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ABAP1 is a novel plant Armadillo BTB protein involved in DNA replication and transcription
2008; Springer Nature; Volume: 27; Issue: 20 Linguagem: Inglês
10.1038/emboj.2008.191
ISSN1460-2075
AutoresHana Paula Masuda, Luíz Mors Cabral, Lieven De Veylder, Miloš Tanurdžić, Janice de Almeida Engler, Danny Geelen, Dirk Inzé, Robert A. Martienssen, Paulo C.G. Ferreira, Adriana Silva Hemerly,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle25 September 2008free access ABAP1 is a novel plant Armadillo BTB protein involved in DNA replication and transcription Hana Paula Masuda Hana Paula Masuda Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, BrazilPresent address: Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-170 Santo André, SP, Brazil Search for more papers by this author Luiz Mors Cabral Luiz Mors Cabral Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, Brazil Search for more papers by this author Lieven De Veylder Lieven De Veylder Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, Belgium Department of Molecular Genetics, Ghent University, Ghent, Belgium Search for more papers by this author Milos Tanurdzic Milos Tanurdzic Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Janice de Almeida Engler Janice de Almeida Engler Unité Mixte de Recherche ‘Interactions Plantes-Microorganismes et Santé Végétale’, Institut National de la Recherche Agronomique-Université de Nice-Sophia Antipolis-Centre National de la Recherche Scientifique, Sophia Antipolis, France Search for more papers by this author Danny Geelen Danny Geelen Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, BelgiumPresent address: Department of Plant Production, Faculty of Bioengineering, Ghent University, 9000 Gent, Belgium Search for more papers by this author Dirk Inzé Dirk Inzé Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, Belgium Department of Molecular Genetics, Ghent University, Ghent, Belgium Search for more papers by this author Robert A Martienssen Robert A Martienssen Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Paulo CG Ferreira Paulo CG Ferreira Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, Brazil Search for more papers by this author Adriana S Hemerly Corresponding Author Adriana S Hemerly Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, Brazil Search for more papers by this author Hana Paula Masuda Hana Paula Masuda Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, BrazilPresent address: Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-170 Santo André, SP, Brazil Search for more papers by this author Luiz Mors Cabral Luiz Mors Cabral Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, Brazil Search for more papers by this author Lieven De Veylder Lieven De Veylder Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, Belgium Department of Molecular Genetics, Ghent University, Ghent, Belgium Search for more papers by this author Milos Tanurdzic Milos Tanurdzic Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Janice de Almeida Engler Janice de Almeida Engler Unité Mixte de Recherche ‘Interactions Plantes-Microorganismes et Santé Végétale’, Institut National de la Recherche Agronomique-Université de Nice-Sophia Antipolis-Centre National de la Recherche Scientifique, Sophia Antipolis, France Search for more papers by this author Danny Geelen Danny Geelen Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, BelgiumPresent address: Department of Plant Production, Faculty of Bioengineering, Ghent University, 9000 Gent, Belgium Search for more papers by this author Dirk Inzé Dirk Inzé Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, Belgium Department of Molecular Genetics, Ghent University, Ghent, Belgium Search for more papers by this author Robert A Martienssen Robert A Martienssen Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Paulo CG Ferreira Paulo CG Ferreira Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, Brazil Search for more papers by this author Adriana S Hemerly Corresponding Author Adriana S Hemerly Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, Brazil Search for more papers by this author Author Information Hana Paula Masuda1,2,‡, Luiz Mors Cabral1,2,‡, Lieven De Veylder3,4, Milos Tanurdzic5, Janice de Almeida Engler6, Danny Geelen3, Dirk Inzé3,4, Robert A Martienssen5, Paulo CG Ferreira1,2 and Adriana S Hemerly 1,2 1Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, DIPEC, Rio de Janeiro, Brazil 3Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, Belgium 4Department of Molecular Genetics, Ghent University, Ghent, Belgium 5Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA 6Unité Mixte de Recherche ‘Interactions Plantes-Microorganismes et Santé Végétale’, Institut National de la Recherche Agronomique-Université de Nice-Sophia Antipolis-Centre National de la Recherche Scientifique, Sophia Antipolis, France ‡The authors contributed equally to this work *Corresponding author. