Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function
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WRKY transcription factors have been shown to play a major role in regulating, both positively and negatively, the plant defense transcriptome. Nearly all studied WRKY factors appear to have a stereotypic binding preference to one DNA element termed the W-box. How specificity for certain promoters is accomplished therefore remains completely unknown. In this study, we tested five distinct Arabidopsis WRKY transcription factor subfamily members for their DNA binding selectivity towards variants of the W-box embedded in neighboring DNA sequences. These studies revealed for the first time differences in their binding site preferences, which are partly dependent on additional adjacent DNA sequences outside of the TTGACY-core motif. A consensus WRKY binding site derived from these studies was used for in silico analysis to identify potential target genes within the Arabidopsis genome. Furthermore, we show that even subtle amino acid substitutions within the DNA binding region of AtWRKY11 strongly impinge on its binding activity. Additionally, all five factors were found localized exclusively to the plant cell nucleus and to be capable of trans-activating expression of a reporter gene construct in vivo.
KeywordsArabidopsis promoters Nuclear localization Transactivation W-box element
Transcription factors act in concert with other components of the transcriptional machinery to modulate the expression of target genes in a temporal and spatial manner. In general, they do so by binding to short defined nucleotide motifs (cis-elements) within the regulatory regions of genes that are under their control. Different classes of transcription factors have characteristic DNA-binding domains that discriminate between distinct cis-regulatory elements at target sites within the genome. Our studies have been focused on a family of zinc-finger type transcription factors, designated WRKY. WRKY factors comprise a large family of DNA-binding proteins found in all plants (Eulgem and Somssich 2007). Although not completely restricted to the plant kingdom, this family has expanded enormously in higher plants whereas they appear to have been lost in yeast and animal lineages (Ülker and Somssich 2004). WRKY proteins have been implicated in the regulation of developmental processes such as trichome and seed development and leaf senescence (Hinderhofer and Zentgraf 2001; Johnson et al. 2002; Luo et al. 2005), but their major functions appear to be in helping the plant to cope with various abiotic and biotic stresses (Ülker and Somssich 2004; Journot-Catalino et al. 2006; Li et al. 2006; Wang et al. 2006; Xu et al. 2006; Zheng et al. 2006; Shen et al. 2007).
All WRKY proteins contain a 60 amino acid long peptide region, termed the WRKY domain, which constitutes their DNA binding regions. Apart from the invariant name-giving amino acid residues W R K and Y, this domain also contains conserved cysteine and histidine residues that bind one zinc atom and form a finger-like structure. Both the WRKY residues as well as the zinc finger motif are required for proper DNA binding of the protein (Maeo et al. 2001). Based on the number of WRKY domains and the pattern of the zinc-finger motif, WRKY factors have been classified into three major groups (Eulgem et al. 2000). The number of family members in higher plants can range from 74 in Arabidopsis, 98 to 102 in rice or even more in other species such as tobacco (Ülker and Somssich 2004; Ross et al. 2007).
Gel-shift experiments, random binding site selection, DNA-ligand binding screens, yeast one-hybrid studies and cotransfection assays performed with different plant WRKY proteins have illustrated that the cis-element 5′-TTGAC-C/T-3′, termed the W-box, represents the minimal consensus required for specific DNA binding (de Pater et al. 1996; Rushton et al. 1996; Wang et al. 1998; Chen and Chen 2000; Cormack et al. 2002). Only in the case of the barley WRKY factor SUSIBA2 and the tobacco NtWRKY12 have additional DNA binding sites been identified, although SUSIBA2 also shows strong in vitro binding to the W-box motif (Sun et al. 2003; van Verk et al. 2008). Considering the size of the WRKY family within a given species and the apparent stereotypic binding preference of these proteins to the W-box, it is difficult to foresee how specificity for certain promoters is accomplished. A certain level of specificity may be conferred by additional nucleotide sequences flanking W-box elements with respective gene promoters. Furthermore, various WRKY subgroup family members within a single plant species may also differ somewhat in their DNA binding requirements. Both of these aspects have not yet been directly addressed for WRKY transcription factors. Finally, the involvement of WRKY factors in diverse higher-order nucleoprotein complexes can also be assumed and may in fact be the major criteria in determining promoter selectivity and transcriptional output.
