Yes, And: Looking beyond patterning genes and minimal enhancers in Drosophila segmentation

Abstract

During development, cells divide, move, and adopt specialized differentiated states. Precise spatial and temporal control of gene expression is central to this process and has been highly studied for decades in many model systems. However, the predominant focus has been on the effect of regulatory proteins that are themselves expressed in specific patterns, and minimal cis-regulatory DNA elements, such as enhancers, that drive those patterns. In my dissertation, I interrogated two additional aspects of developmental gene regulation and showed that they too affect spatiotemporal patterns of gene regulation: 1) ubiquitous trans-acting factors that are active throughout development and 2) cis-regulatory sequences beyond the boundaries of minimal enhancers. I pursued my studies in the Drosophila blastoderm embryo, given its substantial experimental advantages and extensive prior knowledge of the gene regulatory network that patterns the embryo.

Using a maternal-shRNAi system, I depleted members of the Polycomb (PcG) and Trithorax (TrxG) genes from embryos and assessed the effect on the patterning of the anterior-posterior patterning gene even-skipped (eve). PcG and TrxG are regulators involved in gene silencing and activation respectively and are often ubiquitously expressed. My results showed that these complexes affect patterning, often in subunit-specific ways (Chapter 2). One of the most dramatic phenotypes was observed after depletion of the PcG gene pleiohomeotic (pho), which impacted not only eve expression but also the upstream gap gene patterning network. These effects could not be readily explained by changes to the zygotic chromatin landscape indicating the phenotype could be not ascribed to the known activity of the Polycomb Group in the early embryo (Chapter 3). Lastly, by removing a critical binding site for the transcriptional repressor Giant from the eve2 minimal enhancer in both a reporter, an extended reporter, and in the endogenous eve locus, we showed that sequences beyond the minimal enhancer are functional and can buffer against mutation (Chapter 4). Together these results encourage us to look beyond minimal definitions of regulatory roles and regulatory sequences.

In Chapter 5, I present my work on biocensorship, a concept and term I developed and defined for my secondary work in Science, Technology, and Society.