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, CCS—bloco DSS, Rio de Janeiro, RJ 21941-590, Brazil. Tel.: +55 21 32042085; Fax: +55 21 25626789; E-mail: [email protected] The EMBO Journal (2008)27:2746-2756https://doi.org/10.1038/emboj.2008.191 Present address: Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-170 Santo André, SP, Brazil PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In multicellular organisms, organogenesis requires a tight control of the balance between cell division and cell differentiation. Distinct signalling pathways that connect both cellular processes with developmental cues might have evolved to suit different developmental plans. Here, we identified and characterized a novel protein that interacts with pre-replication complex (pre-RC) subunits, designated Armadillo BTB Arabidopsis protein 1 (ABAP1). Overexpression of ABAP1 in plants limited mitotic DNA replication and decreased cell proliferation in leaves, whereas ABAP1 downregulation increased cell division rates. Activity of ABAP1 in transcription was supported by its association with the transcription factor AtTCP24. The ABAP1–AtTCP24 complex bound specifically to the promoters of AtCDT1a and AtCDT1b in vitro and in vivo. Moreover, expression levels of AtCDT1a and AtCDT1b were reduced in ABAP1-overexpressing plants and they were increased in plants with reduced levels of ABAP1. We propose that ABAP1 participates in a negative feedback loop regulating mitotic DNA replication during leaf development, either by repressing transcription of pre-RC genes and possibly by regulating pre-RC utilization through direct association with pre-RC components. Introduction In multicellular organisms, organogenesis requires a tight control of the balance between cell proliferation and differentiation. An important regulatory mechanism controlling cell division is licensing DNA for replication at late G1, which allows cells to progress into S phase. This process is regulated by the sequential assembly of ORC, CDC6, CDT1 and MCM onto replication origins, forming the pre-replication complex (pre-RC) (Blow and Dutta, 2005). Differentiation of specialized cells is driven by transcriptional regulatory events involving chromatin remodelling proteins and transcription factors. Therefore, during the transition from a dividing to a differentiating state, cells must tightly integrate transcription regulation with cell cycle controls, such as DNA replication licensing. It is expected that both regulatory pathways might share similar components to facilitate coordination during development. Pre-RC components have been associated with the control of diverse developmental processes in eukaryotes. In mammals and Drosophila, ORC seems to have a function in neuronal development (Pinto et al, 1999; Huang et al, 2005). The involvement of pre-RC components in heterochromatin formation and establishment of transcriptionally repressed DNA regions was well characterized in yeasts, Drosophila and mammals (Sasaki and Gilbert, 2007). In animals, a key regulator of pre-RC activity, called geminin, has been implicated in the regulation of cell proliferation and cell differentiation by interacting with components of the pre-RC and transcription factors (Seo and Kroll, 2006). In plants, a concerted action of DNA replication and gene transcription controls during development is still unidentified. A modular and indeterminate construction of the plant body by self-perpetuating meristems, shaped by external and endogenous developmental signals, imposes an accurate balance of cell division and cell differentiation during plant life to generate organs of the correct form (Castellano and Sablowski, 2005). The extent of the involvement of DNA replication controls in plant development is still not clear as meristematic plant cells commonly exit the mitotic cycle and start differentiation without pausing DNA synthesis. These DNA endoreduplication cycles seem to be very important for some plant cells to undergo differentiation programmes and correct organ development (Gutierrez, 2005). Genetic analyses in Arabidopsis have shown that pre-RC components, such as AtCDT1 homologues, AtCDC6, AtORC2 and two AtMCMs can interfere in developmental processes either by directly affecting replication or by indirect effects on endoreduplication and heterochromatin formation (Springer et al, 2000; Holding and Springer, 2002; Castellano et al, 2004; Collinge et al, 2004; Dresselhaus et al, 2006). Here, we addressed whether DNA replication and transcription controls would share regulatory pathways during plant development. We report the identification and characterization of a novel pre-RC-interacting protein with the acronym for Armadillo BTB Arabidopsis protein 1 (ABAP1). ABAP1 is a nuclear G1/early S-specific protein that interacts with members of the pre-RC and the transcription factor AtTCP24 in vitro and in vivo. We show that the ABAP1–AtTCP24 complex can bind site-specifically DNA regulatory elements and control expression of DNA replication genes in vivo. Protein interaction experiments indicated that ABAP1–AtTCP24 might associate with pre-RC subcomplexes or eventually with the full complex in plant cells. The ABAP1–AtTCP24 complex regulates cell proliferation rates during leaf development limiting mitotic DNA replication. ABAP1-overexpressing plants exhibit an overall reduced cell number in leaves and an opposite effect is observed in plants with downregulated levels of ABAP1. Our data demonstrate that ABAP1 function might exemplify a new negative feedback loop regulating mitotic DNA replication during leaf development either by repressing transcription of pre-RC genes and possibly by regulating pre-RC utilization through direct association with pre-RC components. Results ABAP1 is a novel plant protein that interacts with the pre-RC In an attempt to identify protein complexes formed with Arabidopsis pre-RC members, a search for pre-RC-interacting proteins was performed. Because AtORC1a harbours two domains involved in transcription regulation—BAH and PHD domains (Masuda et al, 2004), its complete open-reading frame was assayed in a two-hybrid screen against an Arabidopsis cDNA two-hybrid library. Among the interacting proteins, a novel 737-amino-acid protein (At5g13060) of approximately 81 kDa was identified. It harbours eight predicted repeats of the β-catenin-type Armadillo domain (ARM repeats) in its N-terminal region (amino acids 111–162, 164–212, 213–254, 256–296, 298–338, 340–380, 381–421 and 496–536) as well as a BTB/POZ domain (acronym for Broad complex/Tram-Track/Bric-a-brac/Poxyvirus and zinc-finger), located in the C terminus (amino acids 568–665) (Figure 1A). Therefore, the gene was designated ABAP1. Yeast two-hybrid assays revealed that ABAP1 interaction with AtORC1a was mediated by the ARM repeats and by AtORC1a N terminus (amino acids 1–341) that contained the BAH and PHD domains (Supplementary Figure 1A). β-Catenin ARM repeats are approximately 40-amino-acid-long tandemly repeated sequence motifs and occur in various families of eukaryotic proteins involved in different cellular processes, such as cell–cell adhesion, nuclear import, cell signalling and transcriptional regulation (Coates, 2003; Hatzfeld, 2005). The BTB/POZ domain is usually found in leucine zipper proteins involved in transcriptional repression with a possible role as E3 ligase (Perez-Torrado et al, 2006). Figure 1.Characterization of ABAP1 protein interactions and localization. (A) Schematic representation of ABAP1 protein domains with the eight β-catenin-type Armadillo (white boxes) and BTB/POZ (oval grey) domains. Nuclear localization signal (NLS) is represented as an asterisk. (B) Subcellular localization of GFP::ABAP1 in roots of 6-day-old Arabidopsis seedlings by confocal microscopy. Merged images of rhodamine/DAPI (B1 and B3) and rhodamine/GFP (B2 and B4). White arrowheads in (B1) indicate mitotic cells. (C) GST-pulldown assay between ABAP1–GST and radiolabelled AtORC1a (left panel) and AtORC1b (right panel). (D) GST-pulldown assay with ABAP1–GST and radiolabelled AtCDT1 homologues. (E) GST-pulldown assay with ABAP1–GST and radiolabelled AtORC1a-N'Term and AtORC4. (F) Triple GST pulldown with AtORC1a–GST and radiolabelled ABAP1 and AtCDT1b. (G) Immunoprecipitation of Arabidopsis protoplasts expressing ABAP1, AtORC1a-GST, AtORC3–HA and AtCDT1a–FLAG. Anti-ABAP1 immunoprecipitated proteins were assayed with antibodies against ABAP1 and tags. Download figure Download PowerPoint In agreement with the presence of a putative nuclear localization signal in ABAP1 N terminus (Figure 1A), confocal and fluorescent microscopy of Arabidopsis plants producing GFP::ABAP1 indicated that ABAP1 was exclusively located in the nucleus, homogeneously distributed or enriched in nuclear domains, and GFP::ABAP1 was absent in mitotic cells (Figure 1B). Analyses of endogenous levels of ABAP1 in synchronized Arabidopsis cell cultures indicated that it accumulated during G1 and/or early S phase (Supplementary