Studies on Differential Nuclear Translocation Mechanism and Assembly of the Three Subunits of the Arabidopsis thaliana Transcription Factor NF-Y

Molecular Plant Advance Access published December 22, 2011 Molecular Plant

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Pages 1–13, 2011

RESEARCH ARTICLE

Studies on Differential Nuclear Translocation Mechanism and Assembly of the Three Subunits of the Arabidopsis thaliana Transcription Factor NF-Y Dieter Hackenberg, Yanfang Wu, Andrea Voigt, Robert Adams, Peter Schramm and Bernhard Grimm1 Institute of Biology/Plant Physiology, Humboldt University, Philippstr. 13, Building 12, 10115 Berlin, Germany

Key words: interaction.

gene expression; transcriptional control and transcription factors; nuclear translocation; protein–protein

INTRODUCTION The eukaryotic CCAAT-box binding nuclear factor Y (NF-Y) consists of three different subunits, A, B, and C. The three heterologous human NF-Y subunits and their homologous subunits in yeast, HEME ACTIVATED PROTEIN (HAP) 2, 3, and 5, are encoded by a single gene, respectively (Mantovani, 1999). In addition, yeast possesses a fourth subunit, HAP4, which carries a transactivation domain and is required for the functional protein complex (Forsburg and Guarente, 1989; McNabb and Pinto, 2005). In contrast, several filamentous fungi species, inter alia Aspergillus nidulans, express the HapX protein, which interacts with the trimeric NF-Y complex (Tanaka et al., 2002), but shows only low similarity to the Saccharomyces cerevisiae HAP4 subunit (Hortschansky et al., 2007; Schrettl et al., 2010). The transcriptional activation of the human NF-Y complex is distributed on N- and C-terminal domains of NF-YA and NF-YC (Coustry et al., 1996; de Silvio et al., 1999). The plant NF-Y subunits are encoded by multigene families. In Arabidopsis thaliana, each gene family possesses 10, 13, and 13 genes for the

A, B, and C subunits, respectively (Gusmaroli et al., 2001, 2002; Siefers et al., 2009). A plethora of different compositions of the heterotrimeric NF-Y complex is hypothesized in Arabidopsis (Siefers et al., 2009). In total, 1690 different combinations consisting of always one member of each of the three subunit families can theoretically be formed. This combinational variety enables the specific control of a large number of CCAAT-box containing target genes by the 36 representatives of NF-Y subunits in Arabidopsis. Moreover, the transcriptional control of the same target gene can be modulated due to the different combinations of heterotrimeric NF-Y complexes binding to the corresponding promoter element. 1 To whom correspondence should be addressed. E-mail Bernhard. [email protected], tel. +49 30 20936119, fax +49 30 20936337.

ª The Author 2011. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: 10.1093/mp/ssr107 Received 23 August 2011; accepted 26 November 2011

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ABSTRACT The eukaryotic transcription factor NF-Y consists of three subunits (A, B, and C), which are encoded in Arabidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combinations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NFYB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.

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RESULTS

NF-YA2, NF-YA4, NF-YB3, NF-YB10, and NF-YC2 were expressed with a C-terminal eGFP fusion under the control of the 35S CaMV promoter in Arabidopsis leaves after transformation via particle bombardment. NF-YA2, NF-YA4 (data not shown), and NF-YC2 were exclusively detected in the nucleus of the transformed leaf cells (Figure 1A and 1C). In contrast, the two NF-YBs, NF-YB3 (data not shown) and NF-YB10, are found to be localized in the cytoplasm (Figure 1B). The subcellular localization of NF-YB was modified after co-expression of 35S CaMV::NF-YC9. Upon concomitant cotransformation and expression of NF-YC9 and NF-YB3–GFP (data not shown) or NF-YB10–GFP fusion proteins, NF-YB dominantly accumulated inside the nucleus of transformed cells (Figure 1D). In contrast to NF-YA and NF-YC, all NF-YBs lack NLS. NF-YBs are proposed to be transported into the nucleus after interaction with NF-YC, while NF-YA and NF-YC are imported into the nucleus without auxiliary assistance. In conclusion, the interaction between NF-YB10 and NF-YC9 in the cytoplasm is an essential prerequisite for the translocation of NF-YB into the nucleus and follows a piggyback mechanism. The physical interaction of NF-YB10 as well as NF-YB3 with NF-YC9 was additionally demonstrated (1) in vitro by performing pull-down assays (Figure 2A), (2) in planta using the bimolecular fluorescence complementation (BiFC) method (Figure 3), and (3) in vivo by yeast two-hybrid experiments (Figure 2B; see the next section and Figure 4). In one experimental approach, recombinant GST-fused NF-YC9 was immobilized on glutathione beads and subjected to in-vitro translation extracts with S35-labeled NF-YB3 and NF-YB10. NF-YB10 (Figure 2A) and NF-YB3 (data not shown) were released from the glutathione beads in substantially higher amounts after interaction with GST–NF-YC9 than without this interaction partner. In planta, NF-YB3–YFPN and NF-YB10–YFPN fusions as well as NF-YB3–YFPC and NF-YB10–YFPC fusion proteins were able to interact with NF-YC9–YFPC and NF-YC9–YFPN, respectively, resulting in the reconstitution of the YFP fluorescence signal (Figure 3). The subcellular localization of the heterodimers NF-YB3/NF-YC9 and NF-YB10/NF-YC9 dimers was predominantly inside the nucleus. YFP fluorescence did not merge with the CFP fluorescence emitted by the CFP-tagged-ER marker CD3-953, which was co-transformed with the NF-Y fusion gene constructs. As control, any combination of NF-YB3, NY-B10, or NF-YC9 as YFPN or YFPC fusion protein with the empty corresponding non-recombinant YFPN and YFPC containing vector did not result in detectable YFP fluorescence (Supplemental Figure 1).

