Analysis of transcription factor HY5 genomic binding sites in Arabidopsis

The transcription factor HY5 acts downstream of multiple families of the photoreceptors and promotes photomorphogenesis. Although it is well accepted that HY5 acts to regulate target gene expression, in vivo binding of HY5 to any of its target gene promoters has yet to be demonstrated. Here we used a chromatin immunoprecipitation procedure to verify suspected in vivo HY5 binding sites. We demonstrated that in vivo association of HY5 with promoter targets is not altered under distinct light qualities or during light-to-dark transition. Coupled with DNA chip hybridization using high density 60-nucleotide oligomer microarray that contains one probe for every 500 nucleotides over the entire Arabidopsis genome, we mapped genome wide in vivo HY5 binding sites. This analysis showed that HY5 binds preferentially to promoter regions in vivo and revealed over 3 thousand chromosomal sites as putative HY5 binding targets. HY5 binding targets tend to be enriched in the early light responsive genes and transcription factor genes. Our data thus supports a model in which HY5 is a high hierarchical regulator of the transcriptional cascades for photomorphogenesis. Keywords: ChIP-chip To determine the in vivo HY5 binding sites in Arabidopsis genome, four biological ChIP-chip replicate assays were performed using HA specific antibody for HA:HY5 lines. The DNA from each ChIP was labeled with Cy5 and the sonicated input DNA was labeled with Cy3, and the pooled probes were used for hybridization with 60mer probes on a microarray chip that covers the sequenced Arabidopsis genome.