In this study, we selected five Arabidopsis WRKY transcription factors representing the three major groups of WRKY proteins including three from the largest, namely group II. These factors were analyzed with respect to their in vitro binding capabilities to various W-box variants and promoter sequences containing W-box motifs. This analysis already revealed clear as yet not observed binding site preferences between certain representatives. Furthermore, we show that all studied WRKY factors are localized exclusively to the nucleus and that all have in vivo transactivation capabilities.
Materials and methods
Recombinant fusion proteins
The AtWRKY cDNA fragments were obtained with specific Gateway-primers in RT-PCR reactions and cloned in-frame into the modified bacterial expression vector pGEX2T (Pharmacia) or into the vector pQE30a (Qiagen) and verified by sequencing. The C-terminal protein construct of WRKY11 (WRKY CTD) consisted of 98 amino acids encompassing the WRKY DNA binding domain and in addition carrying a 6×His tag at its N-terminus MRGSHHHHHHMKRTVRVPAISAKIADIPPDEYSWRKYGQKPIKGSPHPRGYYKCSTFRGCPARKHVERALDDPAMLIVTYEGEHRHNQSAMQENISSSGINDLVFASA. Mutations within the WRKY11 CTD were introduced using the megaprimer method (Ke and Madison 1997) and cloned into the Gateway compatible vector pASK5 (IBA). A list of the primers used in this study are given in the supplementary table (S-Table 1). Protein expression was carried out in E. coli strain BL21(DE3) following addition of IPTG (1 mM) or 2 μg/μl anhydrous tetracyclin (IBA) for 3–4 h. Bacteria were harvested by centrifugation. The pellet was resuspended and lysed under native conditions but including protease inhibitors at 4°C (PIERCE, lysis protocol according to the instructions of the manufacturer). Due to technical difficulties encountered in purifying the respective recombinant WRKY proteins all subsequent experiments were performed using total soluble E. coli protein extracts derived from bacteria expressing the WRKY cDNA construct or the control empty vector construct. For western blot analyses proteins were separated by SDS-PAGE and blotted onto a PVDF membrane (Sartorius). The membranes were probed with anti-GST antibody (Pharmacia) or anti-6×HIS antibody and anti-goat or anti-mouse IgG conjugated to alkaline phosphatase, respectively. Anti-strep antibody was directly conjugated to alkaline phosphatase. Membranes were developed with NBT-BCIP solution Sigma-fast (Sigma).
Electrophoretic mobility shift assay (EMSA)
Equivalent amounts of sense and antisense fragments of the respective oligonucleotides (Invitrogen) were annealed in 40 mM Tris–HCl pH 7.5, 20 mM MgCl2, and 50 mM NaCl starting at 95°C and allowing to cool slowly to room temperature. About 1.5 μl of the solution corresponding to 150 ng DNA was used for end-labeling with 5 U of terminal transferase (Roche) and 20 μCi dCTP (Amersham) in a total volume of 25 μl according to the manufacturer’s instructions (Roche). The probes were purified on Sephadex G-25 columns and diluted to 5000 cpm/μl. Binding reactions were performed according to Maeo et al. (2001). Reaction mix which included 25 μg of total soluble bacterial protein extract was incubated for 20−25 min to 25°C after addition of the radioactively labeled probe then applied to the gel. Gel electrophoresis was carried out at 4°C. Sequence of 1×W2: TTATTCAGCCATCAAAAGTTGACCAATAAT. For competition experiments unlabelled DNA was added as competitor to the binding reaction and incubated for 10 min on ice. Labeled probe DNA was added and the sample was incubated at room temperature for another 15 min prior to loading on the gel. For all EMSA experiments described in this paper, the specificity of the observed signals obtained with the labeled probes was also confirmed using an excess (50- to 1000-fold) of unlabeled competitor DNA (data not shown).
Biolistic transformation of leaves by particle bombardment
Arabidopsis thaliana plants (ecotype Columbia-0) were grown under long day conditions (18 h light, 6 h darkness, 20°C). Leaves of 4–5-week-old plants were detached, placed on 0.5×MS plates and incubated in a light chamber for at least 4 h prior to bombardment. On average 40 to 50 leaves were used per assay.
