9186 Nucleic Acids Research, 2012, Vol. 40, No. 18 CBNAC binds to the PR1 promoter by a GCTT core element In cbnac1 knockout plants, the levels of PR1 transcripts were higher compared with those in wild-type plants. To examine whether this effect was attributable to direct binding of the CBNAC to the PR1 promoter, we investi- gate the direct interaction of CBNAC to the PR promoter by electrophoretic mobility shift assays (EMSA). EMSA using recombinant GST-CBNAC fusion protein and PR1 promoter fragments up to 1041-bp upstream of the PR1 start codon were used to demonstrate that CBNAC protein can bind to the PR1 promoter. Using seven 110-bp overlapping fragments of the PR1 promoter sequence (Supplementary Figure S3A), ﬁve regions (E0, E3, E4, E5 and E6) were found to bind to CBNAC (Supplementary Figure S3B). Within the ﬁve regions, smaller overlapping fragments ( 52 bp) were used to deﬁne the speciﬁc target of CBNAC (Supplementary Figure S4A). Six regions (E0-1, E0-4, E3-1, E4-1, E5-3 and E6-1) demonstrated binding to CBNAC (Supplementary Figure S4B). Overlapping oligo- nucleotides within the E0-1, E0-4, E3-1, E4-1, E5-3 and E6-1 fragments were used to further delineate CBNAC- binding sequences (Supplementary Figure S5A). CBNAC bound six PR1 promoter fragments (Supplementary Figure S5B and Table 1). Of these, E0-1-1 exhibited very strong CBNAC binding. E0-4-2, E3-1-2 and E5-3-1 had moderate afﬁnity. The two remaining elements, E4-1-1 and E6-1-1, bound very weakly. The E0-1-1 and E0-4-2 fragments contain the GCTT core sequence that was pre- viously identiﬁed as a CBNAC-binding sequence by the random binding site selection method (26). E3-1-2 has been previously identiﬁed as a negative regulatory element on the PR1 promoter (12). Interactions between CBNAC and the E0-1-1 element were further analyzed. Figure 3A depicts the four mutant E0-1-1 elements (M1–M4) used in the EMSA. As shown in Figure 3B, CBNAC bound strongly and speciﬁcally with radiolabeled E0-1-1. A mobility shift was not observed on incubation of the labeled E0-1-1 element with GST alone. Furthermore, addition of unlabeled E0-1-1 element inhibited binding of labeled E0-1-1 to CBNAC in a concentration-dependent manner (Figure 3B). Compared with the interaction of CBNAC with E0-1-1, its binding with M1 was weaker and was almost negligible with M2, M3 and M4 (Figure 3C). These results indicated that the GCTT core sequence of E0-1-1 is critical for CBNAC binding. Chromatin immunoprecipitation (ChIP) experiments were then used to determine whether CBNAC binds the PR1 promoter in vivo. In these experiments, cross-linked chromatin from leaves of wild-type and 35S:Flag-CBNAC plants were incubated with FLAG-speciﬁc monoclonal antibodies enriching for CBNAC and PR1 promoter complexes. The ﬁve PR1 promoter regions shown to bind CBNAC in vitro (E0, E3, E4, E5 and E6) were then ampliﬁed with speciﬁc primers. Three independent ChIP experiments were performed, and representative results from a single assay are shown (Supplementary Figure S5C). PCR analyses of input chromatin samples veriﬁed that similar quantities of ChIP starting materials were Figure 3. CBNAC interacts with the E0-1-1 element of the PR1 promoter. (A) Nucleotide sequence of the native and mutated (M1– M4) E0-1-1 elements used in EMSA. (B) Analysis of binding speciﬁcity. EMSA was performed using 32P-labeled native E0-1-1 as probe as above except that GST-CBNAC protein was preincubated with 50- (lane 4), 100- (lane 5) or 200- (lane 6) fold molar excess of cold native E0-1-1 (competitor) before addition of probe. (C) EMSA of CBNAC binding. 32P-labeled native (lanes 1–3) and mutated (lanes 4–7) E0-1-1 probes were incubated with equal amounts of E. coli- expressed GST-CBNAC (lanes 3 to 7) or GST alone (lanes 1 and 2) before electrophoresis. Table 1. Putative CBNAC-binding cis-acting elements in the PR1 promoter cis element E0-1-1 E0-4-2 E3-1-2 E4-1-1 E5-3-1 E6-1-1 Sequencea TAATAATGCTTAGTTATAAATTACT TGTTATTGCTTAGAATCACAGATTC CTATTGACTGTTTCTCTACGTCACTATT ATACTCATATGCATGAAACACTAAGAAAC ATATACAATGTTTCTTAATAAACTTCATTT AAAAAAATATATCAACAATGGCAAAGCT Positionb (–) 1209 (–) 994 (–) 715 (–) 618 (–) 340 (–) 288 (–) 1185 (–) 970 (–) 688 (–) 590 (–) 311 (–) 261 Binding afﬁnityc +++ ++ ++ + ++ + aSequences are indicated from 50 to 30. GCTT core sequence is underlined. bPositions of the cis elements with respect to the translation start site (ATG). cThe relative binding afﬁnity (+) was determined by densitometry of autoradiograms of DNA-bound CBNAC.