Developing and assessing the use of demonstration videos in the preclinical simulation laboratory.

PURPOSE: This study aims to report the development of a preclinical simulation laboratory Demonstration Video Series (DVS) for the 2021-2022 academic year, measure its usage and usefulness, and compare these findings to the usage and usefulness of the existing didactic lecture videos. METHODS: The DVS videos were intended to be viewed before each preclinical simulation laboratory session along with the pre-existing didactic lectures (DL) by University of California San Francisco (UCSF) learners. Usage measurements included the percentage of the class that viewed each video, the number of views that each video received, and the average duration of each video that was watched. Usefulness of the videos was measured by a survey that assessed learner perspective on knowledge and ability to apply that knowledge during the simulation lab exercises. Both usage and usefulness of the DVS were then compared to the usage and usefulness of the DL. Both descriptive statistics and independent sample hypothesis tests were performed to compare the differences in proportion between DVS and DL mediums. RESULTS: Statistically significant differences were found in terms of both usage and usefulness of the DVS compared to the DL, with DVS being utilized more overall. With an 81% response rate, survey analysis revealed statistically significant differences among the learners' perspectives on the usefulness of the DVS compared to the DL, with a clear preference for the DVS over the DL and an overwhelmingly positive perception of the DVS. CONCLUSION: The DVS was found to be a valuable addition to the preclinical laboratory sessions for first-year learners.

Two promoters integrate multiple enhancer inputs to drive wild-type knirps expression in the Drosophila melanogaster embryo.

Proper development depends on precise spatiotemporal gene expression patterns. Most developmental genes are regulated by multiple enhancers and often by multiple core promoters that generate similar transcripts. We hypothesize that multiple promoters may be required either because enhancers prefer a specific promoter or because multiple promoters serve as a redundancy mechanism. To test these hypotheses, we studied the expression of the knirps locus in the early Drosophila melanogaster embryo, which is mediated by multiple enhancers and core promoters. We found that one of these promoters resembles a typical "sharp" developmental promoter, while the other resembles a "broad" promoter usually associated with housekeeping genes. Using synthetic reporter constructs, we found that some, but not all, enhancers in the locus show a preference for one promoter, indicating that promoters provide both redundancy and specificity. By analyzing the reporter dynamics, we identified specific burst properties during the transcription process, namely burst size and frequency, that are most strongly tuned by the combination of promoter and enhancer. Using locus-sized reporters, we discovered that enhancers with 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 due both to enhancer preference and a need for redundancy and that "broad" promoters with dispersed transcription start sites are common among developmental genes. They also imply that it can be difficult to extrapolate expression measurements from synthetic reporters to the locus context, where other variables shape a gene's overall expression pattern.

Molecular competition can shape enhancer activity in the Drosophila embryo.

Transgenic reporters allow the measurement of regulatory DNA activity in vivo and consequently have long been useful tools for studying enhancers. Despite their utility, few studies have investigated the effects these reporters may have on the expression of other genes. Understanding these effects is required to accurately interpret reporter data and characterize gene regulatory mechanisms. By measuring the expression of Kruppel (Kr) enhancer reporters in live Drosophila embryos, we find reporters inhibit one another's expression and that of a nearby endogenous gene. Using synthetic transcription factor (TF) binding site arrays, we present evidence that competition for TFs is partially responsible for the observed transcriptional inhibition. We develop a simple thermodynamic model that predicts competition of the measured magnitude specifically when TF binding is restricted to distinct nuclear subregions. Our findings underline an unexpected role of the non-homogenous nature of the nucleus in regulating gene expression.

Enhancer redundancy in development and disease.

Shadow enhancers are seemingly redundant transcriptional cis-regulatory elements that regulate the same gene and drive overlapping expression patterns. Recent studies have shown that shadow enhancers are remarkably abundant and control most developmental gene expression in both invertebrates and vertebrates, including mammals. Shadow enhancers might provide an important mechanism for buffering gene expression against mutations in non-coding regulatory regions of genes implicated in human disease. Technological advances in genome editing and live imaging have shed light on how shadow enhancers establish precise gene expression patterns and confer phenotypic robustness. Shadow enhancers can interact in complex ways and may also help to drive the formation of transcriptional hubs within the nucleus. Despite their apparent redundancy, the prevalence and evolutionary conservation of shadow enhancers underscore their key role in emerging metazoan gene regulatory networks.