The chromatin remodelling complex FACT ... - Wiley Online Library

The Plant Journal (2004) 40, 660–671

doi: 10.1111/j.1365-313X.2004.02242.x

The chromatin remodelling complex FACT associates with actively transcribed regions of the Arabidopsis genome Meg Duroux1, Andreas Houben2, Kamil Ru˚zˇicˇka3, Jirˇı´ Friml3 and Klaus D. Grasser1,* Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark, 2 Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany, and 3 Zentrum fur Molekularbiologie der Pflanzen, Universita¨t Tu¨bingen, Auf der Morgenstelle 3, D-72076 Tu¨bingen, Germany

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Received 4 August 2004; accepted 25 August 2004. * For correspondence (fax þ45 9814 1808; e-mail [email protected]).

Summary The packaging of the genomic DNA into chromatin in the cell nucleus requires machineries that facilitate DNAdependent processes such as transcription in the presence of repressive chromatin structures. Using co-immunoprecipitation we have identified in Arabidopsis thaliana cells the FAcilitates Chromatin Transcription (FACT) complex, consisting of the 120-kDa Spt16 and the 71-kDa SSRP1 proteins. Indirect immunofluorecence analyses revealed that both FACT subunits co-localize to nuclei of the majority of cell types in embryos, shoots and roots, whereas FACT is not present in terminally differentiated cells such as mature trichoblasts or cells of the root cap. In the nucleus, Spt16 and SSRP1 are found in the cytologically defined euchromatin of interphase cells independent of the status of DNA replication, but the proteins are not associated with heterochromatic chromocentres and condensed mitotic chromosomes. FACT can be detected by chromatin immunoprecipitation over the entire transcribed region (5¢-UTR, coding sequence, 3¢-UTR) of actively transcribed genes, whereas it does not occur at transcriptionally inactive heterochromatic regions and intergenic regions. FACT localizes to inducible genes only after induction of transcription, and the association of the complex with the genes correlates with the level of transcription. Collectively, these results indicate that FACT assists transcription elongation through plant chromatin. Keywords: euchromatin, Spt16, SSRP1, chromatin immunoprecipitation, immunofluorescence, transcription.

Introduction In eukaryotic cells, packaging of the large genomic DNA with histones and other proteins into chromatin affects DNAdependent processes, including transcription and recombination. The compaction of the DNA provided by chromatin represses the transcription of genes by restricting the access of DNA-binding regulatory factors to their DNA target sites, and by inhibiting the progression of RNA polymerases. As both initiation and elongation of transcription by RNA polymerase II are inhibited in the chromatin context in vitro, the packaging of the genomic DNA into chromatin represents an important level of gene regulation (Felsenfeld and Groudine, 2003; Narlikar et al., 2002). Numerous nuclear activities have been identified, which can help to overcome the repressive effects of chromatin, and many of these chromatin-modifying factors are conserved among eukaryotic organisms including plants. Post-translational modification of histones (acetylation, methylation, etc.) and 660

ATP-dependent chromatin remodelling complexes are involved in altering the chromatin structure stimulating activator-dependent transcription (Narlikar et al., 2002; Spencer and Davie, 1999). In a well-defined in vitro transcription system using reconstituted chromatin templates, activator-induced ATPdependent chromatin remodelling could promote in the presence of general transcription factors and RNA polymerase II the efficient formation of pre-initiation complexes and transcription initiation, but it was not sufficient for productive transcript elongation. Using this system, a human nuclear activity was purified that could facilitate transcript elongation on chromatin templates, and this activity was termed FAcilitates Chromatin Transcription (FACT) (Orphanides et al., 1998). FACT comprises the Spt16 (also termed Cdc68) and the structure-specific recognition protein SSRP1, and it can interact specifically with ª 2004 Blackwell Publishing Ltd

Arabidopsis FACT chromatin remodelling complex 661 nucleosomes and H2A/H2B dimers suggesting that it may promote transcription by nucleosome disassembly (Orphanides et al., 1999). Recently, FACT was shown to stimulate transcription by RNA polymerase II, destabilizing nucleosomes in the path of the enzyme by facilitating the removal of one histone H2A/H2B dimer (Belotserkovskaya et al., 2003). Thus, FACT is a novel factor facilitating RNA polymerase II passage through chromatin by destabilizing nucleosome structure without requirement for ATP hydrolysis (Belotserkovskaya et al., 2004). In yeast, the FACT complex (also termed CP complex for CDC68/Pob3) consists of Spt16 and Pob3 (which shares significant sequence similarity with SSRP1, but lacks the C-terminal HMG-box domain) (Brewster et al., 1998; Evans et al., 1998; Wittmeyer and Formosa, 1997; Wittmeyer et al., 1999). The HMG-box function of yeast FACT is provided by a small HMGB protein termed NHP6 (Brewster et al., 2001; Formosa et al., 2001). Various genetic screens in yeast have revealed a role of Spt16 and Pob3 in transcription and DNA replication (Formosa, 2003). Moreover, Spt16/Pob3 can physically interact with the DNA polymerase a complex (Wittmeyer and Formosa, 1997; Wittmeyer et al., 1999). The finding that FACT function is not restricted to the regulation of transcription and that it is a rather general chromatin-modifying factor is further supported by experiments indicating a role of FACT in other DNA-dependent processes. Thus, Xenopus FACT (also termed DNA unwinding factor) can act as DNA replication factor presumably by making the chromatin structure more accessible to the replication machinery (Okuhara et al., 1999; Seo et al., 2003). A role in DNA damage repair has been implicated from the interaction of FACT with cisplatin-damaged DNA (Yarnell et al., 2001) and from the functional interaction of FACT with protein kinase CK2 and p53 upon UV irradiation (Keller and Lu, 2002; Keller et al., 2001). In plants, a FACT complex has not been identified. Of the potential subunits of a plant FACT complex, only SSRP1 has been analysed. The 71-kDa maize SSPR1 protein contains a C-terminal HMG-box domain that mediates non-sequencespecific binding of the protein to DNA. It can specifically recognize certain DNA structures (DNA minicircles, supercoiled DNA) and structurally flexible regions in linear DNA. Moreover, maize SSRP1 bends DNA upon binding and facilitates the formation of higher-order nucleoprotein structures (Ro¨ttgers et al., 2000). SSRP1 can bind specifically to mono-nucleosome particles and it is released from maize chromatin by limited micrococcal nuclease treatment, suggesting that the protein is enriched in the less compacted chromatin (Lichota and Grasser, 2001). Maize protein kinase CK2a can phosphorylate several residues in the C-terminal part of SSRP1 and two phosphorylation sites have been mapped to the very C-terminal region next to the HMG-box domain. Phosphorylation of these two residues results in a structural change in the region of the HMG-box domain and ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