Cellular Localization of NF-YA, B, and C in Arabidopsis

Bilateral Protein–Protein Interactions among Members of the Arabidopsis NF-Y Subunit Families

The entire NF-Y complex of Aspergillus nidulans was reported to be translocated into the nucleus (Steidl et al., 2004). It remains open whether this translocation mechanism can also be established for the multiple NF-Y complexes in plants. We intended to demonstrate the subcellular localization of representatives of the three NF-Y subunits from Arabidopsis.

Following the hypothesis of combinatorial diversity of Arabidopsis NF-Y subunits, we assayed the bilateral interactions of Arabidopsis NF-Y proteins as bait and prey fusions in the diploid yeast cells via the expression of the yeast two-hybrid reporter genes HIS and lacZ before the potential combinations of each subunit in a heterotrimeric complex is assessed. A maximum of

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One of the challenging questions is how control of expression and assembly of the different subunits ensures their nuclear functions. Transcript analysis revealed that all of the NF-Y genes display a differential expression pattern during development and as a response to environmental stimuli (Gusmaroli et al., 2001, 2002; Hruz et al., 2008). It is proposed that specific expression pattern of each NF-Y gene and interactions among the complementary subunits are substantial for the specific function of NF-Y complexes in transcriptional control. The focus of our intention was to study the potential of protein– protein interactions of the plant NF-Y. The accurate interaction of two NF-Y subunits depends on the complementary interaction of the corresponding protein. Hypothetically, formation of multiple NF-Y heterotrimers is facilitated with a different strength in binding affinity between individual subunits. To understand the interactomic network of the NF-Y complex, we verified the combinational variety of bilateral interactions between all Arabidopsis NF-Y proteins in yeast two-hybrid experiments. Moreover, we extended our prediction on the interconnection among the NF-Y subunits by analysis of the assembly of complete tripartite NF-Y complexes. An additional precondition for formation of functional NF-Y complexes is the translocation of the subunits into the nucleus. As reported for the filamentous fungus Aspergillus nidulans, the complete heterotrimeric NF-Y complex is translocated into the nucleus by a piggyback transport (Steidl et al., 2004). Only HapB (the homolog to Arabidopsis NF-YA) possesses a nuclear localization signal (NLS), whereas HapC and HapE (homologous to NF-YB and NF-YC) lack this domain (Thon et al., 2010). The interaction of HapB with the other subunits requires the combined protein surface of the HapC/HapE heterodimer, as it was also described for rat and yeast (Sinha et al., 1995; McNabb and Pinto, 2005). First, the formation of an initial heterodimer occurs via a head-to-tail annealing of the HFMs of HapC and HapE, which is comparable to the formation of the histone 2A/2B dimer (Liberati et al., 1999; Romier et al., 2003). Then, the HapC/HapE heterodimer assembles with HapB, before the translocation of these subunits is possible into the nucleus (Thon et al., 2010). It is not known, whether complete Arabidopsis NF-Y complexes will be targeted to the nucleus to achieve availability of each subunit in the correct stoichiometric amounts. We examined the translocation mechanism into the nucleus of the three different Arabidopsis subunits.

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Figure 1. Detection of the Subcellular Localization of NF-Y Proteins in Arabidopsis Leaves. NF-Y–eGFP-gene fusions under the control of a 35S::CaMV promoter were used for transient transformation via particle bombardment. The GFP fluorescence was detected by confocal laser scanning microscopy (left row). Location of the nucleus and cell walls was identified after DAPI staining and bright field microscopy, respectively (central rows). Whereas NF-YA2 (A) and NF-YC9 (C) were nuclear-located, NF-YB10 (B) is distributed in the cytoplasm. NF-YB10 is translocated to the nucleus by co-expression of NF-YC2 (D). The right row displays the merge of green fluorescence and the DAPI stain.

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Figure 2. Detection of Protein–Protein Interaction among NF-YB10 and NF-YC9 In Vitro and In Vivo.

666 different combinations of two interacting Arabidopsis NF-Y proteins can be achieved with the total number of 36 members
36  P within the three NF-Y subunit families k=666 . Considerk =1 ing that the coding sequence of each of the 36 Arabidopsis NF-Y subunits is inserted in bait and prey fusion gene constructs, respectively, the proteins can be combined in 1296 different possibilities in a yeast two-hybrid approach. In total, 1224 of the 1296 possible combinations were analyzed in our yeast two-hybrid experiments (Table 1 and Figure 4). The total number of combinations was reduced, because four proteins were not considered for the readout. First, NF-YB12 and NF-Y B13 annotated also as TATA-binding protein-associated phosphoproteins DR1-like proteins were only functional as prey fusions in the yeast two-hybrid experiments and could not be investigated in the opposite combination as a bait fusion protein. Second, NF-YA5 and NF-YC5 evidently did not interact with any other protein in the whole interactome approach. For the evaluation of our interactome assay, protein–protein interactions are accepted as positive, when the protein–protein interactions were reliably induced b-galactosidase levels over 0.2 (Figure 4). Ninety-two combinations were determined in only one combination of bait and prey constructs, whereas 56 interactions among two NF-Y proteins could be proofed in both reciprocal bait/prey combinations resulting in total in 204 protein–protein interactions through unidirectional and reverse combination of bait and prey vectors. Table 1 displays the survey of the NF-Y interactome and Figure 3 highlights the positive protein–protein interactions among the three NF-Y subunits. An additional criterion for reproducible protein–protein interaction was included. Only protein–protein interactions were accepted when they were verified in our yeast two-hybrid experiments in both reciprocal combinations. Based on these settings, 95% of all established bilateral interactions (53 of 56) were determined among proteins of the NF-YC and NF-YB families. Two combinations of NF-YA and NF-YC proteins displayed positive interactions in both

