomics, Volume 13C, February 2014, Pages 21-25. The publisher sincerely apologizes to the readership and in particular to the author of the respective article and deeply regrets the inconvenience caused. DOI of original article: http://dx.doi.org/10.1016/j.margen.2013.11.006. ⁎ Corresponding authors. E-mail addresses: [email protected], [email protected] (L. Tirichine). 1 Current address: State Key Lab of Marine Environmental Science, Xiamen University, China.
The availability of fast, reliable and cheap methods for histone extraction that preserve native PTMs of histones is fundamental for understanding and deciphering the dynamics and complexity of the histone code. Protocols for histone extraction have been developed for many organisms ranging from yeast to human. However, very few protocols are known for eukaryotic photosynthetic single celled algae, with the exception of Chlamydomonas reinhardtii (Morris et al., 1990). We report in the present work a simple and rapid protocol for the isolation and purification of histones in two model eukaryotic diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Diatoms (Bacillariophyceae) are believed to be the most important group of eukaryotic phytoplankton (Bowler et al., 2010). They account for more than 20% of global carbon fixation and 40% of marine primary productivity. Thus, they have a crucial role in the food web and the biological pump that draws down atmospheric carbon dioxide to the ocean interior. With more than 100,000 species estimated, they are likely to be one of the most diverse groups of photosynthetic organisms on Earth (Round et al., 1992). Besides their biogeochemical importance, diatoms are also attracting interest for the nanotechnology, pharmaceutical and biofuel industries (Dismukes et al., 2008; Kroger and Poulsen, 2008). P. tricornutum is a marine diatom whose genome has been fully sequenced (Bowler et al., 2008). It is widely used as a model for pennate diatoms because of its small genome (27 Mb), short life cycle, ease of transformation and growth in laboratory conditions (De Riso et al., 2009), availability of molecular tools for functional genomic characterization (Siaut et al., 2007; Maheswari et al., 2010), and genetic resources such as 12 ecotypes collected worldwide (De Martino et al., 2007), Bowler, unpublished). P. tricornutum is also an emerging model for epigenetic studies in single celled eukaryotes because it has all the components
1874-7787/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.margen.2014.05.003
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
2
L. Tirichine et al. / Marine Genomics xxx (2014) xxx–xxx
of the epigenetic machinery such as DNA methylation (Veluchamy et al., 2013), histones that can be modified (Lin et al., 2012), Veluchamy et al., in preparation), and small RNA-mediated regulation (De Riso et al., 2009; Huang et al., 2011), Angela Falciatore, personal communication). Likewise, T. pseudonana is a model diatom for centric diatoms, and for which a fully sequenced genome and molecular tools are also available (Armbrust et al., 2004; Poulsen and Kroger, 2005; Nicole Poulsen and Kröger, 2006. Histones, their variants and modifications play an important role in transcriptional regulation but also in higher order chromatin structure. Characterization of native histone PTMs by mass spectrometry is a useful technology to survey PTMs prior to the mapping of modifications of interest in the genome by chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). A prerequisite for this is to obtain native purified histones. The majority of protocols published so far use an acid or high salt (NaCl) extraction, which seems to work for most species (Shechter et al., 2007; Jufvas et al., 2011). However, both types of extraction failed in P. tricornutum (data not shown). We therefore adapted a protocol previously described for histone extraction from Chlamydomonas reinhardtii, where it was also reported that acid and salt extraction did not succeed in extracting histones (Morris et al., 1990). Our work describes a protocol for extracting core histones of good quality and pure enough to be used for western blot and mass spectrometry without the need to be purified over a reversed phase HPLC column.
