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Multiple Functions of Kip-Related Protein5 Connect Endoreduplication and Cell Elongation[C][W] Teddy Jégu 1, David Latrasse 1, Marianne Delarue, Christelle Mazubert, Mickaël Bourge, Elodie Hudik, Sophie Blanchet, Marie-Noëlle Soler, Céline Charon, Lieven De Veylder, Cécile Raynaud, Catherine Bergounioux, and Moussa Benhamed* Institut de Biologie des Plantes, Unité Mixte de Recherche 8618 Université Paris-Sud XI, 91405 Orsay, France (T.J., D.L., M.D., C.M., E.H., S.B., C.C., C.R., C.B., M.Be.); Pôle de Biologie Cellulaire, Imagif, Centre de Recherche de Gif, Centre National de la Recherche Scientifique, Institut Fédératif de Recherche 87, 91198 Gif-sur-Yvette cedex, France (M.Bo., M.-N.S.); Department of Plant Systems Biology, VIB, B–9052 Ghent, Belgium (L.D.V.); and Department of Plant Biotechnology and Bioinformatics, Ghent University, B–9052 Ghent, Belgium (L.D.V.) Despite considerable progress in our knowledge regarding the cell cycle inhibitor of the Kip-related protein (KRP) family in plants, less is known about the coordination of endoreduplication and cell differentiation. In animals, the role of cyclindependent kinase (CDK) inhibitors as multifunctional factors coordinating cell cycle regulation and cell differentiation is well documented and involves not only the inhibition of CDK/cyclin complexes but also other mechanisms, among them the regulation of transcription. Interestingly, several plant KRPs have a punctuated distribution in the nucleus, suggesting that they are associated with heterochromatin. Here, one of these chromatin-bound KRPs, KRP5, has been studied in Arabidopsis (Arabidopsis thaliana). KRP5 is expressed in endoreduplicating cells, and loss of KRP5 function decreases endoreduplication, indicating that KRP5 is a positive regulator of endoreduplication. This regulation relies on several mechanisms: in addition to its role in cyclin/CDK kinase inhibition previously described, chromatin immunoprecipitation sequencing data combined with transcript quantification provide evidence that KRP5 regulates the transcription of genes involved in cell wall organization. Furthermore, KRP5 overexpression increases chromocenter decondensation and endoreduplication in the Arabidopsis trithoraxrelated protein5 (atxr5) atxr6 double mutant, which is deficient for the deposition of heterochromatin marks. Hence, KRP5 could bind chromatin to coordinately control endoreduplication and chromatin structure and allow the expression of genes required for cell elongation. Plant development depends on meristem activity. In meristems, stem cells generate cells that undergo an important switch during their lifetime, changing from unspecialized cells undergoing rapid division into specific cell types that will be incorporated into primordia. Nevertheless, differentiated plant cells do not lose their proliferating potentialities. Dedifferentiation is commonly associated with reentry into the cell cycle; its distinguishing feature is the withdrawal from a given differentiated state into a “stem cell”-like state that confers pluripotentiality, a process preceding reentry into the cell cycle (De Veylder et al., 2007). In such a context, both stimulation and inhibition of cell proliferation are intrinsic components for plant development. Core regulators of 1 These authors contributed equally to the article. * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Moussa Benhamed ([email protected]). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.112.212357 1694 the eukaryotic cell cycle are heterodimeric complexes consisting of a catalytic subunit, a cyclin-dependent kinase (CDK), and a regulatory subunit, a cyclin. The kinase activity of CDK is regulated in several ways: by cyclin binding (the cyclin partner being itself highly regulated), by phosphorylation and dephosphorylation of the CDK on evolutionarily conserved residues by specific kinases and phosphatases, and by the binding of CDK inhibitors (called CKIs in mammals). Plant genomes contain genes encoding two different classes of CDK inhibitors: Siamese related and Kip-related protein (KRP; also called Inhibitor of Cyclin-dependent Kinase). As demonstrated by their ability to inhibit CDK activity, plant KRP genes encode functional CKIs. Several reports have established that KRPs bind CDKs of the CDK A type and B type (Lui et al., 2000; De Veylder et al., 2001; Jasinski et al., 2002; Zhou et al., 2002), suggesting that they could all function at the G1/S transition and at the G2/M transition. Consistently, CDK complexes containing cyclin D or A can be inhibited by KRPs in vitro (Coelho et al., 2005), and overexpression of KRPs can restore the growth defects induced by D-type cyclin overexpression (Jasinski et al., 2002; Schnittger et al., 2003; Zhou et al., 2003). Results obtained to date suggest that all KRPs regulate the cell cycle in a similar way. Indeed overexpression of Plant PhysiologyÒ, April 2013, Vol. 161, pp. 1694–1705, www.plantphysiol.org Ó 2013 American Society of Plant Biologists. All Rights Reserved. Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. KRP5 Connects Endoreduplication and Cell Elongation various KRPs induces similar defects in plants, characterized by a stunted growth phenotype and serrated leaf. These defects are attributable to a decrease in cell proliferation as evidenced by reduced leaf cell number (Wang et al., 2000; Jasinski et al., 2002). However, KRPs may well play additional roles together with their function in the regulation of CDK activity. This hypothesis is supported by data obtained on CKI in mammals; it is now clear that these proteins fulfill many functions during development and regulate not only cell proliferation but also cell differentiation, transcription, and many other processes (Besson et al., 2008). In plants, KRP overexpression does not affect cell division in the root meristem but prevents differentiated cells from progressing into the cell cycle