Formation of NF-YB/C Heterodimers In total, 53 of 169 possible bilateral Arabidopsis NF-YB and NF-YC interactions were detected in both combinations of bait and prey fusions in the yeast two-hybrid experiments. In particular, 10 NF-YB and seven NF-YC proteins interact in a high number of combinations to form different heterodimers, whereas three NF-YB (NF-YB11, B12, and B13) and five NF-YC proteins (NF-YC7, C8, C10, C11, and C13) interacted only with a considerably smaller number or none of the members of the other subunit family (Figure 4). To suggest reasons for their inability to form bilateral interactions, we compared the primary amino acid sequence of each member within the NF-Y families of B- and C-subunits in a sequence alignment (Figure 5). Considering in addition the three-dimensional crystal structure of the human NF-YB/C heterodimer, both proteins dimerise by anti-parallel annealing of their central HFMs, similar to the mechanism proposed for histone subunits (Arents and Moudrianakis, 1995; Romier et al., 2003). Comparison of the peptide sequences of the HFMs of the Arabidopsis NF-YB proteins revealed the high similarity (58–72%) to their human ortholog (Figure 5B) except for the HFMs of NF-YB11, B12, and B13, which show a lower similarity (27–36%). This dissimilarity of the HFM interface of NF-YB11, B12, and B13 to NF-YC could explain the restriction of these three B-subunits to interact with NF-YC proteins. As mentioned above, five NF-YC proteins (NF-YC7, C8, C10, C11, and C13) were remarkably limited in their capability to bind NF-YB proteins. Three of them (NFYC10, C11, and C13) show a low similarity (24.7–34.7%) in their conserved HFMs in comparison to the HFM of the human NF-YC subunit (Figure 5C). It is proposed that this lower similarity caused a reduced structural integrity of the HFM and a lower binding capacity to the corresponding NF-YB subunits. According to the three-dimensional-structure model of the human NF-YB/YC heterodimer (Romier et al., 2003), each subunit possesses an intramolecular hydrogen bond within the HFM between the Arg108 and Asp115 of the human NF-YB and Arg93 and Asp100 of NF-YC. Both arginine–aspartate pairs are highly conserved throughout evolution and can be found in NF-YB and NF-YC subunits of several species. The arginine–aspartate pairs

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[35S]-methionine-labeled NF-YB10 can be pulled down from a GST::NF-YC9 matrix (A) (right lane) but not from a GST matrix (left lane). The interaction among NF-YB10 and NF-YC9 can be detected in the yeast two-hybrid system by the activation of the reporter genes HIS3 and lacZ, which lead to an improved growth on SC-HLTU media (B) (upper panel) and increased b-galactosidase activity (lower panel) compared to the control experiment (expression of NF-YB10 and an empty prey vector).

reciprocal combinations of bait and prey vectors, whereas no interaction among a single NF-YA and NF-YB could be established. Related to the maximum number of possible NF-YB/NF-YC combinations, 31% of all possible interactions among each member of the two subunit families were verified with the yeast two-hybrid system. Only two out of 130 possible NF-YA/NF-YC interactions were detected in our interactome assay and no NF-YA/NF-YB interaction. Moreover, except for the combination NF-YB4/NF-YB7, no single interaction among members of the same NF-Y subunit family, as well as no homodimerization of Arabidopsis NF-Y proteins, could be verified (Table 1 and Figure 4), indicating that the NF-Y subunits do not tend to associate with other members of the same subunit family or to form homodimers.

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For specification of the specific bimolecular interaction of the NF-Y subunits in the nucleus, endoplasmic reticulum that also surrounds the nucleus was visualized by co-transformation of a specific CFP-fused ER-marker (ABRC stock: CD3-953). Detection of the YFP (left row), CFP (second row from the left side), merge of YFP and CFP fluorescence (second row from the right side), and bright field microscopy (right row) of NF-YC9_YFPN/NF-YB10_YFPC/CD3-953_CFP (A), NF-Y B10_YFPN/NF-Y C9_YFPC/CD3-953_CFP (B), NF-YC9_YFPN/NF-YB3_YFPC/CD3-953_CFP (C), and NF-Y B3_YFPN/NF-Y C9_YFPC/CD3-953_CFP (D) are displayed. NF-YB3 as well as NF-YB10 interact with NF-YC9. The resulting dimer formation is localized inside the nucleus.

