Figure 1.

CO Binds DNA as a Trimer with At-NF-YB2/NF-YC3 and Recognizes the CORE Element.

(A) CO forms a trimer with At-NF-YB2/NF-YC3 HFD binding to the FT CORE2 element. EMSAs were performed using fluorescently labeled FT CORE2 (lanes 1–14) or FT CCAAT (lanes 15–28) 31-mer oligonucleotide DNA probes (20 nM) by addition of the indicated proteins. CO-CCT (CO) was incubated at increasing concentrations (90, 180, 270, and 360 nM) with the CORE2 probe in the absence (lanes 2–5) or presence (lanes 9–12) of the At-NF-YB2/NF-YC3 HFD dimer (At-NF-YB2/YC3, 60 nM). As controls, At-NF-YA2 or -YA6 (YA2, YA6) was incubated with the CORE2 probe at the highest concentration of the dose curve (360 nM), with or without (−) the HFD dimer (60 nM) (YA2: lanes 13, 6; YA6: lanes 14, 7, respectively). Lane 1: CORE2 probe alone, without protein additions. DNA binding of CO or At-NF-YAs, as indicated (lanes 16–27), was assayed on the FT CCAAT probe in the presence of At-NF-YB2/NF-YC3 (60 nM), with the same protein concentration dose curve (90, 180, 270, and 360 nM). As controls, the FT CCAAT probe was incubated with the HFD dimer alone (60 nM, lane 15) or with At-NF-YA2 protein (360 nM, lane 28). NF-CO and NF-Y/DNA complexes are indicated by closed or open arrowheads. fp, free probe.

(B) EMSA competition analysis of the At-NF-YB2/NF-YC3/CO complex specificity on the labeled CORE2 probe. Top panel: Sequences of the 31-mer CORE2 probe and unlabeled competitor derived from the FT promoter (−172/−141 from ATG). Oligos 1 to 6: CORE2 30-mers and 25-mer, the wild type or mutant was used as unlabeled competitors. The 31-mer derived from the FT enhancer CCAAT sequence and the FT mutant competitor (Cao et al., 2014) are listed below, together with the Hsp70 CCAAT competitor. Sequence identity with the probe is indicated by dots, and 5′ or 3′ sequence extensions, or mutated nucleotides are indicated in capital letters. The previously described TTGTGGTT CORE element (Tiwari et al., 2010) and the CCAAT pentamer are highlighted in bold letters. Bottom panel: EMSA competition analysis was performed by incubation of the CORE2 probe with the trimer composed of indicated subunits (At-NF-YB3/NF-YC3, 60 nM; CO, 180 nM -At-NFY-B2/YC3/CO-: lanes 2–27) in the presence of TE buffer alone (lanes 2 and 27) or with the addition of increasing concentrations of the indicated unlabeled competitors (5× or 25× molar excess; lanes 3–26). Lanes 1 and 28: CORE2 probe alone, without protein addition. The NF-CO/DNA complex is indicated by an arrowhead.