Promoter architecture and sex-specific gene expression in the microcrustacean Daphnia pulex revealed by profiling of 5’-mRNA ends

Large-scale identification of transcription start sites (TSSs) using Cap Analysis of Gene Expression (CAGE) has yielded insight into promoter location, architecture, and regulation for a small set of taxa representing major model organisms. However, comparative and evolutionary genomics studies of cis-regulatory control of transcription initiation for a wider spectrum of Metazoa are still outstanding. To broaden our understanding of core promoter structure in species from metazoan clades with currently scant genome data, we sought to characterize the landscape of cis-regulatory elements in the microcrustacean water flea Daphnia pulex, an important model organism for studies in ecology, toxicology, and genetics. We performed CAGE from total RNA derived from three separate developmental states: sexual females, asexual females, and males, reflecting distinct sexes and modes of reproduction. We mapped over 120 million CAGE reads to the D. pulex, genome and generated a 'Daphnia Promoter Atlas' containing 12,662 unique promoters. Characterization of the transcription initiation sites showed the expected enrichment of the CA-dinucleotide at TSSs [-1,+1] (associated with Initiator-motif containing promoters) but also significant over-representation of GN-dinucleotides. Overall, these data suggest that D. pulex initiation sites are among the most GC-rich yet observed for any organism. Computational de novo motif discovery around CAGE-identified TSSs revealed eight putative core promoter elements, including the canonical TATA (TATAWAA) and Initiator (CAGWY) motifs, as well as statistically significant motifs with no obvious orthologs in other metazoans. Finally, we compared promoter activity across the three sampled developmental states, revealing sex- and condition-specific transcription initiation. Analysis of the deferentially expressed genes suggests that a significant number of cell cycle genes (each with net negative regulatory effects on meiosis) are upregulated in asexual females, providing a glimpse of the molecular events that underpin the cyclical parthenogenesis in D. pulex. Taken together, this work provides the first picture of transcription initiation and promoter architecture within Crustacea. The Daphnia Promoter Atlas we present here provides a basis for future study among Daphnia spp. as well as for comparative genomic analyses of metazoan transcriptional control.