consequently seem to be important for the mutual interaction between both NF-Y subunit families. It is suggested that the peptide motif is functional in stabile structural conformations of NF-YB and NF-YC through the HFMs (Romier et al., 2003). Sequence alignments revealed, with the exception of NF-YB12 and B13, that all Arabidopsis NF-YB subunits possess the highly conserved aspartate and arginine residues at the corresponding positions in their HFMs. This structural consistency correlates with the abilities of NF-YB subunits to interact with NF-YC subunits. All Arabidopsis subunits of the NF-YC family, which interact with a large number of NF-YB subunits,

also contain the two highly conserved amino acid residues in their HFM. The remaining members of the NF-YC family, which are characterized with a lower NF-YB binding capacity in our yeast two-hybrid experiments, like NF-YC8, C10, C11, and C13, have a substitution at least at one of the conserved aspartate or arginine residues. NF-YC7 is characterized by a specific feature of an additional insertion of six amino acid residues in the HFM bordered by the conserved aspartate and arginine residues. In conclusion, these sequence comparisons suggest an essential role of the intramolecular H-bond formation formed by conserved amino acid residues in the HFM of Arabidopsis

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Figure 3. BiFC Visualization of Arabidopsis NF-YB10-NF-YC9 and NF-YB3-NF-YC9 Dimers in Nicotiana benthamiana Leaf Epidermis Cells after Agrobacterium-Mediated Transient Transformation.

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Table 1. Statistics of the Performed Yeast Two-Hybrid Analysis of Bilateral Interactions among Arabidopsis NF-Y Proteins.

Used NF-Y proteins AtNF-Y

Number of possible combinations

Number of tested combinations (total/positive)

Number of possible interactions in plants

Number of detected interactions (uni/bi–directional)

Percentage of proven interactions (bi–directional) 8

1296

1224/204

666

92/56

AtNF-YA

620

600/46

315

42/2

1

AtNF-YB

767

695/158

390

50/54

14

AtNF-YC

767

741/193

390

87/55

14

AtNF-Y A/A

100

100/0

55

0/0

0

AtNF-Y A/B

260

240/3

130

3/0

0

AtNF-Y A/C

260

260/43

130

39/2

2

AtNF-Y B/B

169

143/8

91

6/1

1

169

41/53

31

91

3/0

0

AtNF-Y B/C

338

312/147

AtNF-Y C/C

169

169/3

NF-YB and NF-YC subunits for the capability of protein–protein interaction.

NF-YA Requires the Combined Protein Surface of the NF-YB/C Heterodimer The evaluation of successful interactions between NF-YB and NF-YC subunits by yeast two-hybrid experiments revealed a substantial list of candidates of NF-YB und NFYC subunits (Figure 4). The final assembly of heterotrimeric

complexes of plant NF-Y requires stable interaction of NF-YB and NF-YC proteins with the NF-YA subunit. We assessed positive interactions of three different NF-Y subunits in a modified yeast two-hybrid approach. After transformation of yeast cells with NF-YA/NF-YB prey and bait gene fusions, the additional vector construct pVT-U102::NF-YC2 was introduced. Our yeast-two hybrid experiments showed that NF-YA2 and NF-YA4 did not interact with NF-YB10 independently from their respective expression as bait or prey

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Figure 4. Overview about the Bilateral Interactions among Two NF-Y Proteins Tested in Yeast Two-Hybrid Experiments. The mean b-galactosidase activities of three biological replica of diploid yeast cells possessing bait and prey constructs for the expression of singular NF-Y proteins were plotted as a color-coded diagram. The used bait constructs are positioned on the x-axis and the prey constructs on the y-axis. Successful protein–protein interactions were plotted as heat color-encoded mean values of all three biological replicas exceeding 0.2 relative b-galactosidase activity units. Interaction among two NF-Y proteins, which were only detectable in one of both reciprocal bait/prey combinations were plotted as different intense gray shades of color.

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Figure 5. Sequence Alignments among the Highly Conserved Domains of the Arabidopsis thaliana, Saccharomyces cerevisiae, and Homo sapiens NF-Y Subunits. Functional domains are labeled in black boxes above the sequence alignment of NF-YA (A), NF-YB (B), and NF-YC (C). Conserved cysteine residues of NF-YB subunits are labeled as C1, C2, and C3 (red) and putative H-bonds between arginine and aspartate are labeled with two black arrows (Romier et al., 2003). Amino acid residues highlighted yellow are strictly conserved in all representatives of the corresponding subunit family, turquoise-labeled amino acids are identical at those positions, and green-labeled amino acid residues possess side chains with similar biochemical properties.

fusion-peptide (Figure 4). By means of additional expression of the recombinant NF-YC2, the interaction between NF-YA2 and NF-YB10 as well as NF-YA4 and NF-YB10 was shown by the successful expression of HIS3 and lacZ reporter genes (Figure 6). As already previously shown for the assembly of the trimeric human NF-Y complex in vitro (Mantovani, 1999), the initial formation of NF-YB/NF-YC heterodimer is predicted to be

obligatory for a stable interaction of Arabidopsis NF-YA with the other two subunits. In accordance with a stepwise assembly mechanism described for NF-Y assembly of Aspergillus nidulans, Saccharomyces cerevisiae, and Homo sapiens (Steidl et al., 2004; Kahle et al., 2005; McNabb and Pinto, 2005), the plant NF-YA apparently requires the combined protein surface of the NF-YB/NF-YC dimer.