Figure 2B–E). This result was corroborated by the studies on BY2 tobacco cells producing GFP::ABAP1 after treatment with inhibitors of cell cycle progression (Supplementary Figure 2A and Supplementary Video 1). A genomic search revealed the existence of at least 108 ARM domain and 80 BTB domain proteins in the Arabidopsis genome (Mudgil et al, 2004; Gingerich et al, 2005). ABAP1 has the unique feature to harbour both Armadillo and BTB domains in its primary sequence. Arabidopsis has an ABAP1 homologue (At5g19330), characterized earlier as ARIA (ARM repeat protein interacting with ABF2) that shares 59% identity of the deduced amino-acid sequence with ABAP1 and is involved in abscisic acid response through interaction and regulation of the transcription factor ABF2 (Kim et al, 2004). Remarkably, no ABAP1 homologue was found in eukaryotes other than plants. In a yeast two-hybrid assay against all other pre-RC proteins, except MCM2–7, ABAP1 associated directly with AtORC1b, AtCDT1a and AtCDT1b (Supplementary Figure 1B). The interactions between ABAP1 and the pre-RC components were confirmed in GST-pulldown experiments with ABAP1–GST and radiolabelled AtORC1a35S, AtORC1b35S, AtCDT1a35S and AtCDT1b35S. The pre-RC components bound to ABAP1 but not to GST alone (Figure 1C and D). The specificity of the interactions was demonstrated in an assay where ABAP1–GST did not bind to radiolabelled AtORC435S and bound to AtORC1a N terminus35S (Figure 1E). The formation of a triple complex between AtORC1a, ABAP1 and AtCDT1b was shown in a GST-pulldown experiment with AtORC1a–GST, ABAP135S and AtCDT1b35S (Figure 1F). As AtCDT1b and AtORC1a did not bind directly, the presence of two radioactive bands indicated that ABAP1 can associate with pre-RC subcomplexes or eventually with the full complex. The association between ABAP1 and pre-RC was further confirmed in vivo by immunoprecipitations with the polyclonal anti-ABAP1 antibody (Supplementary Figure 3) and protein extracts of Arabidopsis cell suspension LMM-1 protoplasts that produced ABAP1, AtORC1a–GST, AtORC3–HA and AtCDT1a–FLAG (Figure 1G). In two-hybrid assays with pre-RC proteins, AtORC3 did not interact either with ABAP1 (Supplementary Figure 1B) or with the AtCDT1 homologues, but with all AtORCs, except AtORC1a and itself, possibly having a central function in maintaining ORC associations (Masuda et al, 2004). Similar interaction profile of AtORC3 with the other ORC proteins was reported by GST-pulldown experiments (Diaz-Trivino et al, 2005). In immunoprecipitation experiments with anti-ABAP1, ABAP1 interacted in vivo with AtORC1a–GST, AtCDT1a–FLAG and also with AtORC3–HA (Figure 1G), suggesting that ABAP1 might associate with pre-RC subcomplexes or eventually with the full complex in plant cells. Possibly, ABAP1 can form different complexes with different affinities to anti-ABAP1, as the immunoprecipitations depleted the interacting AtORC3 and AtCDT1a, but not AtORC1a and ABAP1 itself. ABAP1 controls cell proliferation in leaves Studies on localization of ABAP1 expression in plant tissues showed that ABAP1 was weakly expressed in the emerging lateral roots (Figure 2A; Supplementary Figure 2B), being mainly expressed in the shoot apex, young leaves (Figure 2B; Supplementary Figure 2C) and flower buds (Supplementary Figure 4A and D), suggesting a possible function in these organs. Strong GUS expression was detected in developing leaves of 7- and 9-day-old seedlings, as observed for other pre-RC components, such as PROLIFERA (AtMCM7), AtCDC6, AtCDT1 homologues and AtORC1b (Springer et al, 2000; Castellano et al, 2001; Castellano et al, 2004; Diaz-Trivino et al, 2005). As the leaf grew, GUS expression decreased in a tip-to-base gradient (Figure 2C and D). In agreement, ABAP1 mRNA levels were higher in developing leaves during proliferation stage (9-day-old plants) and rapidly declined as observed in leaves of 13 till 21-day-old plants (Figure 2E). GUS expression in the meristemoid and young stomatal cells illustrates ABAP1 expression during stomatal cell differentiation (Supplementary Figure 4E). Figure 2.ABAP1 expression and function in plants. (A–D) Detection of GUS activity of proABAP1::GUS in Arabidopsis. (A) Emerging lateral root of a 22-day-old plant. (B) Shoot apex of 7-day-old seedling. (C) Leaf of 9-day-old seedling. (D) Leaf of 11-day-old plants. (E) Real-time RT–PCR analyses of ABAP1 expression in leaf number 2 at different developmental stages. The transcript level is represented as a ratio of the absolute value of the studied gene to the absolute value of AtUBI14 gene. The data are the result of at least two experiments and representative results