Genome-wide discovery of the daily transcriptome, DNA regulatory elements and transcription factor occupancy in the monarch butterfly brain

PLoS Genet. 2019 Jul 23;15(7):e1008265. doi: 10.1371/journal.pgen.1008265. eCollection 2019 Jul.


The Eastern North American monarch butterfly, Danaus plexippus, is famous 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 in Drosophila and the mouse, little work has been undertaken in the monarch to identify rhythmic genes at the genome-wide level and elucidate the regulation of their diurnal 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 of monarchs with impaired clock function, including Cryptochrome 2 (Cry2), Clock (Clk), and Cycle-like loss-of-function mutants. We identified 217 rhythmically expressed genes in the monarch brain; many of them were involved in the regulation of biological processes key to brain function, such as glucose metabolism and neurotransmission. Surprisingly, we found no significant time-of-day and genotype-dependent changes in chromatin accessibility in the brain. Instead, we found the existence of a temporal regulation of TF occupancy within open chromatin regions in the vicinity of rhythmic genes in the brains of wild-type monarchs, which is disrupted in clock deficient mutants. Together, this work identifies for the first time the rhythmic genes and modes of regulation by which diurnal transcription rhythms are regulated in the monarch brain. It also illustrates the power of ATAC-sequencing to profile genome-wide regulatory elements and TF binding in a non-model organism for which TF-specific antibodies are not yet available.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Brain / metabolism
  • Butterflies / genetics*
  • Chromatin / genetics
  • Circadian Clocks
  • Circadian Rhythm
  • Gene Expression Profiling / veterinary*
  • Gene Expression Regulation
  • Insect Proteins / genetics
  • Regulatory Sequences, Nucleic Acid*
  • Sequence Analysis, RNA / veterinary
  • Transcription Factors / genetics*


  • Chromatin
  • Insect Proteins
  • Transcription Factors

Grants and funding

This work was supported by start-up funds from Texas A&M University and the National Science Foundation (IOS-1456985 and IOS-1754725 to CM). CM is also supported by a Klingenstein-Simons award in the Neuroscience. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.