Arabidopsis coactivator ALY-like proteins, DIP1 and DIP2, interact ...

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The animal PARP1 is a nuclear protein of 113±120 kDa composed of three main domains: an N-terminal DNA- binding domain, a central automodification ...
Journal of Experimental Botany, Vol. 52, No. 359, pp. 1375±1380, June 2001

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Arabidopsis coactivator ALY-like proteins, DIP1 and DIP2, interact physically with the DNA-binding domain of the Zn-finger poly(ADP-ribose) polymerase1 Sergei Storozhenko, Dirk InzeÂ, Marc Van Montagu2 and Sergei Kushnir Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie ( VIB), Universiteit Gent, KL Ledeganckstraat 35, B-9000 Gent, Belgium Received 10 July 2000; Accepted 19 February 2001

Abstract Two novel homologous Arabidopsis proteins, DIP1 and DIP2, interact with the DNA-binding domain of plant poly(ADP-ribose) polymerase (PARP) in the yeast two-hybrid system and in vitro. Their homology to the transcriptional coactivator ALY suggests that plant PARP may play a role in the regulation of transcription. Key words: Arabidopsis thaliana, coactivators, poly(ADPribose) polymerase, protein interaction, transcription.

Introduction Poly(ADP-ribosyl)ation is a type of reversible secondary protein modi®cation, found in most eukaryotes with the exception of yeast. This modi®cation is catalysed by poly(ADP-ribose) polymerase (PARP) and involves the attachment of ADP-ribose moiety from NADq to glutamic acid residues of the modi®ed protein, followed by further transfer of the ADP-ribose monomers to the newly formed adduct, resulting in the formation of long branched polymers (de Murcia and MeÂnissier de Murcia, 1994; Lindahl et al., 1995; D'Amours et al., 1999). PARPs represent a diverse family of enzymes (Babiychuk et al., 1998; Smith et al., 1998; Ame et al., 1999; Kickhoefer et al., 1999) that are involved in modulation of different processes connected to the nucleic acid metabolism by means of poly(ADP-ribosyl)ation (D'Amours et al., 1999). Studies with speci®c competitive inhibitors of the PARP activity have shown that, in plants, poly(ADPribosyl)ation may play a role in the maintenance of the 1 2

genome stability (Puchta et al., 1995) and in the tracheal element differentiation in Zinnia elegans and Helianthus tuberosum (Hawkins and Phillips, 1983; Phillips and Hawkins, 1985; Sugiyama et al., 1995; Shoji et al., 1997), a process that is known to be an example of the programmed cell death during plant development (Jones and Dangl, 1996). Two different PARPs have been identi®ed in plant cells: ZAP, a homologue of the animal PARP1 (Babiychuk et al., 1998; Mahajan and Zuo, 1998) and NAP, found only in plants until now (Lepiniec et al., 1995; Babiychuk et al., 1998). The domain organization of the ZAP protein (Babiychuk et al., 1998; Mahajan and Zuo, 1998) and its properties resemble those of the animal PARP1, suggesting that ZAP and PARP1 may be functional orthologues in plant and animal cells, respectively. The animal PARP1 is a nuclear protein of 113±120 kDa composed of three main domains: an N-terminal DNAbinding domain, a central automodi®cation domain, and a C-terminal catalytic domain (Kameshita et al., 1984; de Murcia and MeÂnissier de Murcia, 1994; Lindahl et al., 1995; D'Amours et al., 1999). The DNA-binding domain (DBD) contains two Zn-®ngers that have a high af®nity to V-shaped DNA generated by a singlestrand break. Binding to the nicked DNA stimulates the catalytic activity up to 500-fold, resulting in a rapid and extensive modi®cation of the nuclear proteins and the enzyme itself. PARP1 is a multifunctional enzyme that has been shown to be involved in the control of the genome integrity, base excision DNA repair, modulation of the chromatin structure, and cell death (for a review, see de Murcia and MeÂnissier de Murcia, 1994; Lindahl et al., 1995; D'Amours et al., 1999). To a large extent, the link between the PARP1 function and many processes of