either for the formation of lateral roots or for in vitro callus induction (Le Foll et al., 2008). In addition, although strong overexpression of KRPs inhibits endoreduplication, moderate overexpression of KRP2 promotes it (Verkest et al., 2005). Consistently, the expression of KRPs is high in endoreduplicating tissues such as maize (Zea mays) endosperm or tomato (Solanum lycopersicum) fruit (Coelho et al., 2005; Nafati et al., 2011). Together, these results suggest that KRPs could act to maintain differentiation in addition to their role in the inhibition of cell proliferation. Another unresolved issue is the respective functions of the seven members of the KRP family. Overexpression analyses seem to indicate that all KRPs have similar functions in planta. However, they do not inhibit the kinase activity of the CDKA complex in vitro with the same efficiency (Wang et al., 1997), suggesting that they could have different behaviors. Indeed, systematic analysis of the core cell cycle interactome revealed that all KRP proteins interact with CDKA;1 but appear to bind preferentially to distinct cyclins (Van Leene et al., 2010); for example, KRP5 was found to interact with CYCD2;1, CYCD4;1, and CYCD4;2. Likewise, although all KRPs have a nuclear localization, they can be grouped into two categories based on their subnuclear distribution: KRP2, KRP6, and KRP7 show a homogeneous pattern throughout the nucleoplasm, while KRP1, KRP3, KRP4, and KRP5 display a punctate pattern of distribution (Bird et al., 2007). This spotted pattern has been associated with chromocenters (Jakoby et al., 2006), suggesting a role of some KRPs in heterochromatin function. In this work, we studied the function of one heterochromatin-associated KRP, KRP5, in Arabidopsis (Arabidopsis thaliana). Here, we show that KRP5 is expressed in endoreduplicating cells and that loss of KRP5 function affects endoreduplication in dark-grown seedlings. Although KRP5 overexpression induces the formation of serrated leaves, as observed for other KRPs, it does not inhibit plant growth and promotes endoreduplication, indicating that KRP5 does not play the same role as other KRPs in planta. The spotted nuclear localization reported previously is due to heterochromatin localization, and chromatin immunoprecipitation sequencing (ChIP-seq) data combined with analyses of mRNA level provide evidence for a role of KRP5 not only in the inhibition of CYCD/CDK complexes but also in the regulation of transcription. Altogether, our results suggest that KRP5 could link cell elongation, endoreduplication, and chromatin structure. RESULTS KRP5 Is Preferentially Expressed in Endoreduplicating Cells Plasmids developed by Deal and Henikoff (2011) allowing the expression of a nucleus-targeted GFP under the control of a promoter of interest were used to study KRP5 expression pattern. The KRP5 promoter was cloned upstream of a Nuclear Targeting Fusion protein (NTF) tagged with GFP and used for Arabidopsis transformation. We next used these transgenic lines to correlate the expression of KRP5 with the distribution of DNA content in rosette leaves, which show the highest level of endoreduplication in Arabidopsis. DNA content per nucleus from whole rosettes of T2 lines was measured and analyzed by flow cytometry simultaneously with GFP fluorescence at two different stages of development. In rosettes of 14-d-old plants, expression of KRP5 was observed in about 20% of the cells and was specifically enriched in endoreduplicating nuclei: 33% of GFP-positive nuclei had a DNA content of 8C or higher (compared with 15% in the total population; Fig. 1A). At later stages, when most leaves had reached maturity, 84% of nuclei were labeled, suggesting that KRP5 expression is associated with differentiation. Furthermore, the distribution of DNA content in GFPpositive nuclei was similar to the distribution observed in the whole population, which contains a high proportion of 4C and higher DNA contents (more than 65%), whereas the majority of GFP-negative nuclei have a 2C DNA content (about 65%; Supplemental Fig. S1). A profiling of plants expressing a 35S::GFP construct was done as a control to make sure that GFP expression did not alter DNA content distribution and that GFP did not accumulate preferentially in some nuclei (Supplemental Fig. S2). Overexpression of KRP5 Induces Endoreduplication To examine the roles of KRP5 on plant growth and development, Arabidopsis transgenic lines were generated in which the KRP5 complementary DNA (cDNA) was expressed under the control of the constitutive cauliflower mosaic virus 35S promoter (KRP5OE plants). Their effective overexpression was monitored using reverse transcription and quantitative PCR (qRT-PCR) analyses (Fig. 1B). Surprisingly, KRP5OE plants did not display reduced growth, as observed for the overexpression of other KRPs (Supplemental Fig. S3). To analyze the effects of KRP5 overexpression on DNA ploidy distribution, the DNA content of cauline leaf nuclei in the wild type and 35 independent transgenic KRP5OE lines was measured by flow cytometry. In Plant Physiol. Vol. 161, 2013 1695 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. Jégu et al. Figure 1. Expression of KRP5 correlates with endoreduplication. A, DNA content distribution of GFP-positive (GFP+) and GFP-negative (GFP2) nuclei isolated from 14-d-old rosette leaves of Arabidopsis KRP5::NTF-GFP plants. For each sample, more than 5,000 DAPI-stained nuclei were analyzed. The left panel shows how GFP+ nuclei were selected based on the intensity of GFP fluorescence (each nucleus is represented by a red point, and the green box indicates a region of high nuclei density); the right panel shows nuclear DNA content distributions of GFP+, GFP2, and total nuclei. The same results were obtained in two independent experiments. B, Real-time RT-PCR data showing the relative expression of KRP5 in wild-type Columbia (Col) and in three independent KRP5OE lines. Average relative quantities 6 SD (n = 3) are shown for each sample. C, DNA content distribution of nuclei isolated from the first cauline leaf of wild-type Columbia (Col 0) and KRP5OE line 3. The data presented here are representative of 35 independent lines. D, Correlation analysis of the DNA content with GFP fluorescence in KRP5OE lines. The distribution shown in gray corresponds to the DNA content of GFP2 nuclei, whereas the green distribution corresponds to GFP+ nuclei. The dashed line represents the distribution of the whole population. The data presented here are representative of two independent experiments. the wild type, endoreduplication occurs in cauline leaves, but it is not as high as in rosette leaves. In all KRP5OE lines, increases in the 8C and 16C populations was observed, correlated with a decrease in the proportion of nuclei with 2C DNA content, illustrating an activation of the endoreduplication cycle (Fig. 1C; Supplemental Table S1). These results suggest that KRP5 is involved in the activation of endoreduplication. To confirm this result, we asked whether the accumulation of KRP5 in the nucleus was associated with higher DNA content. Flow cytometry allowed us to monitor DNA content and GFP fluorescence simultaneously. KRP5-GFP was detected almost exclusively in endoreduplicated nuclei with an 8C and higher DNA content and in some nuclei with a 4C DNA content, which could correspond to a first round of endoreduplication (Fig. 1D). This observation supports the hypothesis that KRP5 stimulates endoreduplication. KRP5 Promotes Endoreduplication in Etiolated Seedlings To provide further evidence for a role of KRP5 as a positive regulator of endoreduplication, we asked whether the loss of KRP5 may result in a reduction of endoreduplication. A search in Arabidopsis transfer DNA (T-DNA) insertion mutant collections was performed. An Arabidopsis insertion line (SK_27217) was obtained from the SALK collection of T-DNA insertion lines (Robinson et al., 2009; Fig. 2A). The genotype of this krp5 mutant, which contains an insertion in the first exon, was confirmed by PCR. qRT-PCR analysis was used to assess the effect of the T-DNA mutation on the KRP5 transcript in homozygous mutant plants. This analysis showed that the expression of the KRP5 gene in the krp5 mutant corresponds to 10% of that in the wild-type Columbia plants (Fig. 2B), indicating that the mutation is not a full knockout but severely affects the expression of the KRP5 gene. 1696 Plant Physiol. Vol. 161, 2013 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. KRP5 Connects Endoreduplication and Cell Elongation Figure 2. KRP5 mutation induces a decrease of endoreduplication and cell elongation in etiolated seedlings. A, Schematic diagram of the Arabidopsis KRP5 gene. Introns are indicated with a single line, exons with dark gray boxes, and untranslated regions with light gray boxes. The position of the T-DNA is indicated by the dashed arrow, and the positions of primers used for genotyping are indicated by arrows with numbers. The results of a PCR experiment performed for genotyping with the indicated primer combination are shown on the right. B, qRTPCR quantification of KRP5 expression in the wild type (Col 0) and the krp5 mutant. C, qRT-PCR quantification of KRP5 expression in the wild type (Col 0) grown in the light (16 h of light) or in darkness. D, DNA content distribution of nuclei isolated from 14-d-old seedlings of the wild type (WT; blue), KRP5OE (black), krp5 mutants (green), and krp5 mutants retransformed with the 35S:KRP5-GFP construct (krp5 KRP5OE; red) germinated in the dark condition. Data are averages 6 SD from three independent experiments. E, Cortical cell size distribution of etiolated hypocotyls from wild-type (Col; white), krp5 (gray), and KRP5OE (OE; black) plants. For each sample, a total of 55 cells were measured. We did not observe any difference in the DNA content of nuclei isolated from mature leaves (data not shown). We reasoned that KRPs may have partially redundant functions and that expression analysis may help us to identify in which particular cellular context KRP5 could have a prominent role in the regulation of endoreduplication. A survey of publicly available gene expression data (https://www.genevestigator.com/gu/) revealed that KRP5 is highly expressed in dark-grown seedlings. To confirm this, we compared KRP5 expression in dark- and light-grown seedlings. We observed an 8-fold induction of KRP5 expression in dark versus light conditions (Fig. 2C). Accordingly, in etiolated seedlings, we observed that the level of endoreduplication correlated with GFP labeling in the KRP5:: NTF-GFP lines (Supplemental Fig. S4). Hypocotyl growth in etiolated seedlings is mediated only by cell elongation and is associated with increased endoreduplication (Traas et al., 1998; Sugimoto-Shirasu and Roberts, 2003). Together with the increased expression of KRP5 in the dark, this suggests that KRP5 could regulate endoreduplication during etiolation. To test this, the DNA content of nuclei isolated from etiolated 7-d-old plantlets of the wild type and the krp5 mutant was measured by flow cytometry. Etiolated krp5 mutants displayed a moderate but reproducible decrease in the 16C population (Student’s t test, P = 0.0049), providing evidence for an inhibition of the endoreduplication cycle in the mutant context (Fig. 2D; Supplemental Table S2). This result is consistent with the opposite effect of KRP5 overexpression observed in the same condition (Fig. 2D; Supplemental Table S2). This reduction of the proportion of 16C nuclei was reversed by the introduction of the 35S::KRP5-GFP construct in the mutant background, demonstrating that reintroduction of the KRP5 cDNA complements the mutant phenotype (Fig. 2D; Supplemental Table S2). We also checked the level of endoreduplication in krp5 mutants grown under the light condition, and no difference was observed compared with the wild type (Supplemental Fig. S5). Because there is strong evidence that the level of