Interactions and CCAAT-Binding of <em>Arabidopsis thaliana</em> NF-Y Subunits

<div><h3>Background</h3><p>NF-Y is a transcription factor that recognizes with high specificity and affinity the widespread CCAAT box promoter element. It is formed by three subunits: NF-YA and the NF-YB/NF-YC- heterodimer containing histone fold domains (HFDs). We previously identified a large <em>NF-Y</em> gene family in <em>Arabidopsis thaliana</em>, composed of 29 members, and characterized their expression patterns in various plant tissues.</p> <h3>Methods</h3><p>We used yeast Two-hybrids assays (Y2H), pull-down and Electrophoretic Mobility Shift Assay (EMSA) <em>in vitro</em> experiments with recombinant proteins to dissect AtNF-YB/AtNF-YC interactions and DNA-binding with different AtNF-YAs.</p> <h3>Results</h3><p>Consistent with robust conservation within HFDs, we show that heterodimerization is possible among all histone-like subunits, including the divergent and related LEC1/AtNF-YB9 and L1L/AtNF-YB6 required for <em>embryo</em> development. DNA-binding to a consensus CCAAT box was investigated with specific AtNF-YB/AtNF-YC combinations and observed with some, but not all AtNF-YA subunits.</p> <h3>Conclusions</h3><p>Our results highlight (i) the conserved heterodimerization capacity of AtNF-Y histone-like subunits, and (ii) the different affinities of AtNF-YAs for the CCAAT sequence. Because of the general expansion of NF-Y genes in plants, these results most likely apply to other species.</p> </div

<i>E. coli</i> co-expression of LEC1/AtNF-YB9 with AtNF-YC3 allows functional heterodimerization, heterotrimerization and CCAAT-binding.

<p><b>A</b>. Purification of soluble LEC1/AtNF-YB9 or L1L/AtNF-YB6 HFD heterodimers by co-expression with AtNF-YC3. Nickel-affinity purification elution profiles obtained from soluble fractions of 6His-LEC1/AtNF-YB9 or 6His-L1L/AtNF-YB6 with AtNF-YC3. Equal volumes of indicated elution fractions (E) in 100 mM Imidazole of LEC1/AtNF-YB9 or L1L/AtNF-YB6 with AtNF-YC3 were analysed by SDS-PAGE and Coomassie staining. E2, were dialysed and used in Agarose gel non-radioactive EMSAs shown in (B). <b>B</b>. Fluorescence agarose gel EMSAs of trimer reconstitution with mouse NF-YA. 5′-Cy5 labeled CCAAT oligonucleotide probe was incubated with increasing amounts of the indicated 6His-tagged HFD dimers isolated by Ni-affinity purification, or mouse 6His-NF-YB/NF-YC as positive control, in the presence, or absence, of purified mouse NF-YA. Purified (untagged) mouse NF-Y trimer was used as a reference for NF-Y complex migration.</p

AtNF-Y Subunits interactions <i>in vitro</i>.

<p><b>A</b>. The indicated labelled, TnT produced NF-YCs were assayed in affinity assays with recombinant AtNF-YB2 containing an His-tag. Load (L), flow-.through (FT) and bound (B) fractions of NTA Nickel columns, with (Lanes 2 and 3) and without (Lanes 4 and 5) His-AtNF-YB2 were run on SDS-PAGE gels and labelled proteins were revealed by autoradiography. <b>B</b>. Same as A, except that labelled, TnT produced AtNF-YA6 was added to the load fraction.</p

AtNF-YB-AtNF-YC interactions by colony yeast two hybrids assays.

<p><b>A</b>.The indicated AtNF-YCs were fused to the Activation Domain (AD) and tested with AtNF-YB fused to the DNA-binding domain of GAL4. <b>B</b>. Same as A, except that the reverse experiment was tested, namely the AtNF-YBs fused to the Activation Domain were matched to the AtNF-YCs fused to the DNA Binding Domain. ++ refers to robust growth on the selective medium, + weak growth, and − no growth.</p

EMSAs of At NF-Y subunits.

<p>Electrophoretic Mobility Shift Assay of the indicated AtNF-Y subunits with a labelled CCAAT-containing oligonucleotide.</p

EMSAs of AtNF-Y subunits with mouse NF-Y.

