Demystifying the relationship between DNA sequence features and regulatory function

All cellular processes from development to homeostasis depend on precise spatiotemporal gene expression. This precision is mediated by two pieces of regulatory DNA, enhancers and promoters, which integrate signals from activating and repressive transcription factors (TFs). Understanding how gene expression is encoded in these pieces of regulatory DNA is the larger question in the field that we hope to better understand. Here we approach this goal by tackling two questions. First, we consider the complexity of a regulatory task as a potential organizing principle for how expression is encoded in enhancers. We define task complexity as the number of fates specified in a set of cells at once. We hypothesized that more complex regulatory tasks would be encoded in longer enhancers with more binding sites, as more binding sites can be rearranged within an enhancer in more ways. This allows for the specification of a wider variety of expression patterns, and therefore, more complex tasks. To test this hypothesis, we compared ~100 enhancers that specify the complex anterior-posterior (AP) and the simpler dorsal-ventral (DV) axis patterning system. We also validated this hypothesis using a larger dataset of enhancers active across development, where we would expect task complexity to decrease over time. In both cases, we found that increased decision complexity is encoded in longer enhancers with more TF binding sites.Second, we consider the role of multiple promoters in the context of a gene locus. Many genes have multiple enhancers and promoters. However, while the role of multiple enhancers in a gene locus has been studied, little work has been done to explicate the roles of multiple promoters for a single gene, especially when these promoters lead to the production of similar or identical isoforms. Here, we propose that like multiple enhancers, multiple promoters can provide redundancy like shadow enhancers or specificity by preferentially engaging with specific enhancers. To distinguish between these two roles, we chose a case study gene, knirps, that has multiple enhancers and promoters that each has different motif content and thus recruits different sets of proteins. As we expect that specificity is mediated by the proteins recruited to the enhancer and promoter, this set of enhancers and promoters allows us to test whether these promoters provide redundancy or specificity. Using synthetic reporter constructs, we found that some, but not all, enhancers in the locus show a preference for one promoter. By analyzing the dynamics of these reporters, we identified specific burst properties during the transcription process, namely burst frequency and size, that are most strongly tuned by the specific combination of promoter and enhancer. Using locus-sized reporters, we discovered that even enhancers that show no promoter preference in a synthetic setting have a preference in the locus context. Our results suggest that the presence of multiple promoters in a locus is both due to enhancer preference and a need for redundancy and that “broad” promoters with dispersed transcription start sites are common among developmental genes. Our results also imply that it can be difficult to extrapolate expression measurements from synthetic reporters to the locus context, where many variables shape a gene’s overall expression pattern