Dynamics and functional aspects of histone modifications in plants

We have studied the replication time, nuclear or-ganization and histone acetylation patterns of distinct chro-matin domains [nucleolus organizers (NORs), centrome-res, euchromatin and heterochromatin] of barley during thecell cycle. The Rabl orientation of chromosomes, withcentromeres and telomeres located at opposite nuclearpoles, was found to be maintained throughout interphase.Replication started at the rDNA loci within nucleoli andthen proceeded from the euchromatic distal chromosomeregions toward the heterochromatic pole. Centromere asso-ciation frequently occurred in midand late S-phase, i.e.,during and after centromere replication. Euchromatin,centromeres and heterochromatin were found to be en-riched in acetylated histone H4 (except for lysine 16) dur-ing their replication; then deacetylation occurred. The levelof deacetylation of H4 in heterochromatin was more pro-nounced than in euchromatin. Deacetylation is finished inearly G2-phase (lysine 8) or may last until mitosis or eventhe next G1-phase (lysines 5 and 12). The NORs werefound to be most strongly acetylated at lysines 5 and 12 ofH4 during mitosis, independently of their potential activityin nucleolus formation and rDNA transcription. The acety-lation pattern of chromosomal histone H3 was character-ized by low acetylation intensity at centromeres (lysines9/18) and pericentromeric regions (lysine 14) and more in-tense uniform acetylation of the remaining chromatin; it re-mained fairly constant throughout the cell cycle. These re-sults have been compared with the corresponding data pub-lished for mammals and for the dicot Vicia faba. Thisrevealed conserved features as well as plantor species-specific peculiarities. In particular, the connection of acety-lation intensity of H4 at microscopically identifiable chro-matin domains with replicational but not with transcrip-tional activity during the cell cycle seems to be conservedamong eukaryotes.Introduction The chromatin of interphase nuclei is highly organized.As early as 1885 Rabl (1885) proposed a model accord-ing to which the anaphase/telophase orientation of chro-mosomes is maintained in interphase nuclei, resulting incentromeres and telomeres being located at opposite nu-clear poles. During the following century, cytogenetic in-vestigation of chromosome structure and compositionwas mainly focused on mitotic/meiotic chromosomes be-cause it was difficult to identify specific chromosometerritories and individual chromatin domains in inter-phase nuclei, where essential processes such as replica-tion, gene expression and DNA repair take place. Duringrecent years, fluorescent in situ hybridization (FISH),immunostaining and flow-sorting of nuclei have been in-troduced and improved. In particular, the painting of en-tire chromosomes by FISH allowed the discovery andanalysis of distinct chromosome territories within inter-phase nuclei in mammals (see, e.g., Eils et al. 1996; Di-etzel et al. 1999; Nagele et al. 1999; Visser and Aten1999; Tajbakhsh et al. 2000). These techniques enabledthe development of complex approaches in combinationwith high resolution image processing for microscopicstudies of the territories and structural composition of in-dividual chromosomes and/or distinct chromatin do-mains in the course of the cell cycle. It thus became pos-sible to examine directly structural/functional interrela-tions, i.e., to correlate spatial and temporal modificationof chromatin structure with specific functions of chroma-tin domains.One of the diverse chromatin modifications is the re-versible acetylation of N-terminal lysine residues of thenucleosomal histones H3 (K9, 14, 18, 23) and H4 (K5, 8,12, 16), which occurs in all eukaryotes studied so far.The degree of histone acetylation varies along the mito-tic chromosomes of insects (Turner et al. 1992), mam-mals (Jeppesen and Turner 1993) and plants (Houbenet al. 1996a; Belyaev et al. 1997, 