are shown. (F–K) Phenotypic analysis of ABAP1OE and ABAP1ET lines. (F) Time course of average rosette area of wild-type ecotype Col (white circles) and ABAP1OE lines (black circles). All trait variables were transformed to Ln. All points of the ABAP1OE plants are significantly different from wild type (ANOVA, P<0.05). (G) Three-week-old soil-grown wild-type Col (upper panel) and ABAP1OE plants (lower panel). (H) Leaf number 4 of 4-week-old wild-type Col (upper leaf) and ABAP1OE (lower leaf) plants. (I) Time course of average rosette area of wild-type ecotype Ler (white circles) and ABAP1ET lines (black circles). All trait variables were transformed to Ln. All points of the ABAP1ET plants are significantly different from wild type (ANOVA, P<0.05). (J) Three-week-old soil-grown wild-type Ler (upper panel) and ABAP1ET plants (lower panel). (K) Leaf number 4 of 4-week-old wild-type Ler (upper leaf) and ABAP1ET (lower leaf) plants. Download figure Download PowerPoint To assess the function of ABAP1 during development, plants with increased or reduced expression levels of ABAP1 were characterized. Seventeen lines of Arabidopsis plants expressing higher levels of ABAP1 mRNA and protein under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter were generated (here denoted as ABAP1OE). The increase in mRNA and protein levels varied among the different overexpressing lines and the developmental stage, ranging from 5- to 17-fold and 2- to 5-fold, respectively (Supplementary Figure 5A and B). A heterozygous enhancer trap line (ET13614, here denoted as ABAP1ET) with interference T-DNA inserted into the first exon of ABAP1 gene (10 base pairs after the initial ATG) was analysed (Supplementary Figure 6). The plants showed an average of two- and five-fold reduction in ABAP1 mRNA and protein levels, respectively (Supplementary Figure 6A and B). Homozygous plants could not be rescued in ABAP1ET, suggesting that the absence of ABAP1 causes a lethal phenotype (Supplementary Figure 6C). Comparative phenotype analyses between ABAP1 overexpressor lines (ABAP1OE) and control lines showed that plants with higher levels of ABAP1 developed normally, except that rosette area and leaf growth were moderately diminished during development (Figure 2F–H). Quite the opposite, reduction of ABAP1 levels in ABAP1ET plants caused an increase in the rosette and leaf growth (Figure 2I–K). Kinematics studies of developing leaves showed that cell division rates were higher in ABAP1ET plants and lower in ABAP1OE plants during early leaf development when compared with wild-type controls (Figure 3A and C). Though leaf cell organization (data not shown) and cell sizes were similar to those of control plants, cell numbers were significantly reduced in ABAP1OE and increased in ABAP1ET plants (Figure 3E). Our data suggested that ABAP1 negatively regulated overall cell divisions in leaves, differing from the pre-RC subunits AtCDT1 and AtCDC6 that could specifically affect stomata formation (Castellano et al, 2004). An effect of ABAP1 levels in early leaf development is consistent with its high expression levels in shoot meristem and young leaves. Figure 3.ABAP1 function in DNA replication control. (A, C) Kinematic analysis of cell division rates of (A) wild-type Col (black circles) and ABAP1OE (white circles) rosette leaves and (C) wild-type Ler (black circles) and ABAP1ET (white circles). (B, D) [3H]Thymidine incorporation assay with (B) wild-type Col and ABAP1OE plants and (D) wild-type Ler and ABAP1ET plants. ABAP1OE and ABAP1ET [3H]thymidine incorporation are significantly different from wild type (Student's t-test, P<0.05). (E) Number and size of cells per leaf in leaf number 2 of wild-type (Col), ABAP1OE, wild-type (Ler) and ABAP1ET 21-day-old plants estimated by kinematic analyses. (F) Chromatin fractionation assay. Pipeline of assay (upper panel, left). S1, first low-speed supernatant containing non-chromatin-binding proteins; P1, crude chromatin pellet; S2, supernatant from nuclear fraction; P2, high-speed pellet containing polynucleosomes. Western blot of the fractions obtained from wild-type Col, ABAP1OE, wild-type Ler and ABAP1ET were assayed with anti-PROLIFERA and anti-ABAP1 antibodies (lower panel) and PROLIFERA P2 band intensity was quantified (upper panel, right). As loading control, a Ponceau staining of the nitrocellulose membrane is shown. Download figure Download PowerPoint As ABAP1 interacted with members of the pre-RC and accumulated at G1 and early S phase of the cell cycle, the cell proliferation phenotypes observed in ABAP1OE and ABAP1ET leaves could be at least partly triggered by a misregulation of the DNA replication