Dimerization of the BASIC PENTACYSTEINE Domain in Plant GAGA-Factors is Mediated by Disulfide Bonds and Required for DNA-Binding

GAGA-binding proteins in plants are encoded by the BARLEY B-RECOMBINANT / BASIC PENTACYSTEINE (BBR/ BPC) family, which can be spilt into several groups on the basis of sequence divergence. The proteins of the different groups share an evolutionary conserved BASIC PENTACYSTEINE (BPC) domain at their very Cterminus that is important for DNA binding. Hallmark of this domain are five Cysteines at defined positions and spacing, which are considered to form a zinc-finger like structure that is involved in GAGA-motif recognition. Here, we report the formation of stabile homodimers between Arabidopsis thaliana group I member BPC1 or between group II member BPC6 in SDS-PAGE. Serial mutations of the highly conserved five Cysteines in the BPC domain of Arabidopsis thaliana BPC1 were tested for their capacity to bind to GAGA-motifs by DPI-ELISA. Our results do not support the idea of a direct involvement of these residues in making physical contact with the DNA, e.g. by formation of a zinc-finger structure. Instead, the data implies an indispensable function for the five Cysteines in homodimerization and stabilization of the protein structure by disulfide bonds. Accordingly, protein folding and structure prediction suggests the formation of a scaffold for dimerization that is supported by three intermolecular and one intramolecular S-S bond. The high degree of conservation between the BPC domains from the different groups and from different species denotes that this role for the five Cysteines might be evolutionary retained. DOI : 10.14302/issn.2638-4469.japb-17-1563 Corresponding author: Dierk Wanke; Center for Plant Molecular Biology (ZMBP) Plant Physiology; University of Tübingen Auf der Morgenstelle 32; 72076 Tübingen, Germany; e-mail: dierk.wanke@zmbp.uni-tuebingen.de Running title: BASIC PENTACYSTEINE domain

One hypothesis is that these diverse functions of animal GAFs rely on explicit protein-protein interactions with partners that confer process-dependent specificities [3,9].For example, both Trl and Psq are involved in the sequence-specific recruitment of Polycomb Repressive

Protein Expression
The open reading frames of BASIC PENTACYSTEINE 1  'Rubber bands' were removed and several rounds of 'repack' and 'minimize all' (behavior: start at 0.3 -end at 1.0; wiggle power: medium) was applied.The dimer model structure was entirely 'unfrozen' and several rounds of "repack" and "minimize" were applied on the "unfreeze all' (wiggle power: medium).The setting behavior was gradually increased (start at 0.3 -end at 1.0) to prevent breaking of the freshly formed bonds.
The resulting dimer model structure was exported as PDB file and imported into PyMOL for illustration.

Results
The Arabidopsis thaliana BBR/BPC family consists of

Introduction
GAGA-motif binding factors (GAF) are indispensable eukaryote transcription factors that act through diverse molecular mechanism during growth and development on homeotic gene expression.Trithorax-like (Trl) and Pipsqueak (Psq) protein families are the representatives of animal GAFs, which possess polyvalent functions in activation and repression of gene expression [1-6].