The 4×W2∷GUS reporter contains a tetramer of the W-box motif TTGACC (Eulgem et al. 1999). The SIRKp∷GUS construct was described by Robatzek and Somssich (2002). Full-length cDNAs of WRKY6, WRKY11, WRKY26, WRKY38 and WRKY43 were fused in-frame to the GFP reporter gene and expressed under the control the CaMV35S promoter (p35S-GW-GFP). For transient expression in onion cells, 5 μg of the respective constructs were introduced into epidermal cells via particle bombardment. Subcellular localization was microscopically monitored 20–24 h post bombardment. The expression constructs were introduced into Arabidopsis leaves via particle bombardment (PDS-1000/He Biolistic® Particle Delivery System, Bio-Rad), essentially as described by Robatzek and Somssich (2002). Briefly, 50 μl of gold particles (1 μm diameter) were coated with both the reporter and effector plasmids (3 μg DNA each). When using one reporter plasmid and two different effector plasmids, 2 μg of each DNA was used. About 3 mg of gold particles were delivered per shot. After 24 h bombardments, the leaves were infiltrated with GUS staining solution for 3–5 min and incubated at 37°C overnight followed by clearing of the leaves with 95% ethanol. Reporter gene activity staining was evaluated macroscopically, taking into account all leaves used in the experiment.
The GenBank DNA sequence flat-files were downloaded from the Entrez Plant Genomes Central at NCBI (http://www.ncbi.nlm.nih.gov/; NC_003070.5, NC_003071.3, NC_003074.4, NC_003075.3, NC_003076.4). Sequences of either 1500 or 600 bp upstream of the annotated translation start sites (ATG) were extracted and compiled to FASTA formatted text with the aGenBankSQL script v3.3.4 (Berendzen et al. 2006), using the default code modifications. Number of W-box motifs has been assessed counting their occurrences in all chromosome sequences and the promoters. The fold-differences were calculated on the number of W-box occurrences in the two promoter datasets divided by the average number of W-boxes in the genome (number of W-boxes divided by the total number of bases of all chromosomes).
W-box elements and derived consensus were examined with the Motif Mapper Open Source Scrip Package (http://www.mpiz-koeln.mpg.de/coupland/coupland/mm3/html/) as has been described previously (Berendzen et al. 2006). The Sequence logos for the two W-box consensus motifs were derived from a position weight matrix using WebLogo (http://weblogo.berkeley.edu/; Crooks et al. 2004).
Functional categorization of the gene lists was performed using the Gene Ontology annotation form at TAIR (http://www.arabidopsis.org/portals/genAnnotation/functional_annotation/go.jsp).
To assess values for significance (P-values), we retrieved the GO-annotations for all genes using the TAIR7 release of the Arabidopsis genome. A set of 20669 GO-annotations was returned, which could be used as a background model to calculate the hypergeometric distribution.
DNA binding selectivity of Arabidopsis WRKY factors
To date, all studied plant WRKY transcription factors show high binding preference to the DNA sequence element, 5′-C/TTGACT/C-3′, termed the W-box (Ülker and Somssich 2004; Eulgem and Somssich 2007 and citations therein). However, no systematic study has been reported testing whether members of the different WRKY subgroups within a single plant species all show similar DNA binding requirements. Thus, we choose five selected subgroup representatives of the Arabidopsis thaliana WRKY gene family for qualitative DNA binding studies. These members represent the WRKY groups I (WRKY26; At5g07100), IIb (WRKY6; At1g62300), IIc (WRKY43; At2g46130), IId (WRKY11; At4g31550) and III (WRKY38; At5g22570) (Eulgem et al. 2000). Full-length cDNAs for all representatives served as templates for expression in E. coli and total soluble protein extracts derived from these bacteria were directly used for further analysis. Initially, we encountered several problems attempting to express a large set of WRKY proteins (both as GST fusions or epitope tagged variants) in E. coli. Often, their expression proved detrimental for bacterial growth. Since WRKY factors are zinc-finger proteins, their expression may in some way negatively influence zinc homeostasis. Exogenous addition of zinc however did not relieve this problem. In several instances bacterial growth was not significantly influenced but the specific WRKY proteins were found exclusively in inclusion bodies. No attempts were made to purify the proteins from this source since previous work in our laboratory has shown that during the denaturation and subsequent renaturation steps misfolding of these proteins often occurs resulting on loss of W-box binding ability. Affinity purification of epitope-tagged WRKY proteins under mild condition also proved problematic. Thus, only those five WRKY members were taken into consideration for further analyses for which sufficient protein was present in the total soluble bacterial protein extracts. As controls for all experiments, protein extracts derived from identically treated bacteria harboring only the appropriate empty expression vector cassette were always included. In no case did these control extracts result in detectable sequence-specific binding to the tested DNA sequences (data not shown).