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Diploid yeast cells possessing NF-YA and NF-YB as bait and prey constructs did not show any activation of the yeast two-hybrid reporter genes HIS3 and lacZ. Co-transformation of these diploid yeast cells with a gene encoding a NF-YC subunit that was fused neither to a Gal4AD nor a LexA-BD of the yeast two-hybrid system enables the corresponding yeast cells to grow on SC-HLTU media (A) and exhibit significantly increased b-galactosidase activity (B).

Nuclear Translocation of NF-Y in Plants Is Not Regulated by Cellular Redox A piggyback transport of the complete heterotrimeric NF-Y complex into the nucleus was reported for the filamentous fungus Aspergillus nidulans (Steidl et al., 2004). Thereby, HapC and HapE (homologous to NF-YB and NF-YC, respectively) form first a heterodimer by head-to-tail annealing of their HFMs. An intramolecular disulfide bond between two cysteine residues inside the HFM of HapC controls the heterodimerization. Under oxidizing conditions, this disulfide bond was proposed to affect structurally the three-dimensional conformation of the HFM of HapC and to prevent heterodimerization. Aspergillus nidulans conclusively possesses a redox-depending molecular switch, which controls the spatial distribution of subunits and subcellular localization of the trimeric HAP (NF-Y) complex (Thon et al., 2010). We assessed the probability of intramolecular disulfide bonding of Arabidopsis NF-YB, which could affect the NF-YB/NF-YC heterodimer formation in a redox-dependent manner and their piggyback transport into the nucleus. All Arabidopsis NF-YB subunits have two conserved cysteine residues in the HFM at the positions Cys64 and Cys80 (referred to as NF-YB10; Figure 7B), which correspond to Cys89 and Cys105 of human NF-YB (Figure 5) and Cys78 and Cys84 of A. nidulans HapC (Thon et al., 2010). The NF-YB of Homo sapiens and A. nidulans possess an additional conserved cysteine separated by only three amino acids from C2, which was denominated C1 (Figure 7A). Redoxdependent intramolecular disulfide bonding of the human as well as the fungal NF-YB protein was described among cysteine residues C1 and C2 (Nakshatri et al., 1996; Thon et al., 2010).

Using the crystal structure of the human NF-YB/C heterodimer (Romier et al., 2003), we modeled the three-dimensional Arabidopsis NF-YB structure, mainly its HFM, and defined the possible spatial orientation of the conserved cysteine residues as well as the distances of these residues to each other in the protein structure. As result, the human NF-YB structure (Figure 7A) confirms that cysteine C1 and C2 are perfectly arranged in the first a-helix of the HFM for the preferential formation of an intramolecular disulfide bond (Nakshatri et al., 1996), whereas disulfide bonding between the cysteine C2 and C3 can be excluded, since they would require massive changes in the three-dimensional conformation of NF-YB HFM (Figure 7A). In analogy, the spatial separation of the two cysteine residues of the Arabidopsis NF-YB, Cys64 (C2) and Cys80 (C3), does not support intramolecular disulfide bonding in the HFM region (Figure 7B). In conclusion, an intramolecular disulfide bond of NF-YB necessarily requires the conserved cysteine residues C1 and C2. Both cysteine side chains can be found in NF-YB of heterotrophic organisms, like H. sapiens and A. nidulans. Arabidopsis NF-YB subunits as well as NF-YBs of other photoautotrophic species do not possess a cysteine residue at the corresponding position to C1. Moreover, analyzing the consensus sequence of all NF-YB proteins hosted in the Plant Transcription Factor Database (http:// planttfdb.cbi.pku.edu.cn/) (Zhang et al., 2011), no phototrophic species possesses cysteine residue C1 in their NF-YB sequences (Supplemental Figure 2). As a consequence, the modulation of the NF-YB/NF-YC interaction under oxidizing conditions is not expected in these organisms. To substantiate these assessments, we analyzed the effects of changing redox states on the protein–protein interaction

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Figure 6. Proof of the Detectable Interaction among NF-YA and NF-YB using the Yeast Two-Hybrid System.

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Peptide chains are displayed as flat ribbons and cysteine side chains as ball-and-stick models with red-labeled sulfur atoms. The human NFYB peptide chain (A) exhibits three cysteine residues. The spatial orientation of C1 and C2 would allow an intramolecular disulfide bond. The Arabidopsis NF-YB10 peptide chain (B) possesses only cysteines C2 and C3, which do not allow the formation of an intramolecular disulfide bond. (C) Identification of protein–protein interactions among NF-YB and C under different oxidizing conditions using the yeast two-hybrid system. Determination of the b-galactosidase activity of diploid yeast cells possessing yeast two-hybrid bait and prey constructs for the expression of Arabidopsis or human NF-YB and NF-YC. Heterodimerization of the human NF-YB and NF-YC is inhibited by treatment with more than 500 lM H2O2, whereas Arabidopsis NF-YB10 and NF-YC2 can form heterodimers even if 5 mM H2O2 were added.

among NF-YBs and NF-YCs in vivo. The diploid yeast cells containing bait and prey gene constructs for the Arabidopsis and human NF-YB subunits were incubated with different concentrations of H2O2. As a result of these growth conditions, human and Arabidopsis NF-YBs differ in their capability to interact with NF-YC of the corresponding organism. Whereas the Arabidopsis NF-YB10/NF-YC2 heterodimer was stable under normal as well as oxidizing conditions (with up to 5 mM H2O2), human NF-YB did not stably interact with NF-YC at more than 1 mM H2O2 (Figure 7C). It is suggested that a redox-dependent mechanism perturbs the stable interaction between human NF-YB and NF-YC and it is likely that the side chains of C1 and C2 of NF-YB form a redox-dependent intramolecular disulfide bond that compromises the heterodimer formation of NF-YB and NF-YC.