2. Results and discussion The protocol comprises a step of nuclei extraction before salt extraction of histones, acid precipitation followed by precipitation of histones with trichloroacetic acid (TCA) and several washes for purification of histones, which are then air dried and resuspended for gel visualization and mass spectrometry (Fig. 1). Cultures of P. tricornutum Bohlin clone Pt1 8.6 (CCMP2561) and Thalassiosira pseudonana (CCMP1335) were grown in artificial seawater at 19 °C under cool white fluorescent lights at approximately 75 μmol m−2 s−1 in 12 h light:12 h dark conditions and maintained in exponential phase in semi-continuous batch cultures. Cells were harvested at approximately 1.4 × 106 cells per ml by centrifugation at 4000 rpm for 20 min. Cell pellets can be stored at − 80 °C for several months. Nuclei were then extracted as previously described (Lin et al., 2012). Pellets of nuclei can be stored at − 20 °C for several weeks. Histones were extracted from isolated nuclei with extraction buffer containing 1 M CaCl2, 20 mM Tris HCl pH 7.4 and 1 mini tablet of proteinase inhibitors cocktail (Complete protease cocktail Inhibitor (Roche cat. No. 11873 580 001)). High salt extraction disrupts DNA–histone interactions and precipitates contaminants such as nucleic acids and non-histone proteins while core histones remain soluble in the supernatant and thus can be enriched and purified. Samples were kept on ice for 10 min. HCl was then added to 0.3 N and samples were
Cultures of P. tricornutum or T. pseudonana 2 hours
15 minutes Extraction buffer 1M CaCl2, 20mM Tris HCl pH 7.2, 1 tablet protease inhibitor cocktail 5 minutes Add pure HCl to 0.3N
40 minutes
Add TCA drop wise to 20% and leave on ice for 10 min. Centrifuge for 30 min at 13000 rpm and 4°C
15 minutes 1st wash: 20% TCA 2nd wash with acetone containing 0.2% HCl 3 washes with acetone Dry sample before storage. Fig. 1. Outline of the different steps of the histone extraction protocol. After centrifugation of diatom cultures, pellets can be stored at −80 °C for several months. Isolated nuclei can be stored at −20 °C for several weeks. Likewise, once histones are isolated, they can be stored at −20 °C for several weeks.
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
L. Tirichine et al. / Marine Genomics xxx (2014) xxx–xxx
It is also known that CaCl2 is routinely used because the heat it generates helps permeabilize membranes allowing a better lysis of the cells. The protocol described herein improves the original protocol from Chlamydomonas (Morris et al., 1990) and removes the use of thiodiglycol which was found to be unnecessary. This is of significance because thiodiglycol is a highly toxic solvent and its utilization is subject to strict regulations. Furthermore, thiodiglycol is a solvent that can be resuspended only in ethanol which mimics some histone methylation, thus inducing a bias if PTMs are to be monitored. We indeed observed methylation on some histone residues when thiodiglycol was included which could not be reproduced when it was omitted (data not shown). We have further modified the Chlamydomonas protocol by adding to the extraction buffer a protease inhibitor cocktail from Roche instead of aprotinin and leupeptin used in the original protocol. This modification reduces the cost without compromising the integrity of the extracted proteins and increases the practicality of the protocol. For western blotting, histone pellets were resuspended in loading dye (0.5 M Tris–HCl pH 8.45, 10% LiDS, 1% b-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue) and denatured at 100 °C for 5 min before loading on a 14% tris-tricine gel. Samples were run at 80 V until they migrated from the stacking gel and then the voltage was set to 100 V for 90 min (Fig. 2A, C) for a good separation of the bands. Gels were first washed in distilled water for 5 min before staining with LabSafe GEL Blue™ (786-35) from G-Biosceinces for 30 min. Gels were
centrifuged at 4 °C at 10 000 rpm for 5 min to precipitate the acidinsoluble fraction. TCA was added to 20% to the supernatant, which contains the acid soluble proteins. To precipitate histones, TCA was added drop wise to the sample kept on ice. Samples were kept on ice for a further 10 min and then centrifuged for 30 min at 13 000 rpm at 4 °C to precipitate histones. We added in our protocol an extra centrifugation step compared to the original method, which increased the yield of precipitated histones. The pellet was washed successively with 20% TCA, and then acetone containing 0.2% HCl, followed with three further washes with acetone. During each wash, the histone pellet was resuspended in the wash buffer by flipping the tubes. The pellet was air dried using a speed vacuum or under a hood. The histones can be stored at − 20 °C for several weeks. The samples at this stage can be used for western blotting or mass spectrometry. The reasons for the lack of success of the acid and NaCl high salt extraction of histones from Phaeodactylum are unclear but it is possible that during extraction the histones might stay bound to the DNA and precipitate with other contaminants, giving rise to a liquid phase that is free of histones. More precisely, sulfuric acid and sodium chloride might not be efficient in breaking the DNA–histone bonds, whereas calcium chloride might overcome this bottleneck thanks to either its exothermic activity that would soften DNA binding to histones or its higher ion exchanging activity of multivalent ions compared to that of monovalent ions disrupting thus electrostatic interaction between DNA and histone.