WRKY factor binding to diverse W-box containing Arabidopsis promoters
We next tested whether W-box containing sequence regions of native Arabidopsis gene promoters will also yield additional information concerning selective binding requirements of WRKY factors. For this, four promoters were selected derived from the SIRK/FRK1 (At2g19190), CMPG1 (At3g02840), 4CL4 (At3g21230) and WRKY11 loci. The SIRK/FRK1gene encodes a receptor kinase whose expression is up-regulated during leaf senescence and upon pathogen challenge (Asai et al. 2002; Robatzek and Somssich 2002). Its promoter contains numerous W-box motifs and several WRKY factors have been implicated in its regulation. CMPG1 represents an immediate-early pathogen-responsive gene coding for a U-box protein that is required for efficient activation of defense mechanisms in tobacco and tomato (Gonzalez-Lamothe et al. 2006). The W-box-containing region was shown to mediate this response (Heise et al. 2002). 4CL4 codes for a 4-coumarate:CoA ligase, a key enzyme in general phenylpropanoid metabolism with unusual catalytic properties (Hamberger and Hahlbrock 2004). The occurrence of three TATA-proximal W-box elements is an absolute singularity among all known 4CL gene promoters.
Binding of Arabidopsis WRKY factors to W-box dimers
WRKY factors have been shown to bind to promoter regions often containing closely spaced W-box motifs (Rushton et al. 1996; Eulgem et al. 1999; Yang et al. 1999; Yu et al. 2001; Chen and Chen 2002; Marè et al. 2004; Zhang et al. 2004).
Amino acid residues influencing DNA binding activity of the WRKY domain
Maeo et al. (2001) demonstrated for tobacco WRKY9 that the two cysteine and histidine residues forming the zinc-finger motif and the highly conserved WRKYGQK amino acid stretch within the WRKY domain are important for W-box binding activity. Furthermore, they and others have shown that despite high sequence identity between the two WRKY domains of group I WRKY factors, only the C-terminal WRKY domain (CTD) actually contributes significantly to DNA binding (de Pater et al. 1996; Eulgem et al. 1999; Maeo et al. 2001).
In vivo interaction of WRKY factors with W-box elements
The occurrence of WRKY factor binding sites within the Arabidopsis genome
Again using the Motif Mapper program, we investigated whether the appearance of two closely adjacent W-box elements show enrichment in our selected datasets and if so, is there a certain spacing preference between individual motifs. As a framework, we choose to analyze a spacing range from 0 to 30 bp, a potentially important distance inferred from several previous publications (see above). Figure 6d summarizes our findings for the 600bp promoter dataset versus the whole genome. This data implies that certain defined distances are significantly enriched (P < 0.001), in particular, closely adjacent (N1–N3, N5, N7) and more distantly spaced (N29–30) W-box elements occur substantially more often than expected.
The availability of wholly sequenced genomes in combination with molecular and functional genomic techniques is enabling us to decipher the complete gene-encoded protein set of single organisms. In higher eukaryotes, including plants, 8–10% of such proteins are transcription factors that play a pivotal role in establishing transcriptional regulatory networks governing diverse cellular expression profiles. A key component in understanding such networks is to identify transcription factor binding sites common to the individual genes within defined regulons. Computational methods are currently being applied to assist in locating such sites within entire genomes (Pilpel et al. 2001; Bulyk 2003; Rombauts et al. 2003; Wasserman and Sandelin 2004). However, in order to improve computational predictions of TF sites within promoters and thereby to extract biologically meaningful information, it is absolutely essential to experimentally define more precisely what constitutes a functional cis-regulatory element.