The sequence of the proteins has been deposited in EMBL databases with the accession numbers AJ278492 and AJ278493. To whom correspondence should be addressed. Fax: q32 9 264 5349. E-mail: [email protected]

ß Society for Experimental Biology 2001

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nuclear physiology may be attributed to the complex domain organization and multiple protein±protein interactions. In an attempt to understand the role of ZAP in plant cells, two new proteins, DIP1 and DIP2, were identi®ed in Arabidopsis thaliana, which interact with the DNAbinding domain of ZAP. Similarity of these proteins to the animal transcriptional coactivator ALY (Bruhn et al., 1997) suggests that ZAP may play a role in the transcriptional regulation.

Materials and methods Bacterial and yeast strains For the molecular cloning procedures, Escherichia coli strain JM109 (Promega, Madison, WI, USA) was used. Expression of the GST fusion proteins was carried out in E. coli BL21(DE3) strain (Promega). Agrobacterium tumefaciens strain EHA 101 w71521x was used for the transformation of BY-2 (An, 1985) tobacco suspension cultures. Saccharomyces cerevisiae strain HF7c (Clontech, Palo Alto, CA, USA) was used for the twohybrid screening. Bait construction and two-hybrid screening For the two-hybrid screening, the Matchmaker two-hybrid system (Clontech) was used. Baits were constructed in the pGBT9 vector. Site-directed mutagenesis was carried out with the GeneEditor Kit (Promega). Expression of the baits in the yeast cells was con®rmed by the protein gel blotting of the yeast protein extracts probed with a monoclonal anti-BD antibody (Clontech). A GAL4 activation domain cDNA fusion library from A. thaliana (De Veylder et al., 1999) was used in the screening. The entire procedure for two-hybrid screening, including selection of positive clones, elimination of false positives and plasmid rescue, was carried out according to the manufacturer's instructions. Cloning of the ZAP cDNA and purification of the ZAP functional domains of Arabidopsis

The ZAP cDNA of Arabidopsis was cloned by reversetranscription polymerase chain reaction (RT-PCR) on A. thaliana cv. Columbia mRNA template using the Marathon Kit (Clontech) and corresponding primers. Advantage1 cDNA Polymerase Mix (Clontech) was used for all PCR applications. Full-length cDNA, DNA binding, automodi®cation, and catalytic domains were ampli®ed by PCR with corresponding primers using Arabidopsis ZAP cDNA as a template and cloned in the pGEX-4T expression vector (Amersham Pharmacia Biotech, Little Chalfont, UK). The GST fusion proteins were puri®ed on GST af®nity resin (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. In vitro pull-down assay

cDNAs coding for DIP1 and DIP2 proteins were ampli®ed by PCR with the AD Primer Set (Matchmaker Co-IP Kit, Clontech) and in vitro translated and labelled with 35Smethionine (Amersham Pharmacia Biotech) using the TnT Quick coupled transcriptionutranslation system (Promega). Of the labelled proteins, 10 ml was incubated overnight with 10 ml of

the GST fusion protein immobilized on the GST af®nity resin at 4 8C in 500 ml of phosphate-buffered saline buffer with the addition of 0.1% of Tween 20 and 12.5 mg ml 1 of ethidium bromide. The resin was washed four times with the same buffer and bound proteins were eluted from the beads by boiling in the sodium dodecyl sulphate (SDS) sample buffer. The eluted proteins were separated by SDS-PAGE and visualized on a Phosphorimager model 445SI (Amersham Pharmacia Biotech). Construction of the DIP-GFP fusion protein

Full-length DIP1 and DIP2 cDNAs were ampli®ed with corresponding primers and cloned into the binary vector pSL10 (unpublished data) to produce the C-terminal DIP-GFP fusion protein sequences. Transformation of BY-2 tobacco cell suspension culture