A
3
B MW KDa
1
2
3
1
2
3
4
MW KDa
40
35 25 15
10
C
1
*
*
H1
* * *
* * *
H3 H2A H2B
*
*
H4
MW KDa
2
* * * * *
D
40 35 25 H1 H3 15 H2A H2B
MW KDa H1
* * ** *
H3 H2A H2B H4
10 H4
Fig. 2. Tris–tricine SDS-PAGE of histones extracted from P. tricornutum and T. pseudonana. (A) Samples of P. tricornutum histones extracted from (1) intact nuclei prepared from 300 ml of cultures at a cell density of 1.4 × 106 cells/ml (2) intact nuclei from 300 ml culture with cell density of 6 × 105 cells/ml (3) chromatin isolated from 300 ml culture with cell density of 1 × 106 cells/ml. Page ruler Prestained Protein Ladder from Fermentas (#SM0671) was used. (B) Samples of P. tricornutum histones extracted from intact nuclei using different buffers. If not specified, extraction buffers used elsewhere do not contain any of the detergent used in 2B (1) extraction buffer with 0.1% SDS (2) extraction buffer with 1% thiodiglycol (3) extraction buffer without SDS or thiodiglycol (4) Calf thymus histones used as a control. (C) Histone samples extracted from T. pseudonana nuclei. (1) Calf thymus histones used as control (2) T. pseudonana histones (D) migration on gels of P. tricornutum histones used for mass spectrometry analysis. Histones were extracted from intact nuclei. Samples were run for a short time to have a maximum of histones concentrated in small bands of gel for mass spectrometry purposes to avoid sample dilution. Sample concentrations range between 0.2 and 1 μg. Asterisks indicate histones H4, H2A, H2B, H3 and H1.
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
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visualized after 3 washes during 5 min each with distilled water. No other detergent or solvent is needed for the initial step of histone extraction using high salt buffer. The yield of histones is equally good with and without thiodiglycol but higher compared to samples extracted with 0.1% SDS (Fig. 2B). We found that it was very important to initiate the salt extraction from high cell density but also from intact nuclei because nuclei lysis to extract chromatin prior to high salt extraction decreases the yield of histones, as shown in Fig. 2A. If the samples were to be used for mass spectrometry, migration time was reduced to 30 min (Fig. 2D).
For mass spectrometry analysis, samples were prepared in loading buffer as above and run using the same gel system for approximately 30 min after migrating out of the stacking gel to prevent their dilution. Excised gel slices were washed and proteins were reduced with 10 mM DTT prior to alkylation with 55 mM iodoacetamide. After washing and shrinking of the gel pieces with 100% acetonitrile, in-gel digestion was performed using trypsin and chymotrypsin overnight in 25 mM ammonium bicarbonate at 30 °C. The extracted peptides were analyzed by nano-LC-MS/MS using an Ultimate3000 system (Dionex S.A.) coupled
Fig. 3. Representative MS/MS spectra for identification of histone modifications in P. tricornutum. LC-ESI MS/MS spectra for acetylated H4 peptide GLGKAcGGAKAcR (464.3(2+) m/z) (A) and methylated H3 peptide KMeSAPATGGVKMeKPHR (731.4(2+) m/z) (B). The fragmentation spectra derived from trypsin H4 and H3 peptides are shown. The inset shows peptide sequence and the observed ions obtained. According to peptide fragmentation nomenclature, y ions extend from the N-terminus while b ions are indicative of C-terminus of the peptide. The tandem mass spectra are labelled to show singly and doubly charged b and y ions, as well as ions corresponding to neutral losses of water (°) NH3 (*) and M the parent ion mass. Me, Methylation; Ac, Acetylation.
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
L. Tirichine et al. / Marine Genomics xxx (2014) xxx–xxx
to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) (Fig. 3). Samples were loaded on a C18 pre-column (300 μm inner diameter × 5 mm; Dionex) at 20 μl/min in 2% acetonitrile, 0.1% TFA. After 3 min of desalting, the pre-column was switched on-line with the analytical C18 column (75 μm inner diameter × 50 cm; C18 PepMap™, Dionex) equilibrated in 100% solvent A (2% acetonitrile, 0.1% acide formic). Bound peptides were eluted using a 0 to 30% gradient of solvent B (80% acetonitrile, 0.085% formic acid) during 157 min, then 30 to 50% gradient of solvent B during 20 min at a 150 nl/min flow rate (40 °C). Data-dependent acquisition was performed on the LTQOrbitrap mass spectrometer in the positive ion mode. Survey MS scans were acquired in the Orbitrap on the 400–1200 m/z range with the resolution set to a value of 100 000. Each scan was recalibrated in real time by co-injecting an internal standard from ambient air into the C-trap (‘lock mass option’). The five most intense ions per survey scan were selected for CID fragmentation and the resulting fragments were analyzed in the linear trap (LTQ). Target ions already selected for MS/MS were dynamically excluded for 20 s. Data were acquired using the Xcalibur software (version 2.0.7) and the resulting spectra were then analyzed via the Mascot™ Software created with Proteome Discoverer (version 1.4, Thermo Scientific) using the in-house database containing the sequence of histone