With respect to the WRKY transcription factor DNA binding site, our study demonstrates that the previously defined W-box consensus, T/CTGACC/T (Ülker and Somssich 2004), alone is insufficient to predict that it will be bound by these proteins. Rather, as illustrated in Fig. 1, additional neighboring nucleotides also contribute in determining high affinity binding in vitro. Moreover, these nucleotides partly determine the type of WRKY factor that will be recruited. AtWRKY6 and AtWRKY11 bind well to W-boxes that have a G residue directly 5′ adjacent to the element, whereas AtWRKY 26, 38 and 43 bind to the same motif if this residue is a T, C or A. This is the first time that such discriminatory WRKY factor binding to W-boxes has been observed. Sequences carrying the hexamer, CTGACC, were not bound by any of the five WRKY representatives tested, indicating that a minimal W-box element should rather be defined as, 5′-TTGACC/T-3′. The sequence motif TTGACA has previously been referred to as a W-box-like motif (Maleck et al. 2000; Kankainen and Holm 2004; Navarro et al. 2004). However, neither our data nor earlier work (de Pater et al. 1996 #3286) lends support to this assumption. In addition, our findings imply that the hexamers TTGACC and TTGACT are not functionally identical with respect to WRKY factor binding. This observation is supported by previous studies demonstrating that the affinity of Arabidopsis ZAP1 (=AtWRKY1) to the TTGACC motif is four-fold higher than to TTGACT (de Pater et al. 1996). By using DNA regions derived from different gene promoters we were able to show that the sequence environment into which W-box elements are embedded can significantly influence protein binding. This influence cannot always be explained merely by differences in the immediately adjacent neighboring bases, indicating that additional, as of yet ill-defined parameters, also play a role. Nevertheless, based on our results, we derived at two consensus sequences that currently best define good WRKY factor binding sites (Fig. 6).
In several cases closely adjacent W-box elements have been observed in various gene promoters (Eulgem et al. 1999; Yang et al. 1999; Yu et al. 2001; Chen and Chen 2002; Marè et al. 2004; Zhang et al. 2004). In case of PcWRKY1, the presence of these multiple sites appears to have a synergistic effect on transcription (Eulgem et al. 1999). On the other hand, the barley Hv-WRKY38 factor actually requires two closely adjacent W-boxes for efficient DNA binding (Marè et al. 2004). Scanning the entire Arabidopsis genome for two W-box elements from 0 to 30 nucleotides apart revealed that there is a statistically significant enrichment of such dimers within promoter regions. However, although certain distances appear to be favored, the biological importance of this observation remains to be tested. Some WRKY family members do contain leucine zippers capable of forming homo- and hetero-dimers (Cormack et al. 2002; Robatzek and Somssich 2002; Xu et al. 2006; Shen et al. 2007) but the majority do not and very likely bind as monomers to W-box elements. Our data are in agreement with this and indicate that no synergistic binding effects are observed for the tested WRKY factors in the presence of two closely adjacent W-box elements (Fig. 3). These results are further substantiated by recent surface plasmon resonance and NMR studies demonstrating that AtWRKY4-CTD binds to the W-box element with a stoichiometry of 1:1 (Yamasaki et al. 2005) and by high-resolution crystal structure of AtWRKY1-CTD (Duan et al. 2007). One should note however, that some WRKY factors can influence expression of specific genes without directly contacting DNA. Recently, the rice WRKY factor OsWRKY51, although failing to bind itself, was shown to enhance specific binding of OsWRKY71 to the Amy32b gene promoter thereby synergistically suppressing gibberellic acid-inducibility of a tested reporter gene (Xie et al. 2006).
Another important finding is that Arabidopsis members of all three major WRKY groups show W-box specific binding both in vitro and in vivo. No subgroup specific differences were observed although this may have been expected based on the fact that group III WRKY proteins very likely utilize different basic amino acid residues within their DNA binding domains for contacting DNA phosphate groups compared to the other two groups (Yamasaki et al. 2005). These results are in accord with in silico data implying that the lineage-specific expansion of the WRKY domains in Arabidopsis has diversified primarily in terms of their expression patterns rather than in their target DNA-binding sites (Babu et al. 2006). All tested Arabidopsis WRKY factors localized to the nucleus, which is fully consistent with previous studies on different plant WRKY factors with the exception of AtWRKY46 whose localization appears to be restricted to the nucleolus (Koroleva et al. 2005).