induces the recognition of UV-damaged DNA, whereas non-phosphorylated SSRP1 does not discriminate between UV-damaged DNA and control DNA, suggesting a role in the UV response (Krohn et al., 2003). Here, we demonstrate the existence of a plant FACT complex consisting of SSRP1 and Spt16 in Arabidopsis that associates with actively transcribed genes. Results Existence of a FACT complex in plants To examine whether a FACT complex occurs in plants, we have initially searched the DNA sequence of the Arabidopsis genome (http://www.arabidopsis.org) for sequences encoding the SSRP1 and Spt16 proteins. The 71.6-kDa SSRP1 (termed nucleosome/chromatin assembly factor D, NFD, in ChromDB) is encoded by the AGI locus At3g28730 and it shares 67 and 34% amino acid sequence identity with the previously characterized SSRP1 proteins from maize (Ro¨ttgers et al., 2000) and human (Bruhn et al., 1992) respectively. The 120.6-kDa Spt16 (termed global transcription factor C, in ChromDB), which has not been characterized from plants, is encoded by the AGI locus At4g10710 and it shares 35% amino acid sequence identity with human Spt16 (Orphanides et al., 1998). For the production of specific antisera against both proteins, we have selected the regions V453-N646 of SSRP1 (DSSRP1) and E416-M554 of Spt16 (DSpt16) as antigens. DSSRP1 and DSpt16 were expressed in Escherichia coli, purified by three-step column chromatography, and the purified recombinant proteins were used for immunization. We have used in this study antibodies recognizing the native proteins rather than epitope-tagged recombinant proteins, as is often used in other studies. The specificity of the obtained antisera was initially tested using immunoblotting by reacting them with a mixture of the recombinant DSSRP1 and DSpt16 proteins that have been used for immunization (Figure 1a). Each antiserum reacted exclusively with the protein that was used for immunization and showed no cross-reactivity with the second protein. In addition, the antisera were tested with total protein extracts derived from Arabidopsis suspension-cultured cells (Figure 1b). The antiserum directed against Spt16 reacted with a protein band of approximately 120 kDa and the antiserum directed against SSRP1 reacted with a protein band of approximately 70 kDa, indicating that both antisera recognize the native Spt16 and SSRP1 proteins, while the pre-immune serum showed no specific reaction with Arabidopsis proteins. In addition to the intact proteins, both antisera also react with distinct bands of higher electrophoretic mobility, which are most likely degradation products of SSRP1 and Spt16, as these proteins are sensitive to proteolysis in human, Drosophila and yeast (Hsu et al., 1993; Orphanides et al., 1998; Wittmeyer and Formosa, 1997; Wittmeyer et al., 1999).

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Figure 1. Specificity of the antisera directed against Arabidopsis Spt16 and SSRP1 tested by immunoblotting. (a) A mixture of equal amounts of the recombinant DSpt16 and DSSRP1 proteins used for immunization were separated by SDS-PAGE and subsequently probed by immunoblotting using the antisera directed against Spt16 (a-Spt16) and SSRP1 (a-SSRP1). The electrophoretic migration positions of the recombinant DSpt16 (approximately 18 kDa) and DSSRP1 (approximately 24 kDa) proteins are indicated. (b) Total Arabidopsis protein extracts (20 lg) were separated by SDS-PAGE and probed by immunoblotting with a-Spt16, a-SSRP1 and Spt16-preimmune serum. For comparison, the protein extract was stained with Coomassie. The electrophoretic migration positions of Spt16, SSRP1 (arrowheads) and of protein molecular weight markers are indicated.

Immunoprecipitation experiments were carried out to determine whether a FACT complex consisting of SSRP1 and Spt16 exists in plants. The antiserum directed against Spt16 was used to precipitate Spt16 from native protein extracts of Arabidopsis suspension-cultured cells (Figure 2a). In the immunoprecipitate, Spt16 could be detected by immunoblotting as expected. In addition, the anti-Spt16 serum co-precipitated SSRP1, which was detected in the

Figure 2. Spt16 and SSRP1 form the Arabidopsis FACT complex. (a) Immunoprecipitation experiments (IP) were performed on Arabidopsis protein extracts using the anti-Spt16 serum (a-Spt16; lanes 2 and 3) and preimmune serum (lane 4). Immunoprecipitated proteins were probed by immunoblotting with a-Spt16 (lanes 2 and 4) or a-SSRP1 (lane 3). The electrophoretic migration positions of Spt16 and SSRP1 (arrowheads) and of protein molecular weight markers are indicated. Asterisks indicate protein bands originating from the antisera. (b) Immunoaffinity purification (IP) of Spt16 and SSRP1 from protein extracts using the a-Spt16 (left) or a-SSRP1 (right) antisera immobilized on a sepharose matrix. After extensive washing, proteins were eluted from the matrices and probed by immunoblotting using the a-Spt16 (top) or a-SSRP1 (bottom) antisera.

precipitate by the anti-SSRP1 serum. Neither protein could be precipitated with the pre-immune serum. To confirm this result, immunoaffinity matrices were prepared by immobilizing the antisera against SSRP1 and Spt16. Arabidopsis protein extracts were incubated with the immunoaffinity matrices that were subsequently carefully washed. The proteins eluted from the matrices were probed with both antisera (Figure 2b). Both SSRP1 and Spt16 were detected in the eluate from the anti-Spt16 and the anti-SSRP1 immunoaffinity matrices. The detection of several peptides indicative of Spt16 and SSRP1 in a tryptic digest of the immunopurified proteins by mass spectrometry confirmed the identity of the two FACT subunits (data not shown). Therefore, in plants SSRP1 and Spt16 occur as FACT complex, as the two ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