proteins from P. tricornutum and T. pseudonana. Carbamidomethylation of cysteines, oxidation of methionines, acetylation of lysines and protein N-termini, methylation, dimethylation of lysines, arginines and trimethylation of lysines were set as variable modifications for all Mascot searches. Specificity of trypsin digestion was set for cleavage after Lys or Arg except before Pro, and five missed trypsin cleavage sites were allowed. The mass tolerances in MS and MS/MS were set to 5 ppm and 0.5 Da, respectively. The resulting Mascot files were further processed using myProMS (Poullet et al., 2007). Similarly, mass spectrometry analysis of histones extracted from T. pseudonana detected modifications on several residues (data not shown). In conclusion, we have developed a simple and rapid protocol for extracting histones from two model diatoms and have shown using mass spectrometry that the extracted histones are of good quality and can be used to monitor their PTMs. This is of particular interest considering the role of histones in higher order structures of chromatin and thus in regulating gene expression. Furthermore, the described protocol opens the way for investigating a whole unexplored field of epigenetics in diatoms, which are outstanding models for studying how the environment shapes the evolution and fitness of unicellular marine protists. Future work based on this protocol should therefore allow further insights into the reasons underlying the ecological success of diatoms. Contributions
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analysis. XL and YT assisted technically LT. LT drafted the manuscript. LT and CB wrote the manuscript. All authors read and approved the manuscript. Acknowledgments CB acknowledges support from the EU MicroB3 project and a European Research Council Advanced Award. References Armbrust, E.V., Berges, J.A., Bowler, C., Green, B.R., Martinez, D., Putnam, N.H., Zhou, S., Allen, A.E., Apt, K.E., Bechner, M., et al., 2004. The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306 (5693), 79–86. Bowler, C., Allen, A.E., Badger, J.H., Grimwood, J., Jabbari, K., Kuo, A., Maheswari, U., Martens, C., Maumus, F., Otillar, R.P., et al., 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456 (7219), 239–244. Bowler, C., Vardi, A., Allen, A.E., 2010. Oceanographic and biogeochemical insights from diatom genomes. Ann. Rev. Mar. Sci. 2, 333–365. De Martino, A., Meichenein, A., Shi, J., Pan, K., Bowler, C., 2007. 'Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions'. J. Phycol. 43, 992–1009. De Riso, V., Raniello, R., Maumus, F., Rogato, A., Bowler, C., Falciatore, A., 2009. Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res. 37 (14), e96. Dismukes, G.C., Carrieri, D., Bennette, N., Ananyev, G.M., Posewitz, M.C., 2008. Aquatic phototrophs: efficient alternatives to land-based crops for biofuels. Curr. Opin. Biotechnol. 19 (3), 235–240. Huang, A., He, L., Wang, G., 2011. Identification and characterization of microRNAs from Phaeodactylum tricornutum by high-throughput sequencing and bioinformatics analysis. BMC Genomics 12, 337. Jufvas, A., Stralfors, P., Vener, A.V., 2011. Histone variants and their post-translational modifications in primary human fat cells. PLoS ONE 6 (1), e15960. Kroger, N., Poulsen, N., 2008. Diatoms-from cell wall biogenesis to nanotechnology. Annu. Rev. Genet. 42, 83–107. Lin, X., Tirichine, L., Bowler, C., 2012. Protocol: chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species. Plant Methods 8 (1), 48. Maheswari, U., Jabbari, K., Petit, J.L., Porcel, B.M., Allen, A.E., Cadoret, J.P., De Martino, A., Heijde, M., Kaas, R., La Roche, J., et al., 2010. Digital expression profiling of novel diatom transcripts provides insight into their biological functions. Genome Biol. 11 (8), R85. Morris, R.L., Keller, L.R., Zweidler, A., Rizzo, P.J., 1990. Analysis of Chlamydomonas reinhardtii histones and chromatin. J. Protozool. 37 (2), 117–123. Nicole Poulsen, P.M.C., Kröger, Nils, 2006. Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). J. Phycol. 42 (5), 1059–1065. Poullet, P., Carpentier, S., Barillot, E., 2007. myProMS, a web server for management and validation of mass spectrometry-based proteomic data. Proteomics 7 (15), 2553–2556. Poulsen, N., Kroger, N., 2005. A new molecular tool for transgenic diatoms: control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette. FEBS J. 272 (13), 3413–3423. Round, F.E., Crawford, R.M., Mann, D.G., 1992. 'the Diatoms: biology and morphology of the genera'. Cambridge University Press, New York 758. Shechter, D., Dormann, H.L., Allis, C.D., Hake, S.B., 2007. Extraction, purification and analysis of histones. Nat. Protoc. 2 (6), 1445–1457. Siaut, M., Heijde, M., Mangogna, M., Montsant, A., Coesel, S., Allen, A., Manfredonia, A., Falciatore, A., Bowler, C., 2007. Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406 (1–2), 23–35. Veluchamy, A., Lin, X., Maumus, F., Rivarola, M., Bhavsar, J., Creasy, T., O'Brien, K., Sengamalay, N.A., Tallon, L.J., Smith, A.D., et al., 2013. Insights into the role of DNA methylation in diatoms by genome-wide profiling in Phaeodactylum tricornutum. Nat. Commun. 4, 2091.
