Chimeric activators and repressors define HY5 activity and a feedback control mechanism in Arabidopsis (HY5_ChIPseq)

Transcription regulation requires many protein interactions on chromatin, and only a subset of transcription factors have well-defined activation or repression domains. The Arabidopsis transcription factor HY5 controls critical growth-related gene expression programs during plant development, but it’s primary activity in regulating transcription remains unclear. To address this question, we generated constitutive repressor and activator HY5 fusion proteins to direct the expression of HY5 target genes. We used RNA-seq, ChIP-seq, and multiple phenotypes to demonstrate that HY5 depends on accessory factors to promote transcription and identify high confidence direct targets of HY5. We suggest that this strategy can be used broadly to define the transcription regulation activity and direct targets of transcription factors. Interestingly, this approach also revealed a mechanism by which HY5 promotes the accumulation of its own negative regulators. We show that HY5 directly regulates components of the COP1 E3-ubiquitin ligase complex, and by uncoupling this feedback loop we can induce partial de-etiolation in the dark. This provides a system by which plants can quickly repress growth upon light exposure. Lastly, we show that modulating this system can generate significant phenotypic diversity and provide proof of concept that these fusion proteins can modulate growth in tomato, opening a novel path toward selecting desirable traits in crop species. We performed whole-genome chromatin immunoprecipitation with sequencing (ChIP-Seq) analysis on 35S:FLASH-HY5, 35S:FLASH-HY5-VP16 and 35S:FLASH-HY5-SRDX during de-etiolation (dark+2.5h in simulated white light (blue, red and far-red LED lights- 100 µmol m−2 s−1 )