In vivo contribution of the DPE core promoter motif to transcriptional regulation in developing Drosophila melanogaster embryos

Regulation of transcription by RNA polymerase II (Pol II), a 12-subunit complex, is critical for most biological processes, including embryonic development. Transcription initiation occurs at the core promoter, frequently referred to as “the gateway to transcription”. Core promoters are diverse in their architecture and function, and can contain distinct DNA motifs termed core promoter elements, e.g., the TATA box and the downstream core promoter element (DPE). Interestingly, DPE-containing genes are highly enriched for heart-related and mesoderm development GO terms. Unfortunately, the analyses of endogenous core promoter function in vivo within the developing embryo are largely lacking.

The tinman gene encodes a transcription factor that orchestrates the formation of the dorsal musculature and heart during Drosophila embryonic development. Using in vitro transcription and reporter assays, we previously showed that the tinman promoter contains a functional DPE. To address the in vivo importance of the DPE, we mutated the endogenous DPE of the tinman gene using the co-CRISPR approach. We show that the 7bp mutation of the DPE motif, located within the 5’UTR of the tinman gene, is sufficient to reduce tinman expression at both the RNA and protein levels.

Remarkably, significantly reduced numbers of mDPE tinman flies are obtained when tested in trans to tinman deficiency, demonstrating the in vivo importance of the DPE. Although the heart is formed in mutant embryos, aberrant phenotypes are observed. Using multiple approaches, including nascent transcription dynamics during embryonic development, we provide mechanistic insights into the in-vivo regulatory role of the DPE during Drosophila development