LT designed the study and performed the experiments. LB carried out the MS experimental work. LD supervised MS and proteomic data
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana"☆ Leïla Tirichine a,⁎, Xin Lin a,1, Yann Thomas a, Bérangère Lombard b, Damarys Loew b, Chris Bowler a,⁎ a b
Environmental and Evolutionary Genomics Section, Institut de Biologie de l'École Normale Supérieure (IBENS), CNRS UMR 8197 INSERM U1024, 46 rue d'Ulm 75005 Paris, France Institut Curie, Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, 26 rue d'Ulm 75248 Cedex 05 Paris, France
a r t i c l e
i n f o
Article history: Received 29 July 2013 Received in revised form 27 October 2013 Accepted 21 November 2013 Keywords: Phaeodactylum tricornutum Thalassiosira pseudonana Epigenetics Mass spectrometry Post-translational histone modifications
a b s t r a c t Post-translational modifications of histones affect many biological processes by influencing higher order chromatin structure that affects gene and genome regulation. It is therefore important to develop methods for extracting histones while maintaining their native post-translational modifications. While histone extraction protocols have been developed in multicellular and single celled organisms such as yeast and Arabidopsis, they are inefficient in diatoms that have a silica cell wall that is likely to hinder histone extraction. We report in this work a rapid and reliable method for extraction of large amounts of high quality histones from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. The protocol is an important enabling step permitting downstream applications such as western blotting and mass spectrometry. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Post-translational modifications (PTMs) on the N-terminal tails of histones play important roles in many biological processes, e.g., by regulating chromatin dynamics and transcription. Combinatorial PTMs of histones give rise to the histone code which defines active or repressed chromatin states which in turn regulate gene expression. Chromatin, the site where all these modifications take place and are regulated, is composed of nucleosomes in which 146 bp DNA is wrapped around an octamer composed of two copies of each of the four core histones, H2A, H2B, H3 and H4. Nucleosomes are linked to each other with the linker histone H1 which is important for nucleosome stability and higher order chromatin structures. The protruding tails of histones are subject to several covalent modifications that include methylation, acetylation, phosphorylation, ubiquitination, sumoylation, and adenosine-diphosphate ribosylation.
☆ The publisher would like to inform the readership that this article is a reprint of a previously published article. An error occurred on the publisher’s side which resulted in the publication of this article in a wrong issue. As a consequence, the publisher would like to make this reprint available for the reader's convenience and for the continuity of the papers involved in the Special Issue. For citation purposes, please use the original publication details; Marine Genomics, Volume 13C, February 2014, Pages 21-25. The publisher sincerely apologizes to the readership and in particular to the author of the respective article and deeply regrets the inconvenience caused. DOI of original article: http://dx.doi.org/10.1016/j.margen.2013.11.006. ⁎ Corresponding authors. E-mail addresses: [email protected], [email protected] (L. Tirichine). 1 Current address: State Key Lab of Marine Environmental Science, Xiamen University, China.
