ChIP-seq analysis of GLABRA2 transcription factor DNA-binding sites in wild-type and mutant Arabidopsis seedlings

GLABRA2 (GL2) is a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor that is critical for the differentiation of the epidermis in the plant model Arabidopsis. HD-Zip IV transcription factors are characterized by the presence of a lipid sensing domain known as START (for STeroidogenic Acute Regulatory (StAR)-related lipid Transfer). Genome-wide ChIP-seq assays were performed with seedlings expressing EYFP-tagged GL2 wild-type and mutant proteins to test whether the START domain is dispensible for DNA binding in vivo. These experiments included three START domain mutants (gl2_∆ST (listed here as gl2_dSTART), gl2_Amo, and gl2_E375G;R392M) as well as a mutant deleted for the six terminal amino acids of the HD and the entire leucine zipper (gl2_∆HD6;∆ZLZ (listed here as gl2_dZip)). Data analysis revealed DNA binding proficiency for wild-type GL2 and for all three START domain mutants whereas little or no binding was detected for gl2∆HD6;∆ZLZ and the gl2-5 null mutant control. Genomic distributions of the binding sites were similar for wild-type GL2 and the START domain mutants. Most of the ChIP-seq hits were mapped to promoter or intergenic regions, consistent with the role of GL2 as a transcriptional regulator. The relative positions of the binding sites to the transcription start sites were also similar. Gene ontology (GO) classification revealed overlapping functional classes of target genes bound by wild-type GL2 and the START domain mutants. Chip-seq analysis to identify genome-wide transcription factor binding sites of wild-type GL2 and five mutant versions thereof in 10-day-old Arabidopsis seedlings.