Genome-wide identification of KANADI1 target genes

Abstract

Plant organ development and polarity establishment is mediated by the action of several transcription factors. Among these, the KANADI (KAN) subclade of the GARP protein family plays important roles in polarity-associated processes during embryo, shoot and root patterning. In this study, we have identified a set of potential direct target genes of KAN1 through a combination of chromatin immunoprecipitation/DNA sequencing (ChIP-Seq) and genome-wide transcriptional profiling using tiling arrays. Target genes are over-represented for genes involved in the regulation of organ development as well as in the response to auxin. KAN1 affects directly the expression of several genes previously shown to be important in the establishment of polarity during lateral organ and vascular tissue development. We also show that KAN1 controls through its target genes auxin effects on organ development at different levels: transport and its regulation, and signaling. In addition, KAN1 regulates genes involved in the response to abscisic acid, jasmonic acid, brassinosteroids, ethylene, cytokinins and gibberellins. The role of KAN1 in organ polarity is antagonized by HD-ZIPIII transcription factors, including REVOLUTA (REV). A comparison of their target genes reveals that the REV/KAN1 module acts in organ patterning through opposite regulation of shared targets. Evidence of mutual repression between closely related family members is also shown.

Figure 1. Identification of KAN1 target genes.

A) Constructing an inducible KAN1 expression system. B) Sequence logos for the cis-element, forward and reverse orientation, enriched in the ChIP-Seq dataset C) Distribution of KAN1 binding sites across the five Arabidopsis chromosomes. D) Location of peaks identified by ChIP-Seq. About 25% of all peaks are located in the first 1000bp upstream of the transcriptional start site.

Figure 2. Gene-ontology analysis of KAN1 targets.

A) KAN1 binds to the ASYMMETRIC LEAVES2 (AS2) promoter. Three distinct binding regions were identified but only the second peak contains the VGAATAW motif. The guanine depicted in red is mutated to adenine in the as2-5d mutant. B) and C) Enrichment of GO terms identified in the set of genes located downstream of the KAN1-binding site. Over-representation of genes involved in multicellular organismal development and in the response to stimuli targeted by KAN1.

Figure 3. Genome-wide comparison of genes bound and regulated by KAN1.

A) Venn-diagram showing numbers of genes bound by KAN1 and regulated by KAN1. The overlap contains 211 genes that are both bound and also regulated by KAN1. B) Gene ontology analysis of 211 potential direct KAN1 targets reveals a strong enrichment for genes involved in shoot patterning and the auxin response. Tables 1 and 2 contain these genes including the binding site information.