1998; Vyskot et al.1999) and is generally more intense in euchromatic thanin heterochromatic domains. A high acetylation statusEdited by: D. Schweizer Z. Jasencakova · A. Meister · I. Schubert (✉)Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK),06466 Gatersleben, Germanye-mail: [email protected] (2001) 110:83–92DOI 10.1007/s004120100132 O R I G I N A L A RT I C L E Zuzana Jasencakova · Armin Meister · Ingo Schubert Chromatin organization and its relation to replicationand histone acetylation during the cell cycle in barley Received: 9 October 2000 / In revised form: 11 December 2000 / Accepted: 26 January 2001 / Published online: 29 March 2001© Springer-Verlag 2001 was found to be connected with transcription (reviewed,e.g., in Grunstein 1997; Struhl 1998), recombination(McBlane and Boyes 2000; McMurry and Krangel 2000)and DNA repair (Ikura et al. 2000) at the level of genes,providing, together with other chromatin modifications,in a concerted manner an epigenetic code used to specifyunique downstream functions (for recent reviews seeStrahl and Allis 2000; Turner 2000). Previously, studieswere undertaken to elucidate at the microscopic levelchromatin acetylation patterns during interphase in mam-malian (Sadoni et al. 1999; Taddei et al. 1999) and plantnuclei (Buzek et al. 1998; Jasencakova et al. 2000).Experiments on Vicia faba interphase nuclei haveshown that acetylation of histone H4 (at lysine positions5, 12 and 16) of euand heterochromatin domains (ex-cept for the nucleolus) is correlated with replicationrather than with transcription, while histone H3 acetyla-tion of chromatin domains did not change significantlyduring the cell cycle (Jasencakova et al. 2000).Here we investigate the chromatin organization of mi-totic and interphase chromosomes in barley. The ar-rangement of chromosomes during interphase stages, thesequential order of replication and the intensity of his-tone H3 and H4 acetylation of distinct chromatin do-mains such as nucleolus organizer regions (NORs), eu-chromatin, centromeres and heterochromatin during themitotic cycle was studied. For this purpose, we com-bined FISH and immunolabeling techniques to identifychromosomal domains and their acetylation status in re-lation to the corresponding replicational and transcrip-tional activities in nuclei isolated from unsynchronizedroot-tip meristems and flow-sorted into G1-, early S-,mid S-, late S-, and G2-phase fractions. The results werecompared with those obtained for V. faba and revealedcommon features as well as differences between mono-cot and dicot plants on the one hand and between ani-mals and plants on the other. This led to conclusions asto the evolutionary conservation of the respective phe-nomena and to what degree histone acetylation of dis-tinct chromatin domains is stable or cell cycle dependentand whether or not different intensities of histone acety-lation reflect potential transcriptional activity of the cor-responding domains or are temporally linked with repli-cation processes. Materials and methods All experiments were performed on barley, Hordeum vulgare L.(2n=14), using the line MK14/2034, which is characterized by twohomozygous translocations involving chromosomes 3S/4L (=T3-4ae)and 1S/7Sat (=T1-7an) see http://wheat.pw.usda.gov/ggpages/Barley_physical/Idiograms/. To study the correlation between histoneacetylation and transcriptional activity of the NORs we additionallyused the translocation line T2052 carrying NOR6 and NOR7 on theopposite arms of chromosome 6, and showing nucleolar dominanceof NOR6 and nearly complete suppression of NOR7 (Schubert andKünzel 1990). Seeds were germinated on soaked paper at 24°C. Syn-chronization and fixation of root tip meristems and isolation of chro-mosomes were done according to Lysák et al. (1999).Nuclei from unsynchronized root tips were fixed in 4% formal-dehyde, TRIS buffer (10 mM TRIS, 10 mM Na2EDTA, 100 mMTriton X-100, pH 7.5), washed in TRIS buffer and isolated fromthe