ploidy has some impact on cell size determination (Sugimoto-Shirasu and Roberts, 2003), cortical cell sizes of etiolated hypocotyls were compared between the wild type, KRP5 OE , and the krp5 mutant. As shown in Figure 2E, cell size distribution indicated that, globally, cortical cells are longer in the hypocotyls of KRP5 OE compared with the wild type, whereas these cells are shorter in krp5 mutants. This observation is consistent with differences in the ploidy levels obtained for each genotype. We also measured the total hypocotyl length of the wild type, KRP5OE, and the krp5 mutant, and no significant differences were observed (Supplemental Fig. S6), indicating either that the differences in cell size are not important enough to affect the global organ size or that cell number is altered in these plants. Taken together, our results point to a role of KRP5 as a positive regulator of endoreduplication, raising the question of the molecular mechanisms underlying this function. KRP5 Can Antagonize CYCD3;1 Function The primary role of KRPs is to inhibit CDK/cyclin complexes. Therefore, we asked whether KRP5 could promote endoreduplication via CDK/cyclin inactivation. As stated above, KRPs preferentially bind D-type Plant Physiol. Vol. 161, 2013 1697 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. Jégu et al. Figure 3. KRP5 can antagonize CYCD3;1 function. Phenotype (A) and DNA content distribution (B) in nuclei of rosette leaves isolated from the wild type (WT; blue), AtCYCD3;1 overexpressors (CycD3;1OE; red), and double transformants CycD3;1OE/KRP5OE (green). The data shown in B are representative of three independent experiments. For each sample, a minimum of 5,000 DAPI-stained nuclei were analyzed. cyclins. The synthesis of D-type cyclins is initiated during G1 and drives the G1/S-phase transition. Constitutive overexpression of CYCD3;1 in Arabidopsis plants leads to dramatic changes in plant morphology. CYCD3;1-overexpressing plants also show an increase in the proportion of cells with a 4C DNA content, and CYCD3s have been identified as negative regulators of endoreduplication (Dewitte et al., 2007). To determine whether KRP5 could inhibit CDK/CYCD3;1 activity and rescue the CYCD3;1 overexpression phenotype, we transformed CYCD3;1-overexpressing lines with the 35S:KRP5-GFP construct. The presence of the KRP5 transgene was monitored via qRT-PCR analyses and GFP visualization. Interestingly, the phenotype of the CYCD3OE/KRP5OE cooverexpression plants was similar to that of the wild type (Fig. 3A), suggesting that these two proteins have antagonistic activities in planta. Furthermore, flow cytometry analysis revealed that CYCD3OE/KRP5OE plants displayed wild-type levels of endoreduplication in cauline leaves, demonstrating that KRP5 overexpression overcomes the effect of CYCD3;1 overexpression (Fig. 3B; Supplemental Fig. S7; Supplemental Table S3). Hence, KRP5 could promote endoreduplication by inhibiting CYCD3. However, other mechanisms may also contribute to the KRP5-dependent activation of endoreduplication. For example, in mammals, p21, a KIP protein, regulates differentiation by directly modulating gene expression (Devgan et al., 2005). Because KRP5 has a punctate distribution in nuclei, we investigated its ability to bind chromatin. KRP5 Colocalizes with Heterochromatin Marks and Binds Different Types of Genes To examine the subnuclear distribution of KRP5, we performed immunodetection experiments using an antibody raised against GFP and with an H3K9me2 antibody, which is a cytological mark for visible heterochromatin. Immunostaining showed that the KRP5-GFP signal was located inside and outside chromocenters (Fig. 4A), as reported previously for other KRPs (Jakoby et al., 2006; Bird et al., 2007). These observations prove that KRP5 localizes into euchromatic and heterochromatic regions, suggesting that it could play a role in chromatin structure. To identify KRP5-binding sites, we performed ChIPseq on chromatin isolated from Arabidopsis plantlets. The MACS (for model-based analysis of ChIP-seq) algorithm was used to determine which loci are significantly enriched for KRP5-GFP (Fig. 4B). The MACS algorithm is designed to detect transcription factor-binding events, which typically produce short peaks with strong signal intensity (Zhang et al., 2008). We identified around 1,090 KRP5-bound genomic loci (Supplemental Table S4). First, we analyzed the KRP5 peak distance to the transcriptional start site (TSS); we showed that KRP5 binding was not centered on the TSS and that the Gaussian curve displays its maximum at 1,000 bp downstream of the TSS (Fig. 4C). To annotate the KRP5-binding sites, we used the Genomic Position Annotation Tool (Krebs et al., 2008). Of these loci, 5% were identified as pseudogenes, 1% as tRNA, 69% as transposable elements, and 25% as protein-coding genes (Fig. 4D). These data were consistent with the dual subnuclear localization of KRP5 in chromocenters, which are known to be full of transposable elements, and in euchromatin. To analyze the Gene Ontology (GO) of the 25% protein-coding KRP5 target genes, we used an integrated Web-based GO analysis toolkit, AgriGO, and generated a hierarchical tree graph of overrepresented GO terms in the biological process category, showing a significant enrichment in 1698 Plant Physiol. Vol. 161, 2013 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. KRP5 Connects Endoreduplication and Cell Elongation Figure 4. KRP5 colocalizes with heterochromatin marks and binds different types of genes. A, Immunofluorescence detection of KRP5-GFP and H3K9me2 in an isolated nucleus of a KRP5OE transgenic plant. The same distribution was observed in all GFP-labeled nuclei. B, Distribution of KRP5-binding sites on chromosome 2. KRP5binding sites appear as cyan peaks, genes are shown in blue, and transposons are shown in black. C, Distribution of KRP5 peaks around the TSS. KRP5 preferentially binds to regions situated downstream of the TSS. D, Annotation