Transformation of the BY-2 tobacco culture was performed as described previously (An, 1985). Confocal microscopy analysis For the confocal microscopy analysis, the transformed BY-2 cultured cells were mounted in water. The cells were examined using a 63 3 u1.2Wcorr water immersion lens on a LSM510 laser scanning confocal microscope (Karl Zeiss Inc., Thornwood, NY, USA). Images were acquired at 2048 3 2048 pixel resolution using the 488 nm line of the argon ion laser (458, 488, 514 nm) with the band pass emission ®lter 505±530 nm. Optical sections were taken and maximal brightness projected using the LSM510 software package supplied with the microscope. For differential interference contrast images, the same laser was used as a transmission light source. Fluorescent images were overlaid onto differential interference contrast reference images using the LSM510 software. Sequence analysis For the sequence analysis, GCG (Genetic Computer Group Inc., Madison, WI, USA) software was used. Multiple sequence alignments were carried out with the PILEUP Program. Aligned sequences were edited by using the SeqVu program (The Garvan Institute of Medical Research, Sidney, Australia). Pair-wise amino acid sequence similarities were calculated using the GAP program. For the homology searches, the BLAST program was used, whereas cellular localization prediction was made with the PSORT program at the PSORT www server (http:uupsort.nibb.ac.jp).

Results Novel Arabidopsis proteins DIP1 and DIP2 interact with DBD of ZAP

To ®nd proteins that possibly interact with ZAP, the yeast two-hybrid system was used (Fields and Song, 1989). For this purpose, two baits were prepared. First, the fulllength maize ZAP cDNA was fused to the DNA-binding domain of the yeast GAL4 transcription activator and used as a bait (BD-ZAP). Second, because it has been shown that (i) expression of the active mammalian PARP1 can be toxic for yeast cells (Kaiser et al., 1992),

PARP and transcription

(ii) the active mammalian PARP1 can inhibit the activity of the GAL4 transcription activation domain (Miyamoto et al., 1999), and (iii) the PARP activity can affect the interaction (Masson et al., 1998; Griesenbeck et al., 1997), a mutant bait BD-ZAPmut was constructed, in which a point mutation K861I was introduced into the ZAPcoding sequence. A mutation of the corresponding K893I in animal PARP1 has been shown to abolish the enzyme activity (Simonin et al., 1993). Because no obvious toxic effect of BD-ZAP on yeast cells could be detected upon their transformation, BD-ZAP was used to screen approximately 1.5 3 106 independent clones of the Arabidopsis cDNA library fused to the GAL4 activation domain. No positive yeast clones were selected. However, screening of approximately 2.0 3 106 independent clones of the same library with BD-ZAPmut as bait produced two positive clones, DIP1 and DIP2, which activated both HIS3 and lacZ reporters. Speci®city of the reporter activation was con®rmed by re-transformation of yeast by using the positive clones together with the bait or a set of negative control baits. DIP1 and DIP2 were tested for possible interaction with the presumably active BD-ZAP. Whereas no activation of the lacZ reporter was detected, a slight activation of HIS3 was still observed suggesting that PARP activity might have negatively affected either the reporter promoter activation or the interaction. Additional experiments are required to distinguish between these two possibilities. To check the interaction of DIPs with Arabidopsis ZAP and to de®ne which functional domain of ZAP is involved in the interaction, a deletion analysis coupled with the in vitro pull-down assay was carried out. The Arabidopsis ZAP cDNA was cloned by RT-PCR and was mutagenized to introduce a point mutation K864I. Three different parts of ZAP that comprise either the DBD, the automodi®cation domain, or the catalytic domain as well as the full-length wild-type or mutant ZAP cDNA were produced as GST fusion proteins in E. coli. Unfortunately, it was not possible to purify any substantial amount of the full-length mutant or wild-type ZAP protein because of its poor solubility. However, GST fusion proteins representing the main functional domains of ZAP were highly produced and soluble in E. coli. The puri®ed GST fusion proteins were immobilized on a GST af®nity resin and incubated with 35S-labelled in vitro translated DIP1 and DIP2. After an extensive washing, only the resin with the immobilized DBD retained both labelled proteins, suggesting that the DBD is the main site of the interaction (Fig. 1). Keeping in mind the interaction of the newly identi®ed proteins with the DBD of ZAP, they were designated as DNA-binding domain Interacting Proteins (DIP). Consistently lower amounts of the 35S-labelled DIP2 than of DIP1 were recovered in the in vitro pull-down experiments. This observation