meristematic regions as described previously (Schubert et al.1993). After staining with 4′,6-diamidino-2-phenylindole (DAPI,1 μg/ml) they were flow-sorted into G1-, early S-, mid S-, late S-,and G2-phase fractions using a FACStarPlus flow cytometer andcell sorter (Becton Dickinson) with a Sort Enhancement Module(SEM) and an Argon-ion laser (INNOVA 90C-5) emitting UVlight with 200 mW output power controlled by a Macintosh Com-puter with Cell Quest Software. The gates for sorting were set ac-cording to the histograms obtained for each suspension of nuclei.A representative histogram is given in Fig. 1. About 1000 nucleiof each fraction were sorted onto microscopic slides into a dropcontaining 100 mM TRIS, 50 mM KCl, 2 mM MgCl2, 0.05%Tween 20 and 5% sucrose (Kubaláková et al. 1997), air-dried atroom temperature for several hours and used immediately for im-munolabeling and/or FISH, or stored at –20°C until use. Fluorescent in situ hybridization The following probes were used: BAC7 containing barley centro-mere-specific retroelement sequences (Presting et al. 1998),pVER17 (with a 3.7 kb insert comprising 18S, 5.8S and most ofthe coding region of 25S rRNA genes of V. faba, Yakura andTanifuji 1983), (GAA)10 oligonucleotides (MWG-Biotech), whichlabel the heterochromatin of barley (Pedersen and Linde-Laursen1994; Pedersen et al. 1996), and HvT01, a 118 bp subtelomeric re-peat of barley (Belostotsky and Ananiev 1990; Schubert et al.1998).Centromereand NOR-specific probes were labeled with dig-oxigenin-11-dUTP, biotin-16-dUTP or tetramethylrhodamine-5-dUTP using a nick translation kit, and (GAA)10 oligonucleotidesusing an end-labeling kit (both from Roche Biochemicals) accord-ing to the manufacturer’s instructions. HvT01 repeats were ampli-fied and labeled with digoxigenin-11-dUTP by the polymerasechain reaction with sequence-specific primers.Treatment with RNase A (50 μg/ml in 2×SSC) for 30 min at37°C was applied only prior to FISH with the rDNA probe.(1×SSC is 0.15 M NaCl, 0.015 M sodium citrate.) Preparations onslides were postfixed in 4% paraformaldehyde, 2×SSC for 15 min,washed three times in 2×SSC, dehydrated through an ethanol se-ries (70%, 96%) and air-dried. The hybridization mixture contain-ing probe, 50% formamide, 10% dextran sulfate and 2×SSC washeated for 10 min at 80°C, cooled on ice and denatured again to-gether with target DNA on slides for 1 min at 80°C. When double-color FISH was performed, one of the probes (BAC7) was directlylabeled with rhodamine-5-dUTP. After overnight hybridization at37°C, the slides were washed three times for 5 min each in 2×SSCat 37°C. Digoxigenin-labeled probes were detected with 1:50 anti-digoxigenin-fluorescein isothiocyanate (FITC; Roche Biochemi-cals). DNA was counterstained with DAPI (1 μg/ml in Vecta-shield, Vector).84 Fig. 1 Histogram of the relative DNA content of unsynchronizedbarley nuclei after 4′,6-diamidino-2-phenylindole (DAPI) stainingand flow-cytometric analysis. The gates used for sorting are indi-cated (1 early S-, 2 late S-phase) 5-Bromo-2′-deoxyuridine labeling combined with FISH For pulse-labeling of replicating chromatin, the roots were incu-bated in 5-bromo-2′-deoxyuridine (BrdU, 100 μM), fluorodeoxy-uridine (0.1 μM) and uridine (5 μM) for 30 min in the dark. Afterrinsing, the roots were fixed in 4% formaldehyde, TRIS buffer be-fore isolation and flow-sorting of nuclei.Detection of BrdU incorporation was combined with FISHwith BAC7 to discriminate between the centromeric and telomericpoles. The slides were treated first as described above for FISH(postfixation, dehydration, air-drying, denaturation and overnighthybridization with probe). Incorporated BrdU was then detec-ted together with the probe, using mouse anti-BrdU antibodies(Becton Dickinson, 1:20–1:50) applied together with anti-digoxi-genin-FITC (Roche Biochemicals, 1:50) for digoxigenin-labeledBAC7, followed by anti-mouse-Alexa596 (Molecular Probes,1:500–1:1000) and counterstaining with DAPI.For