The availability of fast, reliable and cheap methods for histone extraction that preserve native PTMs of histones is fundamental for understanding and deciphering the dynamics and complexity of the histone code. Protocols for histone extraction have been developed for many organisms ranging from yeast to human. However, very few protocols are known for eukaryotic photosynthetic single celled algae, with the exception of Chlamydomonas reinhardtii (Morris et al., 1990). We report in the present work a simple and rapid protocol for the isolation and purification of histones in two model eukaryotic diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Diatoms (Bacillariophyceae) are believed to be the most important group of eukaryotic phytoplankton (Bowler et al., 2010). They account for more than 20% of global carbon fixation and 40% of marine primary productivity. Thus, they have a crucial role in the food web and the biological pump that draws down atmospheric carbon dioxide to the ocean interior. With more than 100,000 species estimated, they are likely to be one of the most diverse groups of photosynthetic organisms on Earth (Round et al., 1992). Besides their biogeochemical importance, diatoms are also attracting interest for the nanotechnology, pharmaceutical and biofuel industries (Dismukes et al., 2008; Kroger and Poulsen, 2008). P. tricornutum is a marine diatom whose genome has been fully sequenced (Bowler et al., 2008). It is widely used as a model for pennate diatoms because of its small genome (27 Mb), short life cycle, ease of transformation and growth in laboratory conditions (De Riso et al., 2009), availability of molecular tools for functional genomic characterization (Siaut et al., 2007; Maheswari et al., 2010), and genetic resources such as 12 ecotypes collected worldwide (De Martino et al., 2007), Bowler, unpublished). P. tricornutum is also an emerging model for epigenetic studies in single celled eukaryotes because it has all the components
1874-7787/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.margen.2014.05.003
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
2
L. Tirichine et al. / Marine Genomics xxx (2014) xxx–xxx
of the epigenetic machinery such as DNA methylation (Veluchamy et al., 2013), histones that can be modified (Lin et al., 2012), Veluchamy et al., in preparation), and small RNA-mediated regulation (De Riso et al., 2009; Huang et al., 2011), Angela Falciatore, personal communication). Likewise, T. pseudonana is a model diatom for centric diatoms, and for which a fully sequenced genome and molecular tools are also available (Armbrust et al., 2004; Poulsen and Kroger, 2005; Nicole Poulsen and Kröger, 2006. Histones, their variants and modifications play an important role in transcriptional regulation but also in higher order chromatin structure. Characterization of native histone PTMs by mass spectrometry is a useful technology to survey PTMs prior to the mapping of modifications of interest in the genome by chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). A prerequisite for this is to obtain native purified histones. The majority of protocols published so far use an acid or high salt (NaCl) extraction, which seems to work for most species (Shechter et al., 2007; Jufvas et al., 2011). However, both types of extraction failed in P. tricornutum (data not shown). We therefore adapted a protocol previously described for histone extraction from Chlamydomonas reinhardtii, where it was also reported that acid and salt extraction did not succeed in extracting histones (Morris et al., 1990). Our work describes a protocol for extracting core histones of good quality and pure enough to be used for western blot and mass spectrometry without the need to be purified over a reversed phase HPLC column.
2. Results and discussion The protocol comprises a step of nuclei extraction before salt extraction of histones, acid precipitation followed by precipitation of histones with trichloroacetic acid (TCA) and several washes for purification of histones, which are then air dried and resuspended for gel visualization and mass spectrometry (Fig. 1). Cultures of P. tricornutum Bohlin clone Pt1 8.6 (CCMP2561) and Thalassiosira pseudonana (CCMP1335) were grown in artificial seawater at 19 °C under cool white fluorescent lights at approximately 75 μmol m−2 s−1 in 12 h light:12 h dark conditions and maintained in exponential phase in semi-continuous batch cultures. Cells were harvested at approximately 1.4 × 106 cells per ml by centrifugation at 4000 rpm for 20 min. Cell pellets can be stored at − 80 °C for several months. Nuclei were then extracted as previously described (Lin et al., 2012). Pellets of nuclei can be stored at − 20 °C for several weeks. Histones were extracted from isolated nuclei with extraction buffer containing 1 M CaCl2, 20 mM Tris HCl pH 7.4 and 1 mini tablet of proteinase inhibitors cocktail (Complete protease cocktail Inhibitor (Roche cat. No. 11873 580 001)). High salt extraction disrupts DNA–histone interactions and precipitates contaminants such as nucleic acids and non-histone proteins while core histones remain soluble in the supernatant and thus can be enriched and purified. Samples were kept on ice for 10 min. HCl was then added to 0.3 N and samples were
Cultures of P. tricornutum or T. pseudonana 2 hours
15 minutes Extraction buffer 1M CaCl2, 20mM Tris HCl pH 7.2, 1 tablet protease inhibitor cocktail 5 minutes Add pure HCl to 0.3N
40 minutes
Add TCA drop wise to 20% and leave on ice for 10 min. Centrifuge for 30 min at 13000 rpm and 4°C
15 minutes 1st wash: 20% TCA 2nd wash with acetone containing 0.2% HCl 3 washes with acetone Dry sample before storage. Fig. 1. Outline of the different steps of the histone extraction protocol. After centrifugation of diatom cultures, pellets can be stored at −80 °C for several months. Isolated nuclei can be stored at −20 °C for several weeks. Likewise, once histones are isolated, they can be stored at −20 °C for several weeks.