Arabidopsis FACT chromatin remodelling complex 663 proteins specifically co-purify from Arabidopsis protein extracts. SSRP1 and Spt16 localize to euchromatic regions in replicating and non-replicating interphase nuclei Using indirect immunolabelling with the antibodies directed against Spt16 and SSRP1, the subcellular distribution of FACT was studied in Arabidopsis cells (Figure 3). The genomic DNA in the nuclei was also visualized by 4¢,6diamidino-2-phenylindole (DAPI) staining. While the preimmune sera did not result in a specific immunostaining (data not shown), the anti-SSRP1 and anti-Spt16 sera resulted in similar immunolabelling in the nuclei (compare Figure 3a–c and Figure 3f,g). Both Spt16 and SSRP1 show a similar Figure 3. Spt16 and SSRP1 co-localize to the euchromatin in interphase nuclei. Using the anti-Spt16 (a) and anti-SSRP1 (b) sera, the presence of Spt16 and SSRP1 on mitotic interphase and metaphase chromosomes (indicated by arrows in a–c) was examined by indirect immunofluorescence. The genomic DNA was visualized by DAPI staining. (c) Co-localization of Spt16 and SSRP1 in the same nuclei was studied by immunofluorescence using antibodies covalently labelled with different fluorochromes. (d) To reveal a potential link between FACT localization and DNA replication, a double-immunolabelling was performed using anti-Spt16 and the detection of incorporated BrdU on mitotic interphase nuclei. In the merge, DAPI fluorescence is in blue, antibody in red and BrdU in green. (e) Histogram of the relative DNA content of nuclei isolated from young Arabidopsis leaves after DAPI staining and flow cytometric analysis. The unreplicated (2C) M1-fraction of nuclei was flow-sorted and used for further experiments. The fractions M2, M3 and M4 represent 4C, 8C and 16C nuclei respectively. In selected flowsorted non-replicating (2C) nuclei, the localization of Spt16 and SSRP1 was determined by indirect immunofluorescence using the antiSpt16 (f) and anti-SSRP1 (g) sera.

ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

uniform distribution pattern in interphase nuclei, except that the brightly DAPI-stained heterochromatic chromocentres remained clearly less immunolabelled (indicated by arrows in Figure 3f,g). In mitotic cells, the condensed chromosomes (indicated by arrows in Figure 3a–c) also revealed no immunoreactivity against Spt16 or SSRP1. Thus, Spt16 and SSRP1 are enriched within the cytologically defined decondensed euchromatin during interphase, while during mitosis neither Spt16 nor SSRP1 are chromatin-associated. The presence of both FACT subunits in the same nuclei was demonstrated by co-localization studies using antibodies that were covalently labelled with different fluorochromes (Figure 3c). To elucidate a possible correlation between the nuclear localization of Spt16 and DNA replication, the

664 Meg Duroux et al. immunodetection of Spt16 was combined with labelling the replicating DNA with incorporated 5-bromo-2¢-deoxyuridine (BrdU) (Figure 3d). Independent of the replication status of the cells, Spt16 showed a similar distribution pattern within

the nuclei. In cells with ongoing replication (indicated by an arrow in Figure 3d), Spt16 is localized as in non-replicating cells. The apparent different subcellular distribution pattern of Spt16 was artificially induced by the denaturation step necessary for the detection of incorporated BrdU (see Experimental procedures). To ascertain whether the Spt16 and SSRP1 patterns are DNA replication-independent, we flow-sorted the non-replicating fraction (2C) of formaldehyde-fixed nuclei from young leaf tissue according to their DNA content (Figure 3e). In these nuclei, Spt16 and SSRP1 displayed a similar distribution (Figure 3f,g). Disperse immunostaining was seen in the less intensely DAPI-stained euchromatin, while brightly DAPI-stained heterochromatic chromocentres remained unlabelled. Therefore, Spt16 and SSRP1 appear to be a specific feature of replicating and nonreplicating interphase euchromatin. SSRP1 and Spt16 are co-expressed in many cell types To determine the spatial pattern of expression and localization of the Spt16 and SSRP1 proteins, we performed indirect, whole-mount immunofluorescence detection using the anti-Spt16 and anti-SSRP1 sera (Figure 4). Embryos of different stages (Figure 4a–h), as well as 4-day (Figure 4i,j) and 8-day-old seedlings (Figure 4k,l) were probed. The observed expression and localization pattern of the Spt16 and SSRP1 proteins were identical. The nuclear localization of both FACT subunits is evident from the comparison of the immunofluorescence signal and the DAPI staining of the DNA (Figure 4a–l). Both proteins could be detected in all cell types from very early stages of development (Figure 4a–d) and also in later embryo stages (Figure 4g,h). In meristematically active, more dividing and less differentiated tissues, both proteins were present ubiquitously. This is demonstrated by the localization pattern in different embryo stages (Figure 4a–g), primary root meristems (Figure 4i,j) and post-embryonically established lateral root meristems (Figure 4k,l). Once cells stopped dividing and underwent terminal differentiation, the immunofluorescence signals

Figure 4. Expression pattern of the Spt16 and SSRP1 proteins examined by whole-mount immunofluorescence. The expression and localization of Spt16 and SSRP1 were examined by whole-mount indirect immunoflorescence detection using the anti-Spt16 and anti-SSRP1 sera. Arabidopsis embryos of different stages were analysed with both antisera and cell nuclei were visualized by DAPI staining: eight-cell embryos (a,b), 16-cell embryos (c,d), globular embryos (e,f), heart stage (g), and torpedo stage (h). Analysis of the primary root meristems of 4-day-old seedlings (i,j) and of post-embryonically established lateral root meristems of 8-day-old seedlings (k,l) by immunofluorescence and DAPI staining. Similarly, mature trichoblasts were examined with both antisera and DAPI staining (merge shown in m,n), but (like in cells of the root cap) no immunofluorescence signal was obtained for Spt16 and SSRP1. Root meristems of 4-day-old seedlings were analysed using an antibody directed against histone H3 dimethylated at lysine 4 (dimethK4 H3) (o) and by DAPI staining (p), while the merge is shown in (q). Arrows indicate some selected cells that display clear nuclear DAPI fluorescence, but no antibody signal for Spt16/SSRP1 (i–n) or for dimethK4 H3 (o,p).

ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

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Figure 5. Spt16 and SSRP1 localize to the transcribed UBQ5 gene but not to non-transcribed heterochromatic regions. ChIP analysis of the UBQ5 gene and two regions of the heterochromatic knob hk4S using anti-Spt16 and antiSSRP1 sera for immunoprecipitation of solubilized chromatin. The DNA purified from the immunoprecipitated chromatin was analysed by PCR with primers specific for the UBQ5 gene (primers 8f þ 8r), or specific for the hk4S regions (primers 9f þ 9r and 10f þ 10r). For comparison, the same regions were amplified by PCR using as template the DNA that was purified from the chromatin prior to the immunoprecipitiation procedure (input).

became weaker and then expression ceased completely. This tendency was observed during the differentiation of root caps at mature embryo stages (not shown) in primary (Figure 4i,j) as well as secondary (Figure 4k,l) root tips. Other differentiated cell types such as mature trichoblasts did not display any Spt16 or SSRP1 signals (Figure 4m,n). As histone H3 dimethylated at lysine 4 (dimethK4 H3) is typically associated with the cytologically defined euchromatin in Arabidopsis (Gendrel et al., 2002; Houben et al., 2003; Soppe et al., 2002), the root tips were also examined using an antibody directed against this histone variant (Figure 4o–q). Similar to the result with the FACT antisera, no dimethK4 H3 immunofluorescence could be detected in root cap cells. The Arabidopsis FACT complex is ubiquitously present in the nuclei of the majority of cell types, whereas it could not be detected in terminally differentiated, non-dividing cells. FACT associates with actively transcribed genes Chromatin immunoprecipitation (ChIP) is a powerful method to examine the in vivo association of proteins with DNA and chromatin (Orlando, 2000) that has been recently adapted for use in plants (Gendrel et al., 2002; Johnson et al., 2003; Wang et al., 2002). To study the chromatin association of FACT, we performed ChIP experiments with the antisera directed against Spt16 and SSRP1 using Arabidopsis suspension cultured cells. Initially, the presence of FACT was examined at the ubiquitin house-keeping gene (UBQ5) in comparison with two non-transcribed regions of the well-defined heterochromatic knob hk4S on the short arm of chromosome 4 (Fransz et al., 2000; Gendrel et al., 2002). Amplification by PCR with primers specific for the coding sequence of the UBQ5 gene revealed that in both the Spt16 and SSRP1 chromatin immunoprecipitates, the UBQ5 sequence is enriched (Figure 5). In contrast, with primers ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

specific for the two non-transcribed heterochromatic regions (Gendrel et al., 2002) the sequences of the heterochromatic knob hk4S (Cinful-like retrotransposon and another transposon) could not be amplified from the immunoprecipitates. Therefore, both FACT subunits localize to the RNA polymerase II transcribed UBQ5 gene, but not to non-transcribed heterochromatin. The association of FACT with genes transcribed by RNA polymerase II was further analysed at the heat shockinducible gene HSP70 (Sung et al., 2001; Zhang et al., 2003). Suspension-cultured cells used as starting material for ChIP with the Spt16 and SSRP1 antisera (Figure 6) were incubated either at normal temperature (24
C, )HS) or under conditions inducing heat shock (37
C, þHS) (Zhang et al., 2003). As examined by RT-PCR, the expression of the HSP70 gene was approximately threefold increased upon heat shock relative to the control cells grown at normal temperature, whereas the elevated temperature had no effect on the expression of the UBQ5 gene (Figure 6f). In the ChIP experiments, association of Spt16 and SSRP1 with the 5¢UTR and different regions of the coding sequence of the HSP70 gene was analysed (Figure 6a). As a control, an intergenic region approximately 3.2 kb downstream of the HSP70 gene was also tested for FACT association. PCR of the different regions of the HSP70 gene from the Spt16 and SSRP1 immunoprecipitates revealed that both subunits of FACT associate with the 5¢-UTR and the coding sequence of the gene in the control cells. In the heat shock-treated cells that show elevated levels of HSP70 expression, an enhanced association of FACT was observed, as evident from the significantly more efficient amplification of the HSP70 gene regions in the ChIP experiment (compare )HS and þHS in Figure 6b,c). The relative difference seen between the ChIP experiments with the Spt16 and SSRP1 antisera reflects most likely the more efficient immunoprecipitation with the Spt16 antiserum that we have consistently observed. No marked effect of the heat shock treatment was detected on the PCR amplification from the input DNA prior to ChIP (Figure 6d). The control of the ChIP procedure performed without addition of specific antisera (but after pre-adsorption with pre-immune serum, see Experimental procedures) resulted in no significant PCR amplification (Figure 6e). When the Spt16 and SSRP1 immunoprecipitates were tested by PCR for the intergenic region (Figure 6b,c), no amplification of this DNA fragment could be detected, whereas it is efficiently amplified from the input DNA samples (Figure 6d). Hence, FACT associates with the non-induced HSP70 gene and to a larger extent when the gene has been transcriptionally induced by heat treatment, but not with the intergenic region downstream of the HSP70 gene. The PR-1 gene is one of the genes that is strongly induced at the onset of systemic acquired resistance or by exogenous application of salicylic acid (SA) (Johnson et al., 2003; Lebel et al., 1998). We have used the induction of the PR-1