of KRP5binding genes. A total of 74% of KRP5-binding sites correspond to transposable elements and pseudogenes, whereas 25% correspond to protein-coding genes. Among those, the hierarchical tree graph on the right shows a significant enrichment in “plant-type cell wall organization.” “plant type cell wall organization” (Fig. 4D). This result suggests a role of KRP5 in the regulation of genes involved in plant cell wall organization and in heterochromatin structure, raising the question of the mechanisms allowing its recruitment on chromatin. To identify chromatin-related proteins that may facilitate KRP5 binding on chromatin, we searched for interacting partners. AtBAF60, a subunit of the putative SWI/SNF chromatin-remodeling complex, was previously characterized by Tandem Affinity Purification of Tagged Proteins experiment as a KRP5 partner (Van Leene et al., 2010). To confirm that KRP5 proteins can interact with AtBAF60, we used bimolecular fluorescence complementation in protoplasts isolated from BY-2 cell suspensions (Walter et al., 2004). BY-2 cell protoplasts were transformed with constructs encoding a fusion between KRP5 and the N-terminal region of yellow fluorescent protein (YFP; KRP5-YFPN) and between the whole AtBAF60 amino acid sequence and the C-terminal region of YFP (AtBAF60-YFPC). As shown in Supplemental Figure S8, YFP fluorescence could be detected in the nucleus of protoplasts cotransformed with KRP5-YFPN and AtBAF60-YFPC, demonstrating that KRP5 can interact with AtBAF60 in plant cells. These results suggest that KRP5 could bind chromatin and control transcription through the action of chromatinremodeling complexes. KRP5 Binding Correlates with Transcription Activation To determine if a correlation exists between chromatin-bound KRP5 and a transcriptional activation or repression, we first selected a set of KRP5-binding genes: CDC20.1, a gene involved in endoreduplication regulation; AtGRP9, AtRX2, EXT10, and EXT2, which are involved in cell wall structure; and Copia78, a transposable element. All of these target genes were confirmed by chromatin immunoprecipitation (ChIP)PCR (Supplemental Fig. S9). Then, the expression levels of these genes were quantified by qRT-PCR, showing a correlation between the presence of KRP5 and an activation of the transcription (Fig. 5). These results suggest that KRP5 could act directly to promote gene transcription, unraveling a new mechanism by which KRP5 may control endoreduplication or cell differentiation. However, 69% of KRP5-binding sites do not correspond to euchromatin but to heterochromatin regions, which raises the question of the role of KRP5 at these sites. Plant Physiol. Vol. 161, 2013 1699 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. Jégu et al. KRP5 Overexpression Induces Chromocenter Decondensation and Further Endoreduplication in Mutants Deficient for the Deposition of Heterochromatin Marks Previous work showed that ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, two redundant genes encoding histone methyltransferases, play a critical role in maintaining heterochromatin condensation and gene silencing in Arabidopsis. Moreover, these proteins can act to suppress DNA rereplication in heterochromatic regions of the Arabidopsis genome (Jacob et al., 2010). Because we found KRP5 to bind to heterochromatin, we asked whether this association could also participate in the control of endoreduplication. To test this, we transformed the atxr5 atxr6 double mutant with the 35S:KRP5-GFP construct. Then, we measured the DNA content of the first cauline leaf nuclei in the atxr5 atxr6 double mutant, KRP5OE, and in five independent atxr5 atxr6/KRP5OE lines by flow cytometry. We observed that the proportion of 16C nuclei was increased when KRP5 was overexpressed in the atxr5 atxr6 double mutant context compared with KRP5OE alone (Fig. 6A; Supplemental Table S1). To confirm that the increase in endoreduplication could be correlated with a destabilization of the chromatin structure, we performed cytological observation of leaf nuclei. As shown in Figure 6B, overexpression of KRP5 in the atxr5 atxr6 double mutant context Figure 5. KRP5 binding correlates with transcription activation. Quantification of the expression level is shown by qRT-PCR of six KRP5binding genes in the wild type (Col) and in a KRP5 overexpressor (KRP5OE). Each panel shows the position of KRP5 peaks (cyan), a schematic representation of the target gene (dark blue; in which exons are represented by boxes and introns by lines), and the quantification of expression. Error bars represent SD values from at least three repetitions. [See online article for color version of this figure.] 1700 Plant Physiol. Vol. 161, 2013 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. KRP5 Connects Endoreduplication and Cell Elongation dramatically decreased the heterochromatin compaction. Twenty-one percent of KRP5OE atxr5/atxr6 nuclei presented this specific chromatin phenotype; comparatively, 0% of wild-type, KRP5OE, and atxr5 atxr6 nuclei showed this strong phenotype (n = 100). Altogether, our results suggested that condensation of heterochromatin functions as a barrier to endoreduplication and that KRP5 can promote heterochromatin decondensation when the deposition of H3K27me1 is inhibited. DISCUSSION One principle of eukaryotic DNA replication is that the genome is duplicated only once each time a cell divides. However, in response to a variety of physiological signals or developmental cues, cells can exit from the classical mitotic cell cycle to start endoreduplication. This corresponds to a modified cell cycle lacking mitosis, using several regulators. As a general rule, this process is associated with cell elongation and differentiation. Until now, it has been unclear how a dividing cell exits its division program and enters the endocycle. Here, we show that KRP5 is highly expressed in endoreduplicating cells, that it is required to fully induce endocycles in etiolated seedlings, and