Uncovering the role of core promoters of genes in regulating multicellular development

Protein-coding genes are transcribed by RNA polymerase II (RNAPII), which initiates transcription from a DNA template at defined nucleotide positions in the genome at the beginning of the gene. These locations are known as transcription start sites (TSS), and the region surrounding them (approximately 40 bp upstream and downstream) represents the core promoter. Recent studies have uncovered the key role of core promoter architectures in mediating different types of gene regulation. The Cap Analysis of Gene Expression methodology (CAGE) was developed for TSS mapping at single nucleotide resolution (Murata et al. 2014). Across eukaryotes, core promoters can be classified according to the distributions of TSSs into two distinct modes of transcriptional initiation (Haberle and Lenhard 2016). These comprise “sharp” promoters with a single well-defined TSS, usually associated with tissue-specific genes, and “broad” promoters that are characterized by closely spaced TSSs distributed across a wide region. Diverse promoter usage between maternal and zygotic stages has been identified throughout development. Our recent work (Haberle et al. 2014) demonstrated that thousands of promoters, including those of most housekeeping genes, have at least two overlapping transcription initiation codes used in different cell types.

Broad TSSs result in the variable length of the 5’-untranslated regions of mRNA coding transcripts. The functional consequences of this variability are unknown. The main goal of this project is to investigate how broad TSS patterns and the resulting transcript’s 5’UTR variability influence the transcript’s translation and protein composition throughout embryonic development.

The project includes both experimental and bioinformatic components and provides training in both. Drosophila melanogaster will be used as a model organism. Since there is evidence for functional equivalence of promoter classes between Drosophila and vertebrates (Lenhard et al. 2012), insights gained on Drosophila will enable understanding of promoter function in other animal genomes, including human. Experimental work will encompass development and application of novel CAGE methodology suitable for low-input RNA samples, application of CAGE on nascent RNA and polysome fractions, and application of the novel polysome profiling technique (Poly-Ribo-Seq (Aspden et al. 2014)). The functionality of interesting target genes where TSS switching changes protein composition will be examined through CRISPR-Cas9 knockouts and in vitro biochemical characterization. Pipelines for integrated computational analyses of the obtained datasets will be developed throughout the project. The results are expected to provide an unprecedented level of detail on transcriptional and translational regulation and dynamics in embryonic development, and their analysis will uncover the rules imposed by the underlying molecular mechanisms, advancing the understanding of mechanisms of transcriptional gene regulation.