Figure 4.

HY5 directly binds to the DEWAX promoter elements and regulates its expression. A, RT-qPCR experiment revealed the role of HY5 in transcriptional regulation of DEWAX. The red line indicates the expression profile of DEWAX in the hy5 mutant and the black line represents the corresponding expression in wild-type Col-0. B, HY5 binds to pDEWAX in the eY1H assay. C, ChIP-qPCR showing relative enrichment of the DEWAX regulatory regions bound by HY5. The ChIP assay was performed on pHY5::HY5-YFP hy5 and 35S::GFP in the wild type. DNA-protein complexes were immunoprecipitated using anti-GFP and IgG antibody as negative controls. Error bars represent the mean ± SE of two biological replicates. Same lowercase letters indicate no statistical difference, while different letters denote significant differences within the group using ANOVA analysis followed by uncorrected Fisher’s least significant difference multiple comparisons test (P < 0.01). D and E, E-Box (−60 bp), NC E-Box (−469 bp), and C-Box (−1,421 bp) elements present in the DEWAX promoters, as well as the corresponding mutated cis-elements (preceded by “m”), are highlighted (red letters; D) and their relative position depicted (E). F, EMSAs show binding of recombinant HY5 protein in increasing concentrations to oligonucleotides containing E-Box, NC E-Box, and C-Box regulatory elements. G, HY5 binding to E-Box, NC E-Box, and C-Box was abrogated upon introduction of mutation in the respective cis-elements.