that its overexpression stimulates endoreduplication. Furthermore, posttranscriptional regulations seem to restrict KRP5 accumulation to endoreduplicating cells, since we observed that the KRP5-GFP fusion protein, Figure 6. KRP5 acts in heterochromatin condensation. A, DNA content distribution of nuclei isolated from the first cauline leaf of the wild type (WT; blue), the atxr5 atxr6 double mutant (green), and KRP5OE (black) and atxr5 atxr6 KRP5OE (red) lines. Endoreduplication caused by KRP5 overexpression was much increased in the atxr5 atxr6 background compared with the wild-type background. Results are averages 6 SD calculated from five different measures for each line. For each sample, a minimum of 5,000 DAPI-stained nuclei were analyzed. B, Immunofluorescence detection of KRP5-GFP in isolated nuclei of wild-type Columbia, double mutant atxr5 atxr6, KRP5OE, and atxr5 atxr6/KRP5OE. Plant Physiol. Vol. 161, 2013 1701 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. Jégu et al. although it was expressed under the control of a constitutive promoter, accumulated in endoreduplicated nuclei. This result is in apparent contradiction to previously reported KRP overexpression studies (De Veylder et al., 2001; Jasinski et al., 2002; Zhou et al., 2002; Schnittger et al., 2003) but is in agreement with the observed stimulation of the endoreduplication cycle in weak KRP2OE plants (Verkest et al., 2005). In addition, although KRP5 overexpression does not affect overall plant size, some results reported here suggest that KRP5 can negatively influence cell proliferation. Indeed, the smaller cell size observed in the krp5 hypocotyl did not correlate with a reduction of hypocotyl length. Likewise, the bigger cells observed in KRP5 OE plants did not affect the overall organ length. Together, these observations suggest that KRP5, like other KRPs, can inhibit cell proliferation, but maybe with a lower efficiency. Whether these contrasting outcomes of KRP overexpression reflect specificities of functions remains to be fully established, but these observations, together with the recent finding that KRPs appear to bind preferentially to different cyclins (Van Leene et al., 2010), hint at a previously unsuspected specialization of KRPs in plants. The data reported here suggest that KRP5 can promote endocycles via at least three different modes of action. The first mechanism is the inhibition of CDK/ cyclin D3 complexes, as evidenced by the ability of KRP5 overexpression to overcome the effects of CYCD3;1 overexpression in planta. CYCD3;1 expression rises as cells reenter the cell cycle, and its ectopic expression in cell cultures results in the accumulation of cells in G2 (Menges et al., 2006). Plants overexpressing CYCD3;1 exhibit ectopic divisions in leaves (Dewitte et al., 2003). Furthermore, cellular expansion and endoreduplication are inhibited in CYCD3;1OE plants, whereas the cycd3 triple mutant has a reduced number of cells and increased endoreduplication levels (Dewitte et al., 2007), pointing to a role of CYCD3 as a negative regulator of endoreduplication. Hence, the ability of KRP5 to inhibit the activity of cyclin D3 could represent a mechanism to promote endoreduplication. In addition to its role in the regulation of CDK/ cyclin activity, our results point to a role of KRP5 in the regulation of gene expression, highlighting another mechanism by which it may stimulate the switch from proliferation to endocycles. Indeed, our results demonstrate that KRP5 binds to various loci on chromatin, possibly via an interaction with the chromatin-remodeling factor AtBAF60, to modulate gene expression. Such a mechanism has been reported in mammalian cells, where the cell cycle inhibitor p16 interacts with the SWI/ SNF complex Brg1 (Becker et al., 2009). Among KRP5 targets, we identified CDC20 in our ChIP-seq experiment. CDC20 is an activator of APC/C, a multisubunit protein complex acting in the degradation process of mitotic cyclins (Marrocco et al., 2010). Overexpression of CDC20.1 promotes endoreduplication in the uvi4 mutant, which is deficient for an inhibitor of APC/C (Iwata et al., 2011). Hence, KRP5-dependent induction of CDC20 expression may contribute to promote endocycles. Furthermore, the observation that genes associated with plant cell wall organization and with cell elongation (Esmon et al., 2006; Sánchez-Rodríguez et al., 2010) are significantly overrepresented among KRP5 target genes and induced by KRP5 overexpression suggests that KRP5 may regulate simultaneously endoreduplication and cell elongation. In most cases, endoreduplication is associated with cell elongation in plants, as observed in wild-type leaf epidermal cells and etiolated hypocotyls (Traas et al., 1998; Sugimoto-Shirasu and Roberts, 2003), even though cell growth and nuclear DNA content can be uncoupled in some instances (Beemster et al., 2002; Jasinski et al., 2002). Although we cannot rule out the possibility that increased expression of genes related to endocycles or cell elongation is an indirect effect of KRP5 overexpression, the concomitant observation of KRP5 binding to these loci strongly suggests that KRP5 directly regulates their expression. This role of KRP5 as a link between cell cycle regulation and the control of cell growth and differentiation is consistent with the situation observed in animals. Indeed, KRPs are putative homologs of mammalian CKI such as p21 and p27 (Inzé and De Veylder, 2006), which have been demonstrated to participate in a number of other specific protein-protein interactions beyond their association with CDK/cyclin complexes. For example, p21 has the potential to physically associate with transcription factors and coactivators and to modulate their function (Dotto, 2000). In particular, p21 can bind to the coactivator p300 and modulate transcription, depending on the nature of the core promoter (Snowden et al., 2000). Recently, Devgan and colleagues have shown that in keratinocytes, p21 functions as a transcriptional regulator that associates physically with the promoter of the Wnt4 gene (Ferrándiz et al., 2012). More generally, it has been demonstrated that p21 can interact with several transcription factors such as CBP, C/ EBPa, E2Fs, Myc, Nrf2, STAT3, and others (for review, Figure 7. Model summarizing the role of KRP5 as a multifunctional protein connecting the transcriptional regulation and/or activity of key genes of the cell cycle, the modulation of the heterochromatin structure, and plant cell wall organization. [See online article for color version of this figure.] 