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(f) PR-1 -HS +HS -HS +HS HSP70 UBQ5 Figure 6. FACT is present at the transcribed region of the HSP70 gene. (a) Schematic representation of the genomic structure of the HSP70 gene showing the regions examined by ChIP. An open box represents the open reading frame of the gene. Bars above the gene show the regions examined in the ChIP analysis by PCR: region 1 (5¢-UTR, primers 1f þ 1r), 2 (coding sequence, primers 2f þ 2r), 3 (coding sequence, primers 3f þ 3r) and 4 (downstream intergenic region, primers 4f þ 4r). (b–e) ChIP analysis of the HSP70 gene in response to heat shock. Suspension culture cells were either incubated at normal temperature (24
C, )HS) or under heat shock conditions (37
C, þHS). Chromatin was immunoprecipitated with the anti-Spt16 (b), and anti-SSRP1 sera (c), or with no antibody added (e). DNA purified from the precipitated samples was examined by PCR using the primer combinations indicated in (a), and numbers denote the regions amplified by PCR. DNA was normalized for amplification efficiency relative to the DNA purified from chromatin prior to immunoprecipitation (d). At least two ChIP assays were performed per heat shock experiment. (f) Analysis of the expression of the HSP70 gene upon heat shock. RNA was isolated from aliquots of the cells used for ChIP analysis after heat shock treatment (þHS), and from control cells ()HS). After reverse transcription, PCR was performed with primers specific for the HSP70 and the UBQ5 genes.

gene to study its association with FACT. In suspension culture cells, we did not detect PR-1 gene expression by RT-PCR ()SA), but expression of the gene was clearly induced by SA treatment (þSA), while this treatment had no influence on the expression of the UBQ5 gene (Figure 7f). In ChIP experiments, the association of FACT with the PR-1 gene was examined (Figure 7b,c). The PR-1 gene regions could not be amplified from immunoprecipitates of the untreated cells in the absence of PR-1 gene expression. In

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Figure 7. FACT associates with the PR-1 gene only after transcriptional induction by salicylic acid (SA). (a) Schematic representation of the genomic structure of the PR-1 gene showing the regions examined by ChIP. An open box represents the open reading frame of the gene. Bars above the gene show the regions examined in the ChIP analysis by PCR: region 5 (promoter/5¢-UTR region, primers 5f þ 5r), 6 (coding sequence, primers 6f þ 6r) and 7 (3¢-UTR, primers 7f þ 7r). (b–e) ChIP analysis of the PR-1 gene in response to SA induction. Suspension culture cells were either incubated in the absence of SA ()SA) or in the presence of SA (þSA). Chromatin was immunoprecipitated with the antiSpt16 (b), and anti-SSRP1 sera (c), or with no antibody added (e). DNA purified from the precipitated samples was examined by PCR using the primer combinations indicated in (a), and numbers denote the regions amplified by PCR. DNA was normalized for amplification efficiency relative to the DNA purified from chromatin prior to immunoprecipitation (d). At least two ChIP assays were performed per SA experiment. (f) Analysis of the expression of the PR-1 gene upon SA induction. RNA was isolated from aliquots of the cells used for ChIP analysis after SA treatment (þSA), and from control cells ()SA). After reverse transcription, PCR was performed with primers specific for the PR-1 and the UBQ5 genes.

the cells treated with SA, the regions of the PR-1 gene can be efficiently amplified from the immunoprecipitates using the Spt16 and SSRP1 antisera. Association of FACT can be seen with the promoter/5¢-UTR region, coding sequence and 3¢-UTR of the gene. While the PR-1 gene regions could be amplified from the input DNA both of SA-treated and of control samples (Figure 7d), no amplification was obtained from the control experiment with no antibody added (Figure 7e). The ChIP analysis of the PR-1 gene reveals that FACT associates with the gene only after transcriptional induction by SA, whereas FACT cannot be detected at the PR-1 gene in control cells that do not express the gene. ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

Arabidopsis FACT chromatin remodelling complex 667 Discussion In general, plant chromatin structure closely resembles that of other eukaryotes, although a few features are different in plants. For instance, it was established several years ago that distinct structural differences exist between plant linker histones and histones H2A/B, compared with those of other organisms (Smith et al., 1995; Spiker, 1985), while some special properties of plant histone modifications have been elucidated only recently (Loidl, 2004). A number of plant ATP-dependent chromatin remodelling activities have been identified by mutant analyses or by sequence similarity to their yeast and mammalian counterparts (Reyes et al., 2002; Verbsky and Richards, 2001; Wagner, 2003). For example, Arabidopsis homologues of subunits of the well-characterized yeast ATP-dependent SWI/SNF chromatin remodelling complex have been identified (Brezeski et al., 1999; Sarnowski et al., 2002; Wagner and Meyerowitz, 2002). Analyses of the chromatin remodelling activities demonstrated that they play a critical role in various aspects of plant development (Goodrich and Tweedie, 2002; Wagner, 2003). Here we have identified a novel plant chromatin remodelling complex consisting of the Spt16 and SSRP1 proteins. The two subunits of the FACT complex specifically co-purify from Arabidopsis protein extracts by immunoaffinity chromatography using antisera recognizing the native proteins. In line with previous findings, showing that SSRP1 is associated with the highly nuclease-sensitive chromatin in maize nuclei (Lichota and Grasser, 2001), Arabidopsis SSRP1 and Spt16 co-localize to the cytologically defined euchromatin of interphase cells, as demonstrated by indirect immunofluorescence. Consistent with a role in transcription, FACT is not associated with the heterochromatic chromocentres and condensed mitotic chromosomes. Spt16 and SSRP1 also share an overall expression pattern, as examined by whole-mount immunofluorescence detection during embryogenesis and root development. Both nuclear proteins can be detected in the majority of analysed cell types with the exception of terminally differentiated cells such as mature trichoblasts or cells of the root caps. The absence of FACT in root cap cells mirrors that found in immunolocalization studies using an antiserum against histone H3 dimethylated at lysine 4. As H3 lysine 4 dimethylation is typically located in the cytologically defined euchromatin in Arabidopsis that contains transcriptionally active genes (Gendrel et al., 2002; Houben et al., 2003; Soppe et al., 2002), this suggests that in these cells euchromatic features are reduced. Based on RT-PCR experiments, the SPT16 and SSRP1 genes are expressed in shoots, roots and flowers (data not shown). The co-purification of SSRP1 and Spt16, the very similar distribution of both proteins in nuclei and their co-expression in the plant provides sound evidence for the existence of the FACT complex in plants. ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