DISCUSSION Interactomic Network of the Arabidopsis NF-Y Proteins Using the defined criteria for a successful protein–protein interaction among the representatives of the individual subunit families, 10 NF-YBs (NF-YB1 to NF-YB10) and seven NF-YCs (NF-YC1 to C4, C6, C9, and C12) are strongly mutually interconnected and form an interactomic network among Arabidopsis NF-YB and NF-YC subunits. Considering the possible combinations in the interactome of the 10 NF-YB and seven NF-YC subunits, more than 74% of all theoretical pairs of interactions among these proteins were detected via the yeast two-hybrid experiments (52 of 70). Members of this network are capable of interacting with multiple representatives of the other

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Figure 7. Models of the Three-Dimensional Structure of the Human NF-YB as well as Arabidopsis NF-YB10 on the Base of the Crystal Structure of the Human NF-YB/C Heterodimer (PDB ID 1N1J; Romier et al., 2003).

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complementary as well as overlapping functions of several NF-Y proteins in planta (Kumimoto et al., 2008, 2010). Thus, specific roles could be assigned to some of these subunits, but the composition of contributing complete NF-Y is not predictable, yet. One exception among the functionally characterized NF-Y subunits belonging to the combinational network is Arabidopsis NF-YA5, which has been reported to lead to tolerance against drought stress (Li et al., 2008) upon overexpression. For unknown reasons, NF-YA5 was not expressed in our yeast two-hybrid-assays.

Comparison of Nuclear Piggyback Import of Arabidopsis and A. nidulans NF-YB The lack of NLS most likely explains the localization of the Arabidopsis NF-YB–GFP fusions in the cytoplasm instead of its final destination in the nucleus (Figure 1B). These results are consistent with the cytoplasmic localization of NF-YB3 in Arabidopsis root cells, while NF-YA4– and NF-YC2–eGFP fusion proteins were detected in the nucleus (Liu and Howell, 2010). In our refined experimental set-up, the concomitant co-expression of a NF-YC subunit enabled translocation of NF-YB to the nucleus (Figure 1D). As reported previously, the nuclear translocation of the entire A. nidulans NF-Y complex is enabled by a redoxcontrolled piggyback transport (Steidl et al., 2004). The intramolecular disulfide bonding between the cysteines at position C1 and C2 of NF-YB prevents the cytosolic assembly of all three fungal subunits and trafficking of the heterotrimeric complex (Thon et al., 2010). The authors also reported about in vitro homodimer stability through an intermolecular bond at the C3 cysteines, which apparently did not affect the nuclear import (Thon et al., 2010). In contrast, we propose that the cotransport into the nucleus of an Arabidopsis NF-YB subunit with the NLS-containing NF-YC depends rather on the stability of the heterodimer, but is hindered neither by the formation of intramolecular disulfide bonds nor by NF-YB homodimerization under oxidizing conditions. Infiltration of transformed Arabidopsis leaves co-expressing NF-YB–GFP and NF-YC with hydrogen peroxide did not result in lower rates of nuclear translocation of NF-YB into the nucleus (data not shown). In the same line, interactions between identical Arabidopsis NF-YB subunits were not favored (Figure 4) in the yeast two-hybrid system, even if the yeast cells grow in medic with additional H2O2 as oxidizing agent (Figure 7C). Moreover, heterodimerization of Arabidopsis NF-YB and NF-YC was not hindered under oxidizing conditions in the same experimental approach (Figure 7C). Phototrophic and heterotrophic eukaryotic organisms differ in the number of genes encoding each subunit of the NF-Y complex. Genomes of heterotrophic organisms contain always a single gene for each NF-Y subunit. With the exception of a few algae, all photoautotrophic species have several gene copies encoding all three subunits. The genomes of the green algae Chlamydomonas reinhardtii and Volvox carteri contain only a single NF-YB gene encoding a NF-YB with two conserved cysteines at position C2 and C3, whereas the moss Physcomitrella