1702 Plant Physiol. Vol. 161, 2013 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. KRP5 Connects Endoreduplication and Cell Elongation see Dotto, 2000; Perkins, 2002; Chen et al., 2009). p21 has been described to act both as a repressor and as a coactivator of the expression of genes (Poole et al., 2004; Fritah et al., 2005; Sestáková et al., 2010). Thus, the transcription function of p21 could be conserved in its Arabidopsis homolog KRP5 and provide a molecular link between the onset of endoreduplication and cell growth or differentiation. Finally, KRP5 may modulate nuclear DNA content by acting on heterochromatin structure. Indeed, 75% of KRP5 targets are in heterochromatin regions, suggesting that KRP5 may modulate chromatin organization. Many lines of evidence link chromatin structure and DNA replication: replication timing requires specific chromatin marks (Casas-Delucchi et al., 2012), and recently, the chromatin factor CAF-1 has been implicated not only in the control of meristem structure but also in the control of genome replication at several stages of development (Exner et al., 2006). Another example is that mutations in the genes encoding the two homologous H3K27 monomethyltransferases ATXR5 and ATXR6 lead to the rereplication of specific genomic portions. These data indicate that ATXR5 and ATXR6 are components of a pathway required to suppress rereplication in Arabidopsis and, more importantly, link chromatin structure to rereplication (Jacob et al., 2010). The synergistic phenotype of the KRP5OE atxr5/ atxr6 plants reported here suggests that in a context where heterochromatin is less condensed, endoreduplication is increased, possibly because heterochromatin could function as a barrier against endoreduplication. The ability of KRP5 to bind to heterochromatin regions and to stimulate the expression of transposable elements suggests that it may participate in the formation of a more accessible chromatin structure for the replication complexes, thereby creating a favorable chromatin context for endoreduplication. In agreement with this hypothesis, we found that KRP5 overexpression severely enhances the chromocenter decondensation phenotype of atxr5 atxr6 mutants. In summary, we show that KRP5 is a multifunctional protein that connects cell elongation and endoreduplication through the combination of at least three modes of action: inhibition of CDK/cyclin complexes, transcriptional regulation of genes involved in endoreduplication, and cell wall synthesis and modulation of heterochromatin structure (Fig. 7). Loss-of-function analysis revealed only very mild defects caused by KRP5 deficiency, pointing to high redundancy between KRPs and suggesting that other members of the family can substitute for KRP5 in most cell types and at most developmental stages. Future work will help to clarify whether other KRPs are also multifunctional and what their distinctive roles are in planta. MATERIALS AND METHODS Plant Material and Growth Conditions Arabidopsis (Arabidopsis thaliana) plants were grown in chambers at 20°C on soil or on sterile one-half-strength Murashige and Skoog medium and 7.5% agar in long-day (16 h of light) conditions. Seeds were surface sterilized by treatment with Bayrochlore (Bayrol) for 20 min, washed, and imbibed in sterile water for 2 to 4 d at 4°C to obtain homogeneous germination. The T-DNA insertion line krp5 (SK_27217) from the SALK collection of T-DNA insertion lines was obtained from the Arabidopsis Biological Resource Center. The double mutant atxr5 atxr6 was obtained from Scott D. Michaels. RNA Extraction and Real-Time Quantitative PCR Analysis Total RNA were extracted from seedlings with the RNeasy MiniPrep kit (Qiagen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 2 mg of total RNA using Improm-II reverse transcriptase (A3802; Promega) according to the manufacturer’s instructions. A 1/25th fraction of the synthesized cDNA was mixed with 100 nM of each primer and LightCycler 480 Sybr Green I master mix (Roche Applied Science) for quantitative PCR analysis. Products were amplified and fluorescent signals were acquired with a LightCycler 480 detection system. The specificity of amplification products was determined by melting curves. AtPTF2 was used as an internal control for signal normalization. Exor4 relative quantification software (Roche Applied Science) automatically calculates relative expression levels of the selected genes with algorithms based on the comparative threshold cycle method. Data were from duplicates of at least two biological replicates. The sequences of primers can be found in Supplemental Table S5. Promoter Cloning The 1,617 bp upstream of the ATG was amplified with Pfu DNA polymerase (primers used are listed in Supplemental Table S4) and cloned in pGEM-T Easy vector. After sequencing, a BamHI/SpeI fragment was cloned in the intact system developed by Deal and Henikoff (2011). Confocal Imaging Transformed BY-2 protoplasts were directly imaged on a TCS-SP2 upright microscope (Leica Microsystems). To observe YFP, excitation was at 514 nm, and the spectral detector was set between 520 and 550 nm. Transmitted light was also collected. All images were acquired with similar gain adjustments. ChIP and ChIP-seq Analysis ChIP assays were performed on 10-d-old in vitro seedlings using anti-GFP (Santa Cruz) or IgG control (Millipore) antibodies using a procedure adapted from