ChIP experiments in Arabidopsis cells revealed that SSRP1 and Spt16 localize to actively transcribed genes, but FACT cannot be detected in regions of the heterochromatic knob hk4S and intergenic regions. For the inducible HSP70 gene, an accumulation of FACT is observed in heat shock-treated cells, compared with control cells that show only basal level of HSP70 expression. In the PR-1 gene, FACT only associates with the gene after SA treatment. Therefore, analyses of the inducible genes HSP70 and PR-1 indicate that the association of FACT correlates well with the transcription level of these genes. FACT is found over the entire transcribed regions of the Arabidopsis genes analysed, including 5¢-UTR, coding sequence and 3¢-UTR, suggesting a function in transcript elongation. This is in agreement with recent studies on the yeast and Drosophila FACT complexes indicating that FACT co-localizes with RNA polymerase II transcription along the transcribed region of the genes (Mason and Struhl, 2003; Saunders et al., 2003). Consistent with a function in transcript elongation, the yeast FACT subunits have been found to interact physically and/or functionally with a number of transcription elongation factors (Belotserkovskaya et al., 2004; Formosa, 2003). The histone octamers of plant nucleosomes display an enhanced stability compared with vertebrate nucleosomes. This is most likely due to the specific nature of the plant histones H2A/H2B (Moehs et al., 1992), but its significance in transcription is still unclear. Recent experiments have indicated that FACT facilitates the passage of RNA polymerase II through chromatin by destabilizing the nucleosomal structure without requirement for ATP hydrolysis (Belotserkovskaya et al., 2003; Formosa et al., 2002; Rhoades et al., 2004). In the case of human FACT, one H2A/H2B dimer is transiently removed from the nucleosomes (Belotserkovskaya et al., 2003), while the reorganization of the nucleosome into a less inhibitory conformation by yeast FACT involves primarily the nucleosomal DNA contacting the H3/H4 tetramer (Rhoades et al., 2004). As FACT can reorganize nucleosomes during transcription elongation, independent of ATP hydrolysis and without altering the position of the nucleosomes, it represents a chromatin remodelling activity that is distinct from the ATP-dependent remodelling factors. Experiments in yeast indicate that FACT also plays a role in the initiation of transcription presumably by influencing the formation of pre-initiation complexes (Mason and Struhl, 2003). Interestingly, FACT possesses intrinsic histone chaperone activity and can reassemble the nucleosomes after passage of the polymerase (Belotserkovskaya et al., 2003; Formosa et al., 2002). This is also a likely feature of plant FACT, as both Arabidopsis SSRP1 (residues D456-E514) and Spt16 (residues D938-E1010) contain extensive acidic domains, typical of histone chaperones. The histone chaperone activity of FACT contributes to the fidelity of RNA polymerase II transcription in yeast by maintaining the proper chromatin

668 Meg Duroux et al. structure over transcribed regions during transcription, thereby suppressing inappropriate initiation of transcription from cryptic promoters (Kaplan et al., 2003; Mason and Struhl, 2003). Due to its histone chaperone activity, the FACT complex could also ensure that the epigenetic status of chromatin is not disrupted during transcription (Belotserkovskaya et al., 2004), an aspect that is likely to be essential during plant development.

Experimental procedures Production of recombinant proteins and antisera To produce antisera specific for the Arabidopsis SSRP1 and Spt16 proteins, we have selected the regions V453-N646 of SSRP1 termed DSSRP1 and E416-M554 of Spt16 termed DSpt16 as antigens for expression in E. coli. The corresponding DNA coding sequences were amplified by PCR using Deep Vent DNA polymerase (NEB, Beverly, MA, USA), primers (5¢-AACTGCAGGTTCTTGGGGATAATGAT and 5¢-AATTAAGCTTCTATCCGGAATCGTTTCCTG for SSRP1, and 5¢-AAGGATCCGAATGTGAGTCAGAAAG and 5¢-AATTAAGCTTCAATTCCTTATGGGGCAT for Spt16), and a cDNA library as template. The amplified DNA fragments were digested with BamHI/ PstI and inserted into the expression vectors pET43 (Novagen, Merck Biosciences, Darmstadt, Germany) for SSRP1 and pQE9cm (Grasser et al., 1996) for Spt16, which result in the expression of histidine-tagged fusion proteins. The expression in E. coli and the purification of the proteins by three-step purification (metal-chelate, FastS and ResQ chromatographies) were performed as previously described (Grasser et al., 1996; Ro¨ttgers et al., 2000). The expression vectors were confirmed by DNA sequencing, and the purified recombinant proteins were checked by MALDI/TOF mass spectrometry. Purified DSSRP1 and DSpt16 were used for immunization of rabbits (four immunizations, each with 100 lg of protein) performed by Eurogentec (Seraing, Belgium).

Arabidopsis cell suspension culture For the preparation of protein extracts and for ChIP experiments, an Arabidopsis thaliana suspension cell culture was used, originally derived from seedling roots of the ecotype Col-0 (Mathur et al., 1998). The culture was grown in the dark at 24
C in BM medium (Mathur et al., 1998) containing 1 mg l)1 2,4-dichlorophenoxyacetic acid on a rotary shaker (100 rev min)1). For heat shock treatment, the cultures were incubated for 2 h at 37
C. For SA treatment, 250 lM SA (pH 6.8) was added for 16 h. The heat shock and the SA experiments were each performed twice.

Immunoblot analyses Protein samples (recombinant proteins or Arabidopsis protein extracts prepared as described below) were separated by SDSPAGE in 12% polyacrylamide gels and then electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). The antisera directed against Spt16 and SSRP1 (or pre-immune sera) were used at 1:2000 dilutions, and antibody binding was detected using Alexa-Fluor 647 anti-rabbit IgG antibodies (Molecular Probes, Eugene, OR, USA) at a dilution of 1:1000 and scanning of the blots with a Typhoon 8600 phosphorimager (Amersham Biosciences, Uppsala, Sweden).