Complex 1 ( 1 (
PRC1) or PRC2 components to Polycomb Repressive Elements (PREs), which leads to trimethylation of Lys27 in Histone 3 (H3K27me3) [4-6, 9-13].In plants, the three groups of the BARLEY B-RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) protein family show GAGA-motif binding properties [14-17].They are important developmental regulators that are involved in gene expression control mainly of transcription factor genes and influence ethylene, cytokinin, abscisic acid or auxin signaling [18-21].Although plant and animal GAFs constitute unrelated protein families [14, 16, 22], it was hypothesized that their mode of action at PRE-like motifs might display similarities [15].Indeed, the mechanism of repression of homeotic genes displays striking functional homology: Just like Trl or Psq in animals, the plant BBR/BPC proteins play a role in gene silencing by interaction with repressive complexes.For example, LHP1) to GAGA-motif containing PREs in the promoters of homeotic genes [18].This interaction presumably leads to an association of BPC6 and LHP1 with VERNALIZATION 2 (VRN2), which is a component of the Polycomb Repressive Complex 2 (PRC2) and responsible for H3K27me3 establishment [18, 23].These findings might explain that GAGA-motifs are associated with repressive H3K27me3 modifications close to the transcription start sites [15, 18, 24-26].Likewise, A. thaliana BPC1, a group I BBR/BPC member, interacts with the SEUSS (SEU) -LEUNIG (LUG) transcriptional cosuppressor complex to control the repression of the homeotic SEEDSTICK (STK) locus [27].Recent data suggest a direct interaction of PRC2 component SWINGER (SWN) with BBR/BPC members of both groups, to repress the expression of the transcription factor gene ABSCISIC ACID INSENSITIVE 4 (ABI4) [20].These repressive mechanisms depend on the specific recruitment of protein complexes to defined chromatin loci in the plant genomes by different groups of BBR/BPC members.Hence, a functional analysis of the DNA-binding mechanism is important to understand the specificity of this recruitment.The different BBR/BPC groups differ by their structural features at the N-terminus and the central region of the proteins, which presumably harbor dimerization domains or nuclear and nucleolar localization signals [15-17].Only the BPC DNA-binding domain at the very Cterminus of the proteins is evolutionary conserved and displays a high degree of sequence similarity that extends over the group's boundaries [15, 16, 22].The hallmark of that BPC domain is the presence of five Cysteine residues, which are highly conserved in position or spacing [15-17].These Cysteines are believed to form a zinc-finger like structure and to make direct physical contact with the GAGA-tetranucleotide [17].The purpose of this study was to examine the function of these five conserved Cysteines in Arabidopsis thaliana BPC1 and their role in GAGA-motif recognition by DPI-ELISA or protein structure prediction.

For
3D protein structure prediction the sequence of the BASIC PENTACYSTEINE domain of BPC1 (aa 185 -aa 283 ) was loaded into I-TASSER [34-37].The PDB file of the selected model was subsequently used as input for the standalone version of FoldIt [38-41].The tools 'repack'and 'minimize all' were applied to the monomeric structure (settings: behavior: start at 0.3 -end at 1.0; wiggle power: medium).After several rounds of relaxation, the derived 3D structure was imported as PDB file into PyMOL (http://www.pymol.org).To generate a dimeric model structure, the monomer was duplicated in PyMol.Each monomer of the resulting dimer model was turned and positioned towards each other.Both molecules were exported as PDB file and again imported into FoldIt.'Rubber bands' were applied to the opposing Cysteine residues.To influence unwanted moving in the process, 'freeze' was applied to the entire dimer model, except for the four amino acids surrounding the opposing Cysteine residues.These residues were brought close to each other with the "drag" tool to enable the formation of disulfide bonds.
three groups with seven members in total[15][16][17].The similarities between these proteins are restricted to the BASIC PENTACYSTEINE DNA-binding domain[17].Sequence alignment between A. thaliana group I member BPC1 and BPC6, a group II member, discloses 75 % similarity and 56 % identity over the 99 amino acid long BPC DNA-binding domains (Fig.1A).Two regions of the BPC domains are of highest conservation:On the one hand side, the N-proximal part containing the five conserved Cysteines.On the other hand, a sequence at the very C-terminus, which displays a WA R/K HGTN motif at its center.To investigate the DNAbinding properties by DPI-ELISA, we expressed BPC1 and BPC6 as recombinant His-epitope tagged proteins.By immunological analyses of the crude total protein extracts, we found that both BPC1 and BPC6 formed stabile dimers in SDS-PAGE (Fig.1B).Specific signals exclusively at the molecular weight of the dimers were evident.No signals were detectable at molecular weight of the monomers.We next performed DPI-ELISA experiments with both protein extracts.A strong binding to the positive dsDNA-probe K4 was detected for both recombinant proteins (Fig. 1C).No significant interaction with the negative control dsDNA-probe Kneg was observed.Control extracts from BL21/RIL cells and control wells without dsDNA-probe showed only minor background signals.Given the strong homotypic dimerization (Fig. 1B), one can propose that exclusively BPC-dimers interacted with GAGA-tetranucleotide motifs in the DPI-ELISA.A characteristics of all BBR/BPC proteins is the BASIC PENTACYSTEINE domain that is crucial for DNA-binding [17].The main features of this domain are the highly conserved Cysteines (Fig. 2A).To examine the contribution of the five Cysteines in DNA-binding, we conducted site-directed mutagenesis on Arabidopsis thaliana BPC1 to replace selected Cysteines with Glycine.Six different mutant versions of BPC1 were made.In addition, we generated two truncations of BPC1 (Fig. 2B): BPC1_DBD comprises the conserved BPC domain (aa 185 -aa 283 ), while BPC1_short contains only the Cproximal part of the domain starting just after the fifth conserved Cysteine (aa 218 -aa 283 ).All BPC1 versions were expressed as recombinant His-epitope tagged proteins, which can be detected by immunoblotting (Fig. 2C).Interestingly, all signals were approximately double the expected size, which is indicative of putative dimer formation.Also both of the truncations displayed a much higher molecular weight than expected for the monomers.The DPI-ELISA experiment uncovered that simultaneous mutation of Cys 195 and Cys 197 (BPC1_mut1) did not affect DNA-binding capacity significantly compared with Freely Available Online www.openaccesspub.org| JAPB CC-license DOI : 10.14302/issn.2638-4469.japb-17-1563Vol-1 Issue 1 Pg.no.-30 wildtype BPC1 (Fig. 2D).In contrast, the replacement of the single Cys 204 with Glycine (BPC1_mut2) reduced binding drastically, which is indicative of an important contribution of this Cysteine to GAGA recognition.Only a minor decrease in binding to GAGA-motifs was found with BPC1_mut3, where Cys 216 and Cys 217 were mutated.A similar, but insignificant decrease was discovered with a mutation of a Cysteine, which does not belong to the conserved PENTACYSTEINEs (BPC1_mut4).Surprisingly, a huge increase in signal intensity was repeatedly detected with BPC1_mut5 extracts.Here, four of the conserved Cysteines were mutated and only C 204 was retained.The result, however, is complementary to the very low binding data with extract BPC1_mut2.Consistently, BPC1_mut6 was derived by mutation of Cys 204 from BPC1_mut5 and displayed very low binding affinity to the dsDNA-probes that is similar to BPC1_mut2.Interestingly, BPC1_DBD and BPC1_short