Gendrel et al. (2005). Briefly, after plant material fixation in 1% (v/v) formaldehyde, tissues were homogenized and nuclei were isolated and lysed. Cross-linked chromatin was sonicated using a water bath Bioruptor UCD-200 (Diagenode; 30-s-on/30-s-off pulses, at high intensity for 24 min). Protein/ DNA complexes were immunoprecipitated with antibodies overnight at 4°C with gentle shaking and incubated for 1 h at 4°C with 50 mL of Dynabeads Protein A (Invitrogen; 100-02D). Immunoprecipitated DNA was then recovered using the IPure kit (Diagenode) and analyzed by quantitative real-time PCR. An aliquot of untreated sonicated chromatin was processed in parallel and used as the total input DNA control. The sequences of primers can be found in Supplemental Table S5. After immunoprecipitation of the chromatin, ChIP-seq libraries were generated and sequenced. In this work, we used the anti-GFP antibody for ChIP-seq, and input chromatin was used as a control to minimize biologically irrelevant experimental bias. Alignment was performed using Bowtie (Langmead et al., 2009) version 0.12.7 on the Arabidopsis genome (The Arabidopsis Information Resource 10). Default Bowtie parameters were used except as follows: -best -strata (used to get the best mapping position with the minimum of mismatch). Peak calling was performed with MACS (Zhang et al., 2008) and gene annotation with the Genomic Position Annotation Tool (Krebs et al., 2008). Immunofluorescence Seedlings were fixed in 4% paraformaldehyde in PHEM (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9) during 1 h at room temperature (apply vacuum for 20 min to facilitate uptake of the fixation solution). Seedlings were washed 5 min in PHEM and 5 min in phosphate buffered saline (PBS), pH 6.9, and chopped on a petri dish in PBS supplemented with 0.1% (w/v) Triton. The mixture was filtered (50 mm) and centrifugated 10 min at 2,000g. The supernatant was carefully removed, and the Plant Physiol. Vol. 161, 2013 1703 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2013 American Society of Plant Biologists. All rights reserved. Jégu et al. pellet was washed once with PBS, gently resuspended in 20 mL of PBS, and a drop was placed on a poly-Lys slide and air dried. Slides were rehydrated with PBS and permeabilized two times by 10 min of incubation in PBS plus 0.1% (v/v) Tween 20 (PBST). Slides were placed in a moist chamber and incubated overnight at 4°C with primary antibody anti-H3K9me2 (Millipore; 07-441) or anti-GFP (Santa Cruz) in PBST supplemented with bovine serum albumin (3%, w/v). Slides were washed five times for 10 min each in PBST (at room temperature) and incubated 1 h at room temperature in the dark with the secondary antibody (Alexa Fluor 594 goat anti-rabbit; A11037; Invitrogen) diluted 1:400 (v/v) in PBST and 3% bovine serum albumin. Slides were washed five times for 10 min each in PBST. Slides were mounted with a drop of Vectashield with 49,6-diamino-2phenylindole (DAPI) and observed as described for pollen mitosis analysis with the suitable cube fluorescence filters (Band Pass [BP] 340–380, Dichroic Splitter [DS] 400, BP 450–490 for DAPI; BP 570–590, DS 595, BP 605–655 for A594). Flow Cytometry For flow cytometric nuclei analysis, tissues were chopped with a razor blade in 1 mL of Galbraith buffer supplemented with 1% polyvinylpyrrolidone 10,000, 5 mM metabisulfite, and 5 mg mL21 RNase from a stock solution at 50 units mg21. Propidium iodide was added to the filtered supernatants at 50 mg mL 21. Endoreduplication levels of 5,000 to 10,000 stained nuclei were determined using a Cyflow SL flow cytometer (Partec) with a 532-nm solid-state laser (100 mW) excitation and emission collected after a 590-nm long-pass filter. Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Nuclear DNA content distribution of GFP+ and GFP2 nuclei isolated from rosette leaves of Arabidopsis KRP5::NTFGFP plants. Supplemental Figure S2. DNA content distributions of nuclei isolated from leaves of 35S::GFP plants. Supplemental Figure S3. Phenotype of the KRP5-overexpressing line. Supplemental Figure S4. DNA content distributions of nuclei isolated from KRP5::NTF-GFP hypocotyls of 7-d-old dark-grown seedlings. Supplemental Figure S5. DNA content distributions of nuclei isolated from light-grown wild-type plants, krp5 mutants, and KRP5OE. Supplemental Figure S6. Average hypocotyl lengths of etiolated seedlings of krp5, wild-type, and KRP5OE plants. Supplemental Figure S7. DNA content distribution in nuclei of rosette leaves isolated from the wild type, AtCYCD3;1 overexpressors, and three different double transformant CycD3;1OE/KRP5OE lines. Supplemental Figure S8. Interaction between KRP5 and AtBAF60 visualized by bimolecular fluorescence complementation. Supplemental Figure S9. ChIP from KRP5OE transgenic plants was performed using anti-GFP or anti-IgG antibody. Supplemental Table S1. Quantification of nuclear DNA content distribution in the first cauline leaf of wild-type, KRP5OE, atxr5 atxr6, and KRP5OE atxr5 atxr6 plants. Supplemental Table S2. Quantification of nuclear DNA content distribution in hypocotyls of 7-d-old dark-grown seedlings of the wild type, krp5, krp5 KRP5OE, and KRP5OE. Supplemental Table S3. Quantification of nuclear DNA content distribution in the wild type, CYCD3;1OE, and KRP5OE CYCD3;1OE from 14-d-old growing plantlets. Supplemental Table S4. List of KRP5 target genes. Supplemental Table S5. Primer sequences. ACKNOWLEDGMENTS We are very grateful to Steven Henikoff (Fred Hutchinson Cancer Research Center) for providing INTACT vector and ACT2p::BirA seeds, to James A.H. Murray (Cardiff School of Biosciences) and Catherine Riou (Limoges University) for providing 35S::CYCD3;1, to Scott D. Michaels (Department of Biology, Indiana University) for the atrx5 atrx6 double mutant, and to François Parcy (Commissariat à l’Energie Atomique) for providing pBiFP vectors for bimolecular fluorescence complementation. 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