Immunoprecipitation and immunoaffinity chromatography Suspension-cultured cells were ground in liquid nitrogen using pestle and mortar and resuspended in 1 volume of lysis buffer [50 mM Tris–HCl pH 7.5, 150 mM NaCl, 15 mM EGTA, 15 mM MgCl2, 1 mM dithiothreitol, 0.1% Tween, 1x complete protease inhibitor (Roche Diagnostics, Basel, Switzerland)]. The suspension was centrifuged at 11 000 g for 10 min and the supernatant was used for immunoprecipitation experiments. Approximately 500 lg total protein was used in each immunoprecipitation, and 2 ll antiserum (anti-Spt16, anti-SSRP1 or pre-immune serum was allowed to bind for 16 h at 4
C. The binding reaction was spun at 11 000 g for 2 min and the supernatant was added to 40 ll Protein A Sepharose CL-4B (PAS) (Amersham Biosciences), pre-equilibrated in lysis buffer. After gentle rotation for 3 h, immunocomplexes were eluted with cold 100 mM glycine elution buffer (pH 2.5), neutralized with 1 M Tris–HCl (pH 9) and then precipitated with 25% TCA. Immunoprecipitated proteins were analysed by immunoblot analysis. For immunoaffinity chromatography, antibodies against Spt16 and SSRP1 (5 mg ml)1) were immobilized on CNBr activated Sepharose 4 Fast Flow (Amersham Biosciences) as outlined by the manufacturer. Approximately 7.5 mg of total protein extract was bound to a 1-ml column, washed with 30 ml lysis buffer. Proteins were eluted in a total volume of 3 ml using glycine elution buffer. After precipitation with 25% TCA, proteins were resuspended in 150 ll SDS loading buffer and analysed by immunoblotting.

Analysis of the nuclear and chromosomal localization of proteins by indirect immunofluorescence Suspensions of nuclei isolated from young Arabidopsis Col-0 leaf tissue were prepared as described by Jasencakova et al. (2000) and stained with 1 lg ml)1 DAPI. The relative DNA content was determined with a FACStarPlus flow cytometer (Becton Dickinson, San Jose, CA, USA) and the fraction containing unreplicated (2C) nuclei was sorted onto a microscopic slide into buffer (100 mM Tris–HCl pH 7.0; 50 mM KCl; 2 mM MgCl2; 0.05% Tween 20; 5% sucrose), nearly air-dried and used for immunoabelling (Kubalakova et al., 1997). Preparation of mitotic cells from root tips was carried out as described previously (Manzanero et al., 2000). Slides were blocked for 30 min in 4% (w/v) bovine serum albumin (BSA), 0.1% Triton X100 in phosphate-buffered saline (PBS) at room temperature (RT) prior to two washes in PBS for 5 min each, and incubated with the primary antibodies in a humid chamber. The antisera directed against Spt16 and SSRP1 were diluted 1:400 in PBS with 1% BSA. The corresponding pre-immune sera did not react with the nuclei of the tested plants. After a 12-h incubation at 4
C and washing for 15 min in PBS, the slides were incubated in rhodamine-conjugated antirabbit IgG (Dianova, Hamburg, Germany) diluted 1:100 in PBS, 3% BSA for 1 h at 37
C. After final washes in PBS, the preparations were mounted in antifade containing DAPI as counterstain. Imaging of immunofluorescence was recorded with an Olympus BX61 microscope (Olympus, Tokyo, Japan) equipped with an ORCA-ER CCD camera (Hamamatsu Photonics GmbH, Herrsching, Germany). For co-localization of Spt16 and SSRP1, the corresponding antibodies were covalently labelled with different fluorochromes using a Zenon Tricolor rabbit IgG labelling kit (Molecular Probes). All images were collected in grey scale and pseudocoloured with Adobe Photoshop. For BrdU-labelling, roots of 5-day-old seedlings were incubated in 100 lM BrdU for 60 min. After a short rinse, the roots were immediately fixed and immunostained for Spt16, as described for indirect immunofluorescence. Subsequently, incorporation of BrdU was detected. After washing in PBS the ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

Arabidopsis FACT chromatin remodelling complex 669 chromosomal DNA was denatured with 70% formamide in 2 · SSC for 5 min at 70
C. The slides were dehydrated in ice-cold 70% and 100% ethanol for 5 min each and air-dried. After blocking with 3% BSA in PBS for 20 min at RT the incorporated BrdU was detected by incubation for 1 h at 37
C with a mouse anti-BrdU monoclonal antibody (Becton Dickinson) diluted 1:10 in PBS followed by three washes in PBS and incubation with the secondary FITC-conjugated sheep anti-mouse antibody (Roche) diluted 1:30 in 1 · PBS for 1 h at 37
C. The slides were then washed in PBS, and the DNA was mounted in antifade containing DAPI.

Analysis of the localization pattern by whole-mount immunolocalization Whole-mount localization by indirect immunofluorescence was performed on 4- and 8-days old Arabidopsis seedlings or embryos as previously described (Friml et al., 2003). Anti-Spt16, anti-SSRP1 and anti-histone H3 dimethylated at lysine 4 (Upstate Biotechnology, Upstate, Lake Placid, NY, USA) were diluted 1:400. Secondary CY3-conjugated goat anti-rabbit antibodies (Dianova) were diluted 1:600. Confocal laser scanning microscopy was carried out on a Leica TCS SP (Leica, Bensheim, Germany). Images were processed in Adobe Photoshop and arranged in Adobe Illustrator.