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
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It is also known that CaCl2 is routinely used because the heat it generates helps permeabilize membranes allowing a better lysis of the cells. The protocol described herein improves the original protocol from Chlamydomonas (Morris et al., 1990) and removes the use of thiodiglycol which was found to be unnecessary. This is of significance because thiodiglycol is a highly toxic solvent and its utilization is subject to strict regulations. Furthermore, thiodiglycol is a solvent that can be resuspended only in ethanol which mimics some histone methylation, thus inducing a bias if PTMs are to be monitored. We indeed observed methylation on some histone residues when thiodiglycol was included which could not be reproduced when it was omitted (data not shown). We have further modified the Chlamydomonas protocol by adding to the extraction buffer a protease inhibitor cocktail from Roche instead of aprotinin and leupeptin used in the original protocol. This modification reduces the cost without compromising the integrity of the extracted proteins and increases the practicality of the protocol. For western blotting, histone pellets were resuspended in loading dye (0.5 M Tris–HCl pH 8.45, 10% LiDS, 1% b-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue) and denatured at 100 °C for 5 min before loading on a 14% tris-tricine gel. Samples were run at 80 V until they migrated from the stacking gel and then the voltage was set to 100 V for 90 min (Fig. 2A, C) for a good separation of the bands. Gels were first washed in distilled water for 5 min before staining with LabSafe GEL Blue™ (786-35) from G-Biosceinces for 30 min. Gels were
centrifuged at 4 °C at 10 000 rpm for 5 min to precipitate the acidinsoluble fraction. TCA was added to 20% to the supernatant, which contains the acid soluble proteins. To precipitate histones, TCA was added drop wise to the sample kept on ice. Samples were kept on ice for a further 10 min and then centrifuged for 30 min at 13 000 rpm at 4 °C to precipitate histones. We added in our protocol an extra centrifugation step compared to the original method, which increased the yield of precipitated histones. The pellet was washed successively with 20% TCA, and then acetone containing 0.2% HCl, followed with three further washes with acetone. During each wash, the histone pellet was resuspended in the wash buffer by flipping the tubes. The pellet was air dried using a speed vacuum or under a hood. The histones can be stored at − 20 °C for several weeks. The samples at this stage can be used for western blotting or mass spectrometry. The reasons for the lack of success of the acid and NaCl high salt extraction of histones from Phaeodactylum are unclear but it is possible that during extraction the histones might stay bound to the DNA and precipitate with other contaminants, giving rise to a liquid phase that is free of histones. More precisely, sulfuric acid and sodium chloride might not be efficient in breaking the DNA–histone bonds, whereas calcium chloride might overcome this bottleneck thanks to either its exothermic activity that would soften DNA binding to histones or its higher ion exchanging activity of multivalent ions compared to that of monovalent ions disrupting thus electrostatic interaction between DNA and histone.
A
3
B MW KDa
1
2
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1
2
3
4
MW KDa
40
35 25 15
10
C
1
*
*
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* * *
* * *
H3 H2A H2B
*
*
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D
40 35 25 H1 H3 15 H2A H2B
MW KDa H1
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H3 H2A H2B H4
10 H4
Fig. 2. Tris–tricine SDS-PAGE of histones extracted from P. tricornutum and T. pseudonana. (A) Samples of P. tricornutum histones extracted from (1) intact nuclei prepared from 300 ml of cultures at a cell density of 1.4 × 106 cells/ml (2) intact nuclei from 300 ml culture with cell density of 6 × 105 cells/ml (3) chromatin isolated from 300 ml culture with cell density of 1 × 106 cells/ml. Page ruler Prestained Protein Ladder from Fermentas (#SM0671) was used. (B) Samples of P. tricornutum histones extracted from intact nuclei using different buffers. If not specified, extraction buffers used elsewhere do not contain any of the detergent used in 2B (1) extraction buffer with 0.1% SDS (2) extraction buffer with 1% thiodiglycol (3) extraction buffer without SDS or thiodiglycol (4) Calf thymus histones used as a control. (C) Histone samples extracted from T. pseudonana nuclei. (1) Calf thymus histones used as control (2) T. pseudonana histones (D) migration on gels of P. tricornutum histones used for mass spectrometry analysis. Histones were extracted from intact nuclei. Samples were run for a short time to have a maximum of histones concentrated in small bands of gel for mass spectrometry purposes to avoid sample dilution. Sample concentrations range between 0.2 and 1 μg. Asterisks indicate histones H4, H2A, H2B, H3 and H1.