machinery. Cell proliferation assays with [3H]thymidine in ABAP1OE and ABAP1ET seedlings showed that the DNA replication levels were significantly lower in ABAP1OE plants than those in the wild-type plants (Figure 3B). In contrast, ABAP1ET seedlings showed higher levels of DNA replication than wild type (Figure 3D). A direct role of ABAP1 in pre-RC assembly and association with chromatin was investigated by measuring MCM loading onto chromatin, the hallmark step in the formation of the pre-RC. Cells were separated into cytoplasmic, nucleus-soluble and chromatin-enriched fractions and levels of MCM7 (PROLIFERA) and ABAP1 were assayed by immunoblot analysis (Figure 3F). PROLIFERA association with chromatin was lower in 6-day-old ABAP1OE plants than in wild-type plants, and the excess of ABAP1 was mostly found as a soluble nuclear protein. When ABAP1 levels were reduced in ABAP1ET, higher levels of PROLIFERA were found loaded onto chromatin. Ploidy levels in developing leaves of ABAP1OE plants were similar as control, suggesting that ABAP1 might have a function in mitotic replication but not in endocycles (Supplementary Figure 7). Altogether, the data on the characterization of plants with modified levels of ABAP1 indicated that ABAP1 exert a negative role in cell proliferation in leaves, possibly by inhibiting mitotic DNA replication. ABAP1 interacts with plant transcription factors, the complex binds DNA and regulates gene expression To unravel protein complexes in which ABAP1 takes part, a yeast two-hybrid screen under high stringency selection conditions was performed with an Arabidopsis cDNA library as a bait. Several transcription factors, belonging to the NAC, AP2 and TCP families (unpublished results) were identified. One of the ABAP1-associated transcription factors was AtTCP24 (At1g30210) that belongs to the class-II TCP transcription factor family (Cubas et al, 1999), the members of which negatively regulate plant cell proliferation and leaf morphogenesis (Nath et al, 2003; Palatnik et al, 2003). To address a possible cooperation of ABAP1 and AtTCP24 in the control of gene transcription, the association between both proteins was characterized. The AtTCP24–ABAP1 interaction was confirmed in a GST-pulldown assay with AtTCP24–GST and radiolabelled ABAP135S. ABAP1 bound to AtTCP24–GST but not to GST alone (Figure 4A). In vivo interaction was observed in immunoprecipitation experiments of protein extracts of Arabidopsis cell suspension LMM-1 protoplasts that produced ABAP1 and AtTCP24–FLAG, with the anti-ABAP1 antibody (Figure 4B). The ability of the ABAP1–AtTCP24 heterodimer to recognize the consensus sequence for class-II TCP (TGGGCC/T) (Trémousaygue et al, 2003) was confirmed in electrophoretic mobility shift assays (EMSAs) (Supplementary Figure 8). The data demonstrated that the association of ABAP1 with AtTCP24 did not affect the DNA-binding properties of the latter and that ABAP1 could participate in mechanisms of transcriptional regulation. Other class-II TCP members (AtTCP3, AtTCP5, AtTCP13 and AtTCP17) did not interact with ABAP1 in yeast two-hybrid assays (Supplementary Figure 9A), indicating some specificity in the ABAP1 association with class-II TCP family. Figure 4.Characterization of ABAP1 and AtTCP24 transcription factor interaction and ABAP1 function in transcription. (A) Confirmation of ABAP1–AtTCP24 interaction through the in vitro GST-pulldown interaction assay. (B) Immunoprecipitation of protein extracts of Arabidopsis protoplasts producing ABAP1 and AtTCP24–FLAG. Total protein extracts were immunoprecipitated with anti-ABAP1 and analysed in a protein gel blot with antibodies against FLAG and ABAP1. (C) EMSA of ABAP1, AtTCP24 and the heterodimer AtTCP24–ABAP1 with wild-type (WT) and mutated (mut) radiolabelled probes of AtCDT1b and AtCDT1a promoter regions harbouring TCP recognition box. Anti-GST and anti-ABAP1 were used for ABAP1 and AtTCP24–GST supershift assays (lanes 3, 7–9 and 12, 16–18). (D, E) Real-time RT–PCR analyses of AtCDT1a and AtCDT1b expression in (D) wild-type Col (white bars) and ABAP1OE 6-day-old seedlings (black bars), and (E) in wild-type Ler (white bars) and ABAP1ET 6-day-old seedlings (black bars). The transcript levels are represented as a ratio of the absolute value of the studied gene to the absolute value of AtUBI14 gene. Gene expression was normalized by values obtained for wild-type controls. Asterisks indicate values statistically different from the wild type (Student's t-test, P<0.05). Inset in (D, E), western blot analysis using anti-ABAP1 in total protein extracts of ABAP1OE and wild-type Col 6-day
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