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subunit family. As a consequence, these NF-YB and NF-YC proteins can be combined in a plethora of different composed heterodimers, forming the essential platform for the subsequential association of a NF-YA subunit. Apart from this interactome network, only the additional interaction between NF-YB3 and NF-YC11 was demonstrated in our yeast two-hybrid approach. Thus, dimer formation of NF-YB and NF-YC subunits that do not belong to the subclass of the interactomic network described above seems to occur more rarely. We propose that structural features of the interphase between both subunits determine the stability of the formed heterodimer and emphasize the integrity of the HFM of each of the two NF-Y subunits. While the overall structural identity of the NF-YB-HFMs seems to be essential for the stable contact with NF-YC, the binding capability of NF-YCs depends on the formation of an intramolecular hydrogen bonding between conserved amino acids arginine and aspartate in the HFM. In accordance with the three-dimensional model of the human NF-Y complex, we conclude that these conserved interacting domains of each NF-YB and NF-YC subunit facilitate the broad range of interactions with different members of the corresponding NF-Y subunit family. Minor structural modifications due to amino acid substitutions in the protein sequences of the HFM of each subunit most likely determine the strength of specific bilateral bonds between proteins, while larger structural alterations such as addition/deletions of amino acid residues in the HFM and substitution of the conserved aspartate/arginine residues required for hydrogen bonding compromise the bilateral interaction between NF-YB and NF-YC. As the multiple protein members share a rather low overall similarity within their NF-Y subunit family, it cannot be excluded that these distinctions are the base for the different affinity strength to NF-Y subunits, but also for interaction to a large variety of other nuclear factors. Interestingly, NF-YBs and NF-YCs almost never showed interaction with NF-YA subunits in the yeast two-hybrid system, indicating that the interphase of the NF-YB/C heterodimer is required for stable assembly with the NF-YA subunit. As suggested previously, a large complexity of multiple NF-Y subunits would enable targeting of specific genes containing CCAATboxes (Siefers et al., 2009). Thus, the NF-Y subunit family accomplishes a substantial prerequisite for the action of combinatorial transcription factors. At present, functions of a number of single NF-Y subunits are reported (Lotan et al., 1998; Cai et al., 2007; Chen et al., 2007; Nelson et al., 2007b; Warpeha et al., 2007; Kumimoto et al., 2008, 2010; Liu and Howell, 2010). All of these Arabidopsis NF-YB and NF-YC subunits described in the literature belong to the 17 NF-Y subunits, which are embedded in the NF-Y interactomic network described in the ‘Results’ section (Figure 4). The characterization of the specific functions of single NF-Y subunits of Arabidopsis was possible by their overexpression or gene inactivation when redundancies and complementary functions of related NF-Y subunits will not mask the detection of their function. However, multiple mutant analyses revealed

Hackenberg et al.

METHODS RNA Isolation and Reverse Transcription Total RNA was isolated from 3-week-old Arabidopsis leaf material using TRIsure (Bioline). One microgram of DNAse I (Fermentas)-pretreated total RNA was reverse-transcribed with oligo dT18 using a RevertAid reverse transcription kit (Fermentas).

Construction of the Vector DNA and Yeast Transformation The coding regions of all 36 NF-Y genes from Arabidopsis thaliana were amplified from cDNA by PCR using specific primers (primer sequences on request) containing either a SalI or a NotI restriction site, respectively. Human NF-YB and NF-YC coding sequences were amplified from pET3b_hsNF-YB and pET3bhs_NF-YC constructs kindly provided by Dr Mantovani (University of Milano, Italy). Yeast two-hybrid bait and prey constructs were generated by insertion of the amplified fragments between SalI and NotI restriction sites of modified pBTM117c or pGAD10 vectors possessing identical multiple cloning sites and the correct coding and in-frame sequence was confirmed by DNA sequencing. Yeast strains L40ccu mating type a and type a (Wanker et al., 1997) were used for transformation with either 500 ng of bait (pBTM117c) or prey (pGAD10) plasmids, respectively, according to the method described previously (Gietz and Schiestl, 2007) and selected for leucine or tryptophan prototrophy in synthetic drop-out (SD) medium (containing 0.66% yeast nitrogen base, 2% glucose, and appropriate auxotrophic supplements). Only yeast cells containing prey constructs of NF-YB12 or NF-YB13 were not able to be cultivated on appropriate selective media. For cultivation of yeast cultures in 96-well plates, always 4 ll of the pre-culture were used for inoculating 200 ll of the appropriate media followed

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by incubation of up to 24 h at 30
C and 230 rpm. Interaction of three NF-Y subunits was investigated after co-transformation of diploid yeast cells containing bait and prey constructs with the pVT-U102::NF-YC2 plasmid and subsequent selection for uracil autotrophy on appropriate SD medium. The coding region of NF-YC2 was amplified using 5-GCGCCTCGAGATGGAGCAGTCAGAAGAGGG-3and 5-TTTAAGCTTTTAAGACTCATCAGGGTGTTGCT-3 primers and ligated between XhoI and HindIII restriction sites of pVT-U102 vector.