<p><b>A</b>.Electrophoretic Mobility Shift Assay of the indicated AtNF-YB with recombinant mouse NF-YA and NF-YC using a labeled CCAAT-containing oligonucleotide. <b>B</b>. Same as A, except that At NF-YCs were used with recombinant mouse NF-YA and NF-YB. <b>C</b>. Same as A, except that AtNF-YA were used with recombinant mouse NF-YB and NF-YC. The migration of the mouse NF-Y complex is indicated.</p

<i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> cannot rescue the <i>nf-yb2 nf-yb3</i> late flowering phenotype.

<p>A) Flowering time quantification of 15–20 randomly selected T1 <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> plants in the Col-0 background. B) Flowering time quantification of 15–20 randomly selected T1 <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> plants in the <i>nf-yb2 nf-yb3</i> background. C) Flowering time quantification of two independent, stable T3 <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> plant lines in the <i>nf-yb2 nf-yb3</i> background (n≥12). D) Representative plants of <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> in the <i>nf-yb2 nf-yb3</i> background. E) Relative transcript abundance of <i>NF-YB2</i>, <i>FT</i> and <i>AP1</i> in stable T3 <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> plants in the <i>nf-yb2 nf-yb3</i> background. Asterisks in 3A, 3B and 3C represent significant differences derived from one-way ANOVA (P < 0.05) followed by Dunnett’s multiple comparison post hoc tests against <i>nf-yb2 nf-yb3</i>.</p

NF-YA2 and NF-YA6 bind the <i>FT</i> -5.3kb <i>CCAAT</i> box as a trimer with NF-YB2 and NF-YC3.

<p>NF-Y trimerization and <i>FT CCAAT</i> binding was assessed by EMSA analysis. An <i>FT CCAAT</i> probe was incubated with wild type (WT, lanes 2–8; 20) or E65R mutant (B2^E65R, lanes 15–18; 21) NF-YB2/NF-YC3 dimers (60 nM) in the presence of NF-YA2 (lanes 3–5; 16–18), or NF-YA6 (lanes 6–8) at increasing molar ratios (3, 4.5 or 6 fold), or CO (lanes 20, 21; 6 fold molar ratio). As controls, NF-YA2 (lane 9), NF-YA6 (lane 10), or CO (lane 22) were incubated alone with the probe, at the highest concentration of the dose curve (360 nM), in the absence of NF-YB2/NF-YC3. Lanes 1, 11, 14, 19: probe alone, without protein additions; lanes 12, 13: empty lanes. The NF-Y/DNA complex is indicated by a labelled arrowhead. fp: free probe.</p

NF-YB2^E65R loses interaction with NF-YA subunits.

<p>A) Alignment of the core domain of human and Arabidopsis NF-YB subunits. * marks the position of the conserved glutamic acid required for interaction with NF-YA in humans [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006496#pgen.1006496.ref027" target="_blank">27</a>]. B) NF-YB2 and NF-YB2^E65R interact with NF-YC3, NF-YC4, and NF-YC9 in Y2H assays. C) NF-YB2, but not NF-YB2^E65R, interacts with NF-YA2 when NF-YC9 is expressed using a bridge vector in yeast three-hybrid assays. DBD: DNA binding domain, AD: activation domain, EV: empty vector control.</p

NF-YA2 is a positive regulator of photoperiod dependent flowering.

<p>A) Flowering time quantification of two independent plant lines each (plant lines 1 and 2) for <i>p35S</i>:<i>NF-YA2</i> (white bars), <i>p35S</i>:<i>NF-YA7</i> (light grey bars), <i>p35S</i>:<i>NF-YA8</i> (grey bars), and <i>p35S</i>:<i>NF-YA9</i> (dark grey bars) (n≥12/line). B) Flowering time quantification of two independent <i>pNF-YA2</i>:<i>NF-YA2</i> plant lines (n≥24). C) The expression pattern of <i>pNF-YA2</i>:<i>GUS</i> in leaves of 10 day old plants. D) Relative transcript abundance of <i>CO</i>, <i>FT</i>, and <i>AP1</i>. Asterisks in 1A and 1B represent significant differences derived from one-way ANOVA (P < 0.05) followed by Dunnett’s multiple comparison post hoc tests against Col-0. Asterisks in 1D represent significant differences derived from Student’s T-tests (P < 0.05). All experiments were repeated with identical results.</p