Using an alanine scanning approach to analyze the C-terminal domain of tobacco NtWRKY-9, Maeo et al. (2001) and a site directed mutagenesis approach for the AtWRKY1-CTD (Duan et al. 2007) revealed that not all amino acid residues within the highly conserved WRKYGQK peptide stretch of the DNA binding domain are absolutely essential for W-box binding. The substitutions R→A and R→E, Y→F, G→A, Q→A and Q→K still enabled binding to the DNA element in EMSA, albeit at lower efficiencies. One apparent discrepancy between our results and those from Maeo et al. (2001) relate to the substitution of a critical cysteine involved in forming the zinc finger. Whereas this substitution completely abolished W-box binding of NtWRKY-9 (Maeo et al. 2001), this was not completely the case for AtWRKY11-CTD (Fig. 4). One plausible explanation for this difference is that no additional neighboring cysteine or histidine residues are present in the NtWRKY-9 protein, while two additional histidine residues are present in AtWRKY11-CTD, which may partly allow such a finger to form.
Our analysis, that included substitutions at other conserved amino acid positions within the WRKY domain of AtWRKY11, suggest that in most cases even significantly conservative exchanges perturbed efficient binding to the W-box (Fig. 4). This hints towards a need for a rather stringent conformational structure for high affinity binding that is also supported by the studies of Duan et al. (2007). Consistent with this assumption is the fact that an Arabidopsis mutant encoding an AtWRKY52 protein having a single amino acid insertion within the WRKY domain could no longer bind a W-box element (Noutoshi et al. 2005). It is also partly substantiated by protein structure prediction programs and by the NMR solution structure of the C-terminal AtWRKY4 DNA demonstrating that this region consists of four β-strands forming a compact antiparallel β-sheet (Yamasaki et al. 2005) with many of the introduced substitutions that negatively affect binding located within the β-sheet strands in WRKY11 CTD (for example m10–m15 and m18 in Fig. 4a). Two of the three substitutions that did not affect WRKY11 CTD binding are outside of the predicted β-strand structural elements (Fig. 4b, m7 and m16). A structural model predicts that the β-strand containing the WRKYGQK motif makes contact with a 6 bp DNA region, consistent with the length of the W-box, in the major DNA groove (Yamasaki et al. 2005; Duan et al. 2007). Verification of this model however, awaits a structural determination of the WRKY protein complexed with its DNA binding site.
The high-resolution crystal structure of AtWRKY1-C revealed an additional β-strand N-terminal to the WRKY domain that was missing in the NMR AtWRKY4-C model (Yamasaki et al. 2005; Duan et al. 2007). Within the loop between this and the adjacent β-strand containing the WRKYGQK motif, a pivotal residue, D308, was identified that forms a well-defined salt bridge with a lysine residue and extensive H-bonds with two additional amino acids. All these residues are conserved among the WRKY proteins and may be important in stabilizing this domain structure (Duan et al. 2007). These stabilizing requirement may explain why the D to E substitution in AtWRKY11 significantly affected binding of the protein to DNA (Fig. 4c, m6).
The co-bombardment assays indicate that all tested WRKY factors has intrinsic transactivation capabilities in vivo (Fig. 5). However, as illustrated by AtWRKY11, W-box binding alone is not sufficient for this function. Very likely promoter architecture as well as additional associated factors will in part determine distinct transcriptional outputs. AtWRKY11 along with its closely related family members AtWRKY7 and AtWRKY17, have been demonstrated to act as negative regulators of defense gene expression in vivo (Journot-Catalino et al. 2006; Kim et al. 2006). Thus, similar to what has been observed for AtWRKY6 (Robatzek and Somssich 2002), these factors may possess dual functionalities dependent on promoter context. No role has as yet been identified for the other here studied factors, AtWRKY26, 38 and 43.
It is evident that our studies cannot fully explain the discrete binding site selectivity of the large set of WRKY factors to their in vivo target sites. Still, as a starting point, our analysis provides important information on what actually constitutes a W-box-like element within regulatory sequences that can be predicted to be bound by certain WRKY family members. These studies therefore should help to improve whole genome in silico analyses regarding WRKY protein-DNA interactions.
We thank Dr. Aifen Zhou for providing the WRKY6-GFP and WRKY11-GFP expression constructs, and Dr. Janna Brümmer for providing the WRKY26-GFP construct. This work was supported by the DFG-funded Graduiertenkolleg für Molekulare Analysen von Entwicklungsprozessen IIIGK-GRK 306/1 and by the DFG/AFGN grants SO 235/3-2/5-4 to Imre E. Somssich.
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