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Fig. 1. In vitro pull-down experiments of the 35S-labelled DIP1 and DIP2 proteins with the GST fusions of the main ZAP domains puri®ed and immobilized on resin. Auto, automodi®cation domain; bd, DNA-binding domain; cat, catalytic domain; input, 20% of the labelled protein used in the pull-down reaction.

might re¯ect functional differences between the proteins. The interaction most probably is not mediated via contaminating nucleic acids because the pull-down experiments were carried out in the presence of ethidium bromide at a concentration that has been shown to prevent interaction between nucleic acids and proteins (Lai and Herr, 1992). It is noteworthy that functional domains of ZAP are relatively independent protein folds (Kameshita et al., 1984). Moreover, the DBD retains its main characteristics even when expressed in E. coli without the other two domains of the protein (Gradwohl et al., 1989). This observation allows the assumption that DIPs would interact not only with DBD as part of the GST fusion protein but also as a part of the full-length ZAP. Thus, DIP1 and DIP2 interact with DBD of ZAP in vitro and in yeast. DIP1 and DIP2 are homologous to the transcriptional coactivator ALY

Sequence analysis showed that DIP1 and DIP2 cDNAs encoded 295 amino acid-long (DIP1) and 288 amino acid-long (DIP2) homologous proteins with predicted molecular masses of 31.2 and 30.3 kDa, respectively. They share 73% identity and 80% similarity. Both DIP1 and DIP2 sequences show 42% and 46% identity and 58% and 55%, respectively, to the mouse transcription coactivator ALY required for the TCRa-enhancer function (Bruhn et al., 1997). In addition, a hypothetical Arabidopsis protein (accession number CAB85990) was also found in the Arabidopsis genome database that shared 47% identical and 56% similar amino acids with DIP1 and DIP2, and even higher homology to ALY (57% identity and 64% similarity) (Fig. 2). BLAST searches with both cDNA sequences against the Arabidopsis DNA database produced a 100% match to a number of expressed sequence tags (more than 10),

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con®rming that the cDNAs were highly expressed. Moreover, sequences of the putative genes coding for the DIP1 and DIP2 proteins were found on chromosomes 1 and 5, respectively.

Both DIP1 and DIP2 sequences contained a putative RNA-binding domain (Fig. 2), which was found in a number of RNA-binding proteins and was shown to be responsible for the RNA binding (Nagai et al., 1995). However, two conserved RNA-binding motifs, octamer RNP1 and hexamer RNP2, lacked the aromatic residues important for the RNA binding (Nagai et al., 1995) in both DIPs and ALY. It has been shown that ALY does not bind RNA or single-stranded DNA (Bruhn et al., 1997). Both DIP1-GFP and DIP2-GFP fusion proteins are localized in the nucleus

ZAP is known to be a nuclear protein, therefore, it was important to de®ne the intracellular localization of DIP1 and DIP2. In silico analysis did not reveal any potential nuclear localization signals either in DIP1 or in DIP2 amino acid sequences. On the contrary, the N-terminal sequences of both DIPs were recognized as chloroplast transit peptides by the PSORT software program. To determine the intracellular localization of DIPs, C-terminal DIP-GFP fusion proteins were designed and expressed in the BY-2 tobacco cells. Examination of the transgenic cell lines using confocal laser scanning microscopy showed that GFP ¯uorescence was exclusively localized in the nuclei both in BY-2(DIP1-GFP) and BY-2(DIP2-GFP) cells (Fig. 3). Thus, the intracellular localization analysis showed that DIP1 and DIP2 were localized in the same cellular compartment as ZAP supporting the conclusion that DIP1 and DIP2 may interact with ZAP in vivo.