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
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visualized after 3 washes during 5 min each with distilled water. No other detergent or solvent is needed for the initial step of histone extraction using high salt buffer. The yield of histones is equally good with and without thiodiglycol but higher compared to samples extracted with 0.1% SDS (Fig. 2B). We found that it was very important to initiate the salt extraction from high cell density but also from intact nuclei because nuclei lysis to extract chromatin prior to high salt extraction decreases the yield of histones, as shown in Fig. 2A. If the samples were to be used for mass spectrometry, migration time was reduced to 30 min (Fig. 2D).
For mass spectrometry analysis, samples were prepared in loading buffer as above and run using the same gel system for approximately 30 min after migrating out of the stacking gel to prevent their dilution. Excised gel slices were washed and proteins were reduced with 10 mM DTT prior to alkylation with 55 mM iodoacetamide. After washing and shrinking of the gel pieces with 100% acetonitrile, in-gel digestion was performed using trypsin and chymotrypsin overnight in 25 mM ammonium bicarbonate at 30 °C. The extracted peptides were analyzed by nano-LC-MS/MS using an Ultimate3000 system (Dionex S.A.) coupled
Fig. 3. Representative MS/MS spectra for identification of histone modifications in P. tricornutum. LC-ESI MS/MS spectra for acetylated H4 peptide GLGKAcGGAKAcR (464.3(2+) m/z) (A) and methylated H3 peptide KMeSAPATGGVKMeKPHR (731.4(2+) m/z) (B). The fragmentation spectra derived from trypsin H4 and H3 peptides are shown. The inset shows peptide sequence and the observed ions obtained. According to peptide fragmentation nomenclature, y ions extend from the N-terminus while b ions are indicative of C-terminus of the peptide. The tandem mass spectra are labelled to show singly and doubly charged b and y ions, as well as ions corresponding to neutral losses of water (°) NH3 (*) and M the parent ion mass. Me, Methylation; Ac, Acetylation.
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
L. Tirichine et al. / Marine Genomics xxx (2014) xxx–xxx
to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) (Fig. 3). Samples were loaded on a C18 pre-column (300 μm inner diameter × 5 mm; Dionex) at 20 μl/min in 2% acetonitrile, 0.1% TFA. After 3 min of desalting, the pre-column was switched on-line with the analytical C18 column (75 μm inner diameter × 50 cm; C18 PepMap™, Dionex) equilibrated in 100% solvent A (2% acetonitrile, 0.1% acide formic). Bound peptides were eluted using a 0 to 30% gradient of solvent B (80% acetonitrile, 0.085% formic acid) during 157 min, then 30 to 50% gradient of solvent B during 20 min at a 150 nl/min flow rate (40 °C). Data-dependent acquisition was performed on the LTQOrbitrap mass spectrometer in the positive ion mode. Survey MS scans were acquired in the Orbitrap on the 400–1200 m/z range with the resolution set to a value of 100 000. Each scan was recalibrated in real time by co-injecting an internal standard from ambient air into the C-trap (‘lock mass option’). The five most intense ions per survey scan were selected for CID fragmentation and the resulting fragments were analyzed in the linear trap (LTQ). Target ions already selected for MS/MS were dynamically excluded for 20 s. Data were acquired using the Xcalibur software (version 2.0.7) and the resulting spectra were then analyzed via the Mascot™ Software created with Proteome Discoverer (version 1.4, Thermo Scientific) using the in-house database containing the sequence of histone proteins from P. tricornutum and T. pseudonana. Carbamidomethylation of cysteines, oxidation of methionines, acetylation of lysines and protein N-termini, methylation, dimethylation of lysines, arginines and trimethylation of lysines were set as variable modifications for all Mascot searches. Specificity of trypsin digestion was set for cleavage after Lys or Arg except before Pro, and five missed trypsin cleavage sites were allowed. The mass tolerances in MS and MS/MS were set to 5 ppm and 0.5 Da, respectively. The resulting Mascot files were further processed using myProMS (Poullet et al., 2007). Similarly, mass spectrometry analysis of histones extracted from T. pseudonana detected modifications on several residues (data not shown). In conclusion, we have developed a simple and rapid protocol for extracting histones from two model diatoms and have shown using mass spectrometry that the extracted histones are of good quality and can be used to monitor their PTMs. This is of particular interest considering the role of histones in higher order structures of chromatin and thus in regulating gene expression. Furthermore, the described protocol opens the way for investigating a whole unexplored field of epigenetics in diatoms, which are outstanding models for studying how the environment shapes the evolution and fitness of unicellular marine protists. Future work based on this protocol should therefore allow further insights into the reasons underlying the ecological success of diatoms. Contributions
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LT designed the study and performed the experiments. LB carried out the MS experimental work. LD supervised MS and proteomic data
Please cite this article as: Tirichine, L., et al., Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana", Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.05.003