Figure 1 .
Figure 1.Comparison of group I BPC1 and group II BPC6.(A) Protein sequence alignment of the BASIC PENTACYSTEINE DNA-binding domains.Identical residues are highlighted by grey background.Positions that are evolutionary retained in all BBR/BPC family members are indicated by asterisks (*) above the alignment.The highly conserved Cysteines are emphasized by black background.The conserved WA R/K HGTN signature is indicated by red letters.(B) Gel-blot experiments with immunological detection of the recombinant proteins.The expected molecular weights for monomer (*) and dimer (**) proteins are indicated.Arrows point to unspecific bands detected also in control extracts with anti-His antibody.(C) Specific binding of epitope tagged BPC1 and BPC6 to positive (K4) and negative (Kneg) dsDNA-probes in DPI-ELISA experiments.The histogram bars show normalized signal intensities and error bars represent one standard deviation.Grey background shading indicates level of confidence for significant binding (t-test p < 0.05).Representative wells of the microtiter plate are shown below the graph for visual inspection.

Figure 2 .
Figure 2. Binding capacity of BPC1 mutants.(A) Versions of BPC1 with mutations of the Cysteines in the BASIC PENTACYSTEINE DNA-binding domain.(B) Schematic overview of all 6xHis-epitope tagged BPC1 mutants and truncations.The highly conserved Cysteines are highlighted by yellow boxes.The position of the conserved WA R/K HGTN signature is indicated (red).Mutations in Cysteines are shown as crosses.(C) Gel-blot experiments with immunological detection of all recombinant proteins.(D) Specific binding of 6xHis-epitope tagged BPC1 versions to positive (K4) and negative (Kneg) dsDNA-probes in DPI-ELISA experiments.The histogram bars show normalized signal intensities and error bars represent one standard deviation.Grey background shading indicates level of confidence for significant binding (t-test p < 0.05).The bars annotated with the same letter are not significantly different.

Figure 3 .
Figure 3. Binding capacity of BPC1 under oxidizing or reducing buffer conditions.Specific binding of 6xHis-epitope tagged BPC1 to positive (K4) or negative (Kneg) dsDNA-probes in DPI-ELISA experiments.The histogram bars show normalized signal intensities and error bars represent one standard deviation.Grey background shading indicates level of confidence for significant binding (t-test p < 0.05).The bars annotated with the same letter are not significantly different.(A) Binding capacity under oxidizing conditions.Protein dilution buffer was supplemented with 5% (v/v) H 2 O 2 immediately before the experiment.Asterisks indicate a significant decrease of binding (t-test p < 0.001) by ~ 45%.(B) Analysis of reducing capacity of DTT on DNA-binding.The amount of 5 mM DTT was reduced to 0 mM DTT in the protein extraction buffer (DTT extr. ) or the protein dilution buffer (DTT bind. ) or in both of the buffers.(C) Effect of 100µM ZnCl2 or 100µM EDTA supplements on DNA-binding.