Discussion Fig. 2. Alignment of the sequences of DIP1, DIP2, mouse ALY, and the putative protein CAB85990. Identical amino acids are boxed in gray, the putative RNA-binding domain is underlined, the putative octamer RNP1 and hexamer RNP2 are bold underlined, and the putative modi®cation domain is underlined by a bold gray line.

In this study, two new Arabidopsis proteins, DIP1 and DIP2, have been identi®ed that interact with the maize ZAP in the two-hybrid system and with DBD of the Arabidopsis ZAP in vitro. The interaction of both DIP1

Fig. 3. Confocal images of the transgenic BY-2 cells producing the DIP1-GFP and DIP2-GFP fusion proteins.

PARP and transcription

and DIP2 with ZAPs from different plant species, Arabidopsis and maize, detected in two systems, has seemingly a universal character and relies rather on higherorder structures than on a particular sequence. Moreover, both newly identi®ed proteins and ZAP are localized in the nuclei of tobacco BY-2 cells, suggesting that the interaction of DIPs with ZAP may occur in vivo. Both DIP1 and DIP2 physically interact with DBD of ZAP in vitro. The ability of DBD of the animal PARP1 to interact with other proteins has already been demonstrated. This domain has been shown to interact with retinoid X receptor (RXR) (Miyamoto et al., 1999) and with the catalytic subunit of DNA-polymerase a-primase complex (Dantzer et al., 1998). DIP1 and DIP2 belong to a small multigene family, the members of which are all homologous to ALY, the animal context-dependent coactivator of transcription factors LEF-1 and AML-1 required for the TCRaenhancer function (Bruhn et al., 1977). This ®nding connects the plant PARP with transcriptional regulation. A direct role of the animal PARP1 in transcription has already been demonstrated. It has been shown that PARP1 recognizes and binds together with the transcription enhancer factor 1 (TEF1) to the MCAT1 elements in promoters of muscle-speci®c genes to regulate their transcription, thus, combining features of a transcription factor with those of a chromatin-modifying protein (Butler and Ordahl, 1999). In another report, PARP has been shown to block speci®cally the ligand-dependent transcriptional activity mediated by the thyroid hormone receptor (TR) by interacting directly with RXR, which forms heterodimers with TR (Miyamoto et al., 1999). It has been shown that the mammalian PARP1 is associated with the B-MYB oncoprotein in HL60 cells and plays a role as transcriptional coactivator of the B-MYB-driven transcription, with the transactivation being independent of the PARP enzymatic activity (Cervellera and Sala, 2000). The examples mentioned above illustrate how ZAP may possibly regulate plant transcription. In the yeast genetic tests, a much weaker interaction of DIP1 and DIP2 could be detected with the presumably active BD-ZAP than with BD-ZAPmut. One of the explanations of this result might be the possible poly(ADP-ribosyl)ation of ZAP anduor DIPs, which can modulate the interaction (Griesenbeck et al., 1997; Masson et al., 1998). Consistent with this explanation, a number of Glu residues, which are known to be acceptors of the polymer, have been identi®ed in the sequences of DIPs: 15 residues in DIP1 and 14 in DIP2. Most of them are not only conserved but also clustered, which might serve as respective modi®cation domains (Fig. 2). In conclusion, taken together, these results suggest that ZAP may play a role in the transcriptional regulation in plant cells.

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Acknowledgements The authors thank Dr Elena Babiychuk for help with the two-hybrid screening and BY-2 transformation, Gilbert Engler for the confocal microscopy, Wilson Ardiles, Raimundo Villarroel and Jan Gielen for the DNA sequencing, Magali Lescot for help with the computer analysis, Stijn Morsa and Els van Lerberge for technical assistance, Martine De Cock for help in preparing the manuscript, and Rebecca Verbanck for excellent artwork. This work was supported by a grant from the Interuniversity Poles of Attraction Programme (Belgian State, Prime Minister's Of®ceÐFederal Of®ce for Scienti®c, Technical and Cultural Affairs; P4u15). SK is indebted to the Diensten van de Eerste Minister, Wetenschappelijke, Technische en Culturele Aangelegenheden (DWTC T960226) for a research grant.

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