Chromatin immunoprecipitation For ChIP experiments (Wang et al., 2002), suspension-cultured cells were fixed by adding formaldehyde (1% final concentration) to the culture medium and incubation for 30 min at 4
C with gentle rotation. The reaction was quenched by adding glycine to a final concentration of 0.125 M. After 15 min the cells were pelleted by centrifugation and washed twice with cold PBS and finally with PBS containing 1 mM PMSF. For preparation of nuclei, flash-frozen fixed cells (10 g) were ground to a fine powder in liquid nitrogen with a mortar and pestle. Nuclei were resuspended in 2 ml of buffer (10 mM KPO4 pH 7.0; 0.1 mM NaCl; 10 mM EDTA; 0.5% sarkosyl; 1 mM PMSF), sonicated and centrifuged at 20 000 g for 20 min. As checked by gel electrophoresis, the sonicated chromatin had a size of around 1 kb. Sonicated chromatin samples (containing 2.5 lg DNA) in a total of 1 ml of immunoprecipitation buffer (50 mM Hepes, pH 7.5; 150 mM KCl; 5 mM MgCl2; 10 lM ZnSO4; 1% Triton X-100; 0.05% SDS) were pre-cleared by the addition of 5 ll of pre-immune serum and gentle rotation at 4
C for 16 h. Samples were centrifuged at 11 000 g for 10 min and the supernatant was incubated with preequilibrated PAS for 1 h. After centrifugation at 11 000 g for 2 min, the supernatant was used for immunoprecipitation with specific antibodies. A 2.5-ll volume of the antisera was allowed to bind at gentle rotation for 16 h. Pre-equilibrated PAS (50 ll, 50% packed volume) was added to each sample, and gently mixed for 1 h. Immunocomplexes bound to the resin were recovered by a centrifugation at 7000 g for 2 min, followed by five sequential washes in 1 ml of immunoprecipitation buffer containing 500 mM NaCl. The resin was then eluted three times each with 100 ll of glycine elution buffer pH 2.8 (0.1 M glycine, pH 2.8; 0.5 M NaCl; 0.05% Tween 20) and neutralized with each 50 ll 1 M Tris (pH 9) resulting in a total volume of 450 ll; 350 ll was used for DNA work up and 100 ll for further analysis of the immunoprecipitation steps. RNA and protein were eliminated from samples by treatment with RNase A and proteinase K. Formaldehyde-induced cross-links were reversed by incubation at 65
C for 16 h. Samples were then centrifuged at 14 000 g for 10 min, and the genomic DNA in the supernatant was purified by phenol/chloroform extraction. Upon adding 1 lg of glycogen, DNA was precipitated with ethanol and resuspended in ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 660–671

30 ll of DNAse-free water. ChIP experiments were repeated at least twice for each treatment. DNA purified from ChIP experiments was analysed by PCR carried out in a thermal cycler (T-gradient; Whatman Biometra, Go¨ttingen, Germany) with settings (95
C/45 sec, 50
C/45 sec (primers 1–4 and 8) or 60
C/45 sec (primers 5–7) and 72
C/ 1 min). This cycle was repeated 34 times, optimized to produce unsaturated PCR product accumulation. PCRs performed in a total volume of 25 ll [75 mM Tris–HCl, pH 8.8; 20 mM (NH4)SO4; 0.01% Tween 20; 2.5 mM MgCl2; 0.3 mM dNTPs] contained 2.5 U Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania), 12.5 mM of each primer and 7 ng input DNA or 0.5 ll of 1:10 dilution of the DNA recovered from each immunoprecipitation. PCR products were analysed by gel electrophoresis in 1.5% agarose gels, stained with ethidium bromide and scanned using a Typhoon 8600 phosphorimager (Amersham Biosciences). All PCR experiments were repeated three times and the intensity of the electrophoretic DNA bands corresponding to the PCR products was quantified using Image Quant, revealing that the variability between independent PCRs typically was £5%. The ratio of each gene-specific product to that of the control immunoprecipiation (ChIP) was determined after normalization for amplification efficiency (determined from the input sample signals). The following oligonucleotide primers were used for PCR analyses: For the HSP70 gene (At3g12580): 5¢-UTR, (1f: 5¢-TTGCCGACCCACTCTTCATTCAT) and (1r: 5¢-ACCTTCACCTTTACCCGCCATTA), exon 2, (2f: 5¢-GGTTGTTTCCGGTCCAGGTGAGA) and (2r: 5¢-CGCCAACACTCGACGCCTTCTT), 3¢end of coding sequence, (3f: 5¢-AGGAGCTCGAGTCTCTTTGC) and (3r: 5¢-AGGTGTGTCGTCATCCATTC) and 3¢ intergenic region (4f: 5¢-TGCCATGTGTCAAGTCTATCGATCAG) and (4r: 5¢-GGCGGGTGTAACATATAACTGTAATT). For the PR-1 gene (At2g14610): 5¢- UTR (5f: 5¢-TCGGAGGGAGTATATGTTATTGCTTAGAATCA) and (5r: 5¢-TTGTTTCGTATCGGTAGCTTTGCCAT), coding sequence (6f: 5¢-GCTCTTGTAGGTGCTCTTGTTCTTC) and (6r: 5¢-ACCGCTACCCCAGGCTAAGTT), and 3¢-UTR (7f: 5¢-TGGGAATTATGTGAACGAGAAGC) and (7r: 5¢-TCTCGTAATCTCAGCTCTTATTTGTATT). For the UBQ5 gene (At3g62250) coding sequence (8f: 5¢-CTTGAAGACGGCCGTACCCTC) and (8r: 5¢-CGCTGAACCTTTCAAGATCCATCG). For analysis of the heterochromatic 0.5 Mb knob region on chromosome 4: heterochromatin 1, the silenced Cinful– like retrotransposon (BAC T5L23.29) (9f: 5¢-CTCGATGTCGTATTCGCTGA) and (9r: 5¢-GCAACCTATCAACGCTTCGT) and heterochromatin 2, another transposon (BAC T27D20.5) (10f: 5¢-GCGAGAAAGAAGAAGCTGGA) and (10r: 5¢-ACAACTGCTCTGGCACATTG) (Gendrel et al., 2002).

Reverse-transcribed PCR Total RNA of Arabidopsis cells was isolated using the RNA mini kit for plants (Qiagen, Venlo, The Netherlands). First-strand synthesis was carried out using reverse transcriptase (Revert Aid; MBI Fermentas) and an oligo (dT) primer [5¢-ATT CTA GAG GCC GAG GCG GCC GAC ATG (T)32]. The primer pairs used for the HSP70, PR-1, and UBQ5 genes were as described above (2f and 2r), (6f and 6r) and (8f and 8r) respectively. Conditions for PCR were as described for the PCR amplification of the immunoprecipitated DNA, however, using 32 cycles.

Acknowledgements We would like to thank Dr A. Meister for flow-sorting of nuclei, Dr F. Scho¨ffl for advice concerning the heat shock experiments, Dr C. Koncz for providing the Arabidopsis suspension cell culture,

670 Meg Duroux et al. and Dr V. Colot and Dr A.-V. Gendrel for providing primer sequences of non-transcribed hk4S regions. This work was supported by a grant from the Danish Research Council to KDG.

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