Figure 4 .
Figure 4. Model structure of the BPC1 DNA-binding domain.The monomeric protein model structure was predicted from I-TASSER, relaxed in FoldIt and surface was illustrated in PyMOL.Cysteines at the surface are highlighted in yellow color.The five conserved Cysteine residues are depicted as ribbons.The conserved WA R/ K HGTN signature is shown in red color.The intramolecular disulfide bond between Cys 204 and Cys 217 is indicated.

Figure 5 .
Figure 5. Proposed dimer model structure of two interacting BPC1 DNAbinding domains.(A) Two monomer models were organized as homotypic dimers in PyMOL.Structural modeling and relaxation on the dimer model structure was performed in FoldIt.The surface of each monomer is shown in green or in blue.Cysteines at the surface are highlighted in yellow color.The five conserved Cysteine residues are depicted as ribbons.The conserved WA R/K HGTN signature is shown in red color.(B) Close-up view on the inter-and intramolecular disulfide bonds that are formed in the structural model.Ribbons and labels are displayed in either green or blue to specify the contribution of the monomers.Disulfide bonds are shown in yellow.
Transient bimolecular fluorescence complementation (BiFC) experiments with a BPC6 version that lacks the C-terminus in Nicotiana benthamiana demonstrated that the BPC DNA-binding domain and its conserved Cysteines were not required for homotypic dimerization[16].Spectro-microscopic analysis with FRET-FLIM consolidated a parallel dimer conformation in vivo[16], which is consistent with our data presented in this manuscript on the parallel orientation of the BPC-domain dimers, which are essential for disulfide bond formation.Experiments with group I member BPC1 discovered bands that were shifted and supershifted in EMSA, which is an indication for dimer formation in vitro[17, 20,49].Unfortunately, the use of longer DNA-probes with extended GA/TCrepeats might give inconclusive results, as a single GAGA -tetranucleotide is sufficient for BBR/BPC-binding in vitro[29, 33]: The simultaneous binding of several BBR/BPC proteins independently to the same DNA-probe or the binding of dimers to neighboring sites might provide identical outcomes[17, 20, 33,49].All these data suggest, however, the formation of higher order complexes of BBR/BPC proteins at GAGA-motifs.The high degree of conservation of the five Cysteines in all BBR/BPC family members proposes a conserved mechanism.In the light of our recent findings, we propose that these residues might possibly constitute a general interaction surface between BBR/BPC proteins, which implies a possible dimer formation also between members of different groups.Indeed, there is preliminary data that heterotypic dimerization between different group members might occur.A weak but significant heterodimer formation between Arabidopsis BPC1 and BPC6 was found in yeast two-hybrid experiments[16].This interaction, however, could not be consolidated in BiFC experiments in planta[16].In contrast, spectro-microscopic analyses with ectopically overexpressed BPC1 and BPC6 in heterologous Nicotiana benthamiana cells suggest a very close association of both proteins in the nucleus[50].The proportion of the two proteins, however, that actually underwent a possible heterotypic dimerization compared with those forming homodimers in planta was not resolved the study.Hence, these reports on heterotypic dimerization between members of different groups might constitute artifacts due to ectopic overexpression.To clarify the dynamics between the different groups of BBR/BPC proteins in the cell and their mechanistic function needs further experiments on these issues in the future.ConclusionOur serial analysis of mutants in the conserved BASIC PENTACYSTEINE DNA-binding domain contradicts the Freely Available Online www.openaccesspub.org| JAPB CC-license DOI : 10.14302/issn.2638-4469.japb-17-1563Vol-1 Issue 1 Pg.no.-37 idea of a zinc-finger-like DNA-binding mechanistic.Instead, we propose that the conserved Cysteines form a scaffold for homotypic dimerization of BBR/BPC proteins under native conditions.Inter-and intramolecular disulfide bonds stabilize a parallel conformation of the monomers.Such a conformation will consequentially lead to the recognition and binding of neighboring GAGA -motifs, which will have tremendous impact on target loci selection in vivo.