detection of replicating centromeres, FISH with BAC7 wasperformed after BrdU labeling. In this experiment BrdU detectionwas as described (Jasencakova et al. 2000). Briefly, the slideswere postfixed in 4% paraformaldehyde/PBS, washed in PBS, de-natured in 50% formamide/PBS at 80°C for 1 min and cooleddown in ice-cold PBS. After blocking, the slides were incubatedwith mouse anti-BrdU (Becton Dickinson, 1:20) followed by anti-mouse-Alexa596 (Molecular Probes, 1:500). After counterstainingwith DAPI (1 μg/ml in Vectashield) the slides were checked andimages of nuclei together with their coordinates were recorded.After washing in TNT (100 mM TRIS-HCl pH 7.5, 150 mMNaCl, 0.05% Tween 20), the slides were dehydrated through anethanol series, air-dried, and baked at 60°C for 30 min. Fluores-cent in situ hybridization with BAC7 was then performed as de-scribed above. Biotin-labeled BAC7 was detected using streptavi-din-AMCA (7-amino-4 methylcoumarin-3-acetic acid,Vector,1:50), followed by biotinylated anti-streptavidin (Vector, 1:200)and streptavidin-AMCA (1:50) for signal amplification. Slideswere mounted in Vectashield. The BrdU labeling patterns of nu-clei before FISH were then compared with FISH signals obtainedwith the BAC7 probe. Histone immunolabeling For histone immunolabeling the following rabbit polyclonal anti-sera recognizing histone H3 and H4 isoforms with acetylated ly-sine residues (given in parentheses) were used: R41 (H4Ac5),R232 (H4Ac8), R101 (H4Ac12), R252 (H4Ac16), R243 (bindingpreferentially to triand tetra-acetylated H4), R47 (H3Ac9 and/or18) and R224 (H3Ac14) (Turner and Fellows 1989; Turner et al.1989; Belyaev et al. 1996; Stein et al. 1997; White et al. 1999).The specificity of these antibodies to the corresponding isoformsof plants has been shown on immunoblots (Buzek et al. 1998).Preimmune sera neither reacted with nuclear proteins (Buzek et al.1998) nor with chromosomes of the tested plants (Vyskot et al.1999). The immunolabeling procedure was carried out as de-scribed (Jasencakova et al. 2000): after postfixation in 4% para-formaldehyde in PBS and washes in PBS, the slides were blockedfor 1 h at 37°C, and then incubated with the primary antisera dilut-ed 1:100–1:200 in PBS buffer containing 1% BSA, 10% horse se-rum, and 0.1% Tween 20. Antisera were detected by anti-rabbit-FITC (Sigma, 1:80), and nuclei were counterstained with DAPI(1 μg/ml, Vectashield). Secondary antibodies stained neither mitot-ic chromosomes nor nuclei of barley.When histone immunolabeling and FISH were combined, im-munolabeling was performed first. After checking the slides, cov-erslips were removed and dehydration, baking and FISH weredone as described above. In this case, the probe was either directlylabeled (tetramethylrhodamine-dUTP) or, when digoxigenin-labeled probe was used, it was detected with anti-digoxigenin-rhodamine (Roche Biochemicals, 1:50).Usually, histone immunolabeling signals were preserved afterFISH. In case their intensity decreased considerably, images of nu-clei captured before FISH were compared with those obtained af-ter FISH.Nascent RNA labeling 5′-Bromo-5′-triphosphate (BrUTP) incorporation into isolatednuclei was done on slides according to Thompson et al. (1997)and Abranches et al. (1998). Preparations were incubated with thetranscription mix consisting of 50 μM CTP, 50 μM GTP, 25 μMBrUTP (Sigma), 100 U/ml RNase inhibitor RNA Guard (Pharma-cia) in MPB buffer (100 mM potassium acetate, 20 mM KCl,20 mM HEPES, 1 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol,pH 7.4) for 6 min at room temperature. Nuclei were fixed in 4%paraformaldehyde/PBS for 30 min, washed in PBS and blockedfor 45 min at 37°C. BrUTP was then detected using the same anti-bodies as for BrdU (see above).The slides were inspected using a Zeiss Axiophot 2 epifluores-cence microscope equipped with a cooled CCD camera (Photo-metrics). Images were captured using IPLab Spectrum software,pseudocolored, and merged in Adobe Photoshop.