show Abstracthide AbstractThe Eastern North American monarch butterfly, Danaus plexippus, is notorious for its spectacular seasonal long-distance migration. In recent years, it has also emerged as a novel system to study how animal circadian clocks keep track of time and regulate ecologically relevant daily rhythmic activities and seasonal behavioral outputs. However, unlike Drosophila and the mouse, little work has been undertaken in the monarch to identify clock-controlled output genes and elucidate the regulation of their rhythmic expression. Here, we used RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC)-sequencing to profile the diurnal transcriptome, open chromatin regions, and transcription factor (TF) footprints in the brain of wild-type monarchs and Cryptochrome 2 (Cry2), Clock (Clk), and Bmal1 (named DCyc-like) butterfly mutants with impaired clock function. We identified 366 rhythmic transcripts under circadian clock control belonging to biological processes key to brain function, such as neurotransmission, neuropeptide signaling, and glucose metabolism. Surprisingly, we found no significant time of day and genotype-dependent changes in chromatin accessibility (i.e., cis-regulatory elements) in the brain. Instead, we found the existence of a temporal regulation of TFs occupancy within open chromatin regions in the vicinity of rhythmic genes in the brains of wild-type monarchs, which is abolished in clock deficient mutants. Our data suggest that TFs binding specifically in the middle of the day display pioneer-like activity by increasing the accessibility of the surrounding chromatin, while TFs binding specifically in the middle of the night would bind DNA with a longer residency time without affecting accessibility of the surrounding chromatin. Together, this work identifies for the first time the clock-controlled genes and modes of regulation by which diurnal transcription rhythms are regulated in the monarch brain. It also illustrates the power of ATAC-seq to profile genome-wide regulatory elements and TF binding in unconventional organisms. Overall design: Determine the identify of clock-controlled genes and their cis-regulatory elements in monarch butterfly brains using RNA-Seq and ATAC-Seq open chromatin profiling.