Yeast Two-Hybrid System Diploid yeast cells harboring any possible combination of bait and prey plasmids were generated by mating corresponding haploid yeast cultures of opposite mating types during cultivation in YPAD medium (containing 1% of yeast extract, 2% of peptone, 2% of glucose, 0.01% adenine hemisulphate). After successful mating, yeast cultures were selected by growth in SD medium lacking leucine and tryptophan (SD–LT). Protein–protein interactions were confirmed by growth in medium lacking histidine, leucine, tryptophan, and uracil (SDHLTU) and subsequent determination of the b-galactosidase activity using ortho-nitrophenyl-b-galactoside (ONPG). Autoactivation of the HIS3 reporter genes was suppressed by adding appropriate concentrations of 3-amino-1,2,4-triazole (3-AT) to the SD-HLTU medium. b-galactosidase activity assays were performed similarly to that described before (Franklin, 2002). Two hundred ll diploid yeast cultures were harvested by centrifugation and re-suspended in 200 ll PBS buffer (137 mM NaCl, 2.7 mM KCl, 100 mM Na2PO4 and 2 mM KH2PO4, pH 8.0) before yeast cell walls were disrupted by three freeze–thaw cycles consisting of 15 min at –80
C and 25 min at 37
C. After centrifugation for 15 min at 5000 g, cell debris was re-suspended in 20 ll 250 mM TRIS-HCl, pH 8.0 and OD600 were measured. For developing ONPG colorimetric reaction, 80 ll of z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, pH 7.0) and 17 ll 0.1% (w:v) ONPG were added and incubated at 30
C for 60 min, before the reaction was stopped by adding 125 ll 1 M NaHCO3. After centrifugation for 2 min at 5000 g, the supernatant was used for measuring absorbance at 410 nm and calculation of the relative b-galactosidase activity as described previously (Miller, 1972). The threshold for a positive protein–protein interaction was set at 0.2 relative b-galactosidase activity units, which was determined for interaction between NF-YB10 and NF-YC9 in a yeast two-hybrid assay using filter lift-assays as described previously (Schneider et al., 1996). This interaction was the result of a previous screen for interacting proteins of NF-YC9 as bait and confirmed with an alternative method in a pull-down experiment (Figure 2A). Thus, an interaction between two NF-Y subunits was accepted if relative b-galactosidase activity of at least two of the three biological replicas and the corresponding mean value of all three replicas of both bait–prey combinations exceeded 0.2. Yeast twohybrid analysis under oxidative conditions was performed following the protocol by Franklin (2002).

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patens already exhibits six homologous gene copies coding for NF-YBs (Xie et al., 2008). As suggested before, the development of the NF-YB gene family occurred most likely at the beginning of the diversification of land plants and included several rounds of gene duplication and retrotransposition (Xie et al., 2008). In the course of NF-YB evolution, dicotyledonous plants, like Arabidopsis thaliana, and monocotyledonous species, like Oryza sativa, acquired up to 13 and 10 members of their NF-YB gene family, respectively. When NF-Y assembles by subunits of large protein families, it could evolve to a combinatorial transcription factor to specify most likely transcriptional activation in response to development and stress adaptation. In Aspergillus nidulans, the cellular redox state can change the conformation of NF-YB by intramolecular disulfide bonding between the two cysteines at the positions C1 and C2. Thus, oxidizing conditions can compromise the co-import of the complete fungal NF-Y-complex (Thon et al., 2010). In contrast, NF-YBs of phototrophic organisms cannot form intramolecular or intermolecular disulfide bridges between the conserved cysteine residues and are co-translocated with the aid of NF-YC.

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In Vitro Translation and GST Pull-Down

GFP Constructs and Transient Transformation of Arabidopsis Leaves Coding region of NF-YA2, NF-YA4, NF-YB3, NF-YB10, NF-YC1, and NF-YC2 were amplified with specific primers and ligated between the MfeI and SalI restriction sites of the linearized pRT-100 plasmid, which contains the eGFP sequence among BglII and XbaI restriction sites from the EPFGP N2 plasmid (kindly provided by Bianca Baudisch, Martin-Luther Universita¨t Halle/Saale, Germany). Arabidopsis NF-Y sequences were cloned as c-terminal eGFP fusions. For co-transformation, a pRT-100mod_NF-YC9 construct was used possessing a stop codon between NF-YC9 and eGFP sequence. Three-week-old Arabidopsis Col-0 WT leaves were used for the transient transformation using the PDS 1000/HE particle gun (Biorad) and gold particles according to the manufacturer’s data. Detection of the subcellular localization was performed using the spectral confocal & multiphoton system TCS SP2 (Leica Mikrosysteme). The GFP fluorescence signal was recorded between 500 and 600 nm after excitation at 488 nm. Cell walls were identified by bright field microscopy and localization of nuclei was determined after staining with 4,6-diamidino2-phenylindole-dihydrochloride (DAPI).

Bimolecular Fluorescence Complementation and Transient Transformation of Nicotiana benthamiana NF-YC9, NF-YB3, and NF-YB10 coding sequences were cloned between the SpeI and XhoI restriction sites of the BiFC plasmids pSPYNE and pSPYCE (Walter et al., 2004), respectively. Twoweek-old Nicotiana benthamiana leaves were used for transient transformation using Agrobacterium tumefaciens strain GV2260 (Bendahmane et al., 2000) carrying NF-Y pSPYCE and pSPYNE constructs. Cell walls were identified by bright field microscopy and localization of nuclei was determined by identification of the nucleus surrounding endoplasmic reticulum using a CFP-tagged ER marker (ABRC stock no. CD3-953), possessing the signal peptide of AtWAK2 at the N-terminus and the ER

In Silico Modeling As a template, the high-resolution crystal structure of the human heterodimer NF-YB/NF-YC (1.67 A˚) was chosen (PDB ID 1N1J) to model the homologous Arabidopsis NF-YB10 structure (derived from TAIR, AT3G53340.1-related protein sequence). The sequence alignment of the amino acid sequence of AT3G53340.1 (NF-YB10) with NF-YB of the human structure showed a sequence identity of 70% with 91% similar amino acids in a gapless alignment ensuring a reliable structural modeling. The sequence alignment and the structural superposition were carried out using version 4.01 of the Swiss PDB viewer software (Guex and Peitsch, 1997). The modeled structure was energy minimized applying the CharmM force field (Brooks et al., 2009) using the Accelrys Discovery Studio 2.5.

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING This work was financial supported by the Deutsche Forschungsgemeinschaft, DFG (GR 936/9–1, 9–2). No conflict of interest declared.

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