The role of aureochromes in transcriptional and circadian regulation

Abstract

Like most organisms on earth, diatoms are exposed to environmental conditions that regularly change on a daily basis. To anticipate those changes, many organisms have evolved circadian clocks. While it is known that diatoms must possess such circadian clock, very little is known about the molecular mechanism behind it. Two proteins, the bHLH-PAS transcription factor RITMO1 and the dual function photoreceptor/transcription factor PtAUREO1a were implicated as potential clock elements in the mode diatom Phaeodactylum tricornutum. RITMO1 has two paralogs, PtbHLH1b-PAS and PtbHLH2-PAS, while PtAUREO1a has three paralogs, PtAUREO1b, PtAUREO1c and PtAUREO2. This work focused on the role of PtAUREO1a in the circadian clock and could provide additional evidence that it partakes in circadian regulation. By monitoring the circadian rhythmicity of single-cell chlorophyll fluorescence, it is shown that PtAUREO1a is essential for maintaining rhythmicity under constant (free-running) conditions. Analyzing transcript rhythmicity analysis in a 24 h entrainment and a 48 h free-running RNA-Seq time series, it was determined that ~60% of the P. tricornutum transcriptome exhibit a rhythmic pattern under entrainment conditions and ~40% of the transcriptome maintain circadian rhythmicity under free-running conditions. The transcriptomic data also suggests that PtAUREO1a knockout (KO) affects the diel expression patterns of a large portion of the transcriptome, including other aureochromes and bHLH-PAS genes. It is shown that the effect of RITMO1 KO on the transcriptome is smaller. Applying DNA Affinity Purification and Sequencing (DAP-Seq) the genomic DNA binding landscapes of the four aureochrome isoforms of P. tricornutum and their binding motifs are revealed. It is further shown that ~15% of all genes possess a PtAUREO1a binding site in their promotor, a number much lower for the other three aureochromes. Further, it was attempted to generate a knockout of PtAUREO1b and a double knockout of PtAUREO1a and RITMO1 using the CRISPR/Cas9 system, both of which appear to be lethal, although hypomorphic PtAUREO1a - RITMO1 mutants were generated. This work provides valuable data for advancing the understanding of the diatom circadian clock while suggesting an essential role of PtAUREO1a in diel and circadian regulation of P. tricornutum as well as a possible feedback loop between PtAUREO1a and RITMO1.