More than just on and off: exploring enhancer function and regulation during early embryonic development.

Enhancers are cis-regulatory elements that regulate when and where genes are expressed. These functions are mediated by transcription factors that bind to sites within the enhancer sequence. It is still an open question how transcription factors find their binding sites in an expanse of genomic DNA or how they facilitate enhancer activity. My doctoral work aims to address these questions in part, through the lens of Drosophila melanogaster embryogenesis. In the early Drosophila embryo, enhancers regulate gene expression in refined spatial patterns, setting a foundation for the adult body plan. In chapter 1, I discuss enhancer based regulation of gene expression - what is currently known about how enhancers function and how they themselves are regulated in the context of embryonic development./nIn the nucleus, genomic DNA forms higher-order chromatin structures. However, enhancers and promoters that are transcriptionally active are often highly accessible. In chapter 2, I seek to understand how chromatin accessibility is established around active enhancers and promoters by asking if chromatin accessibility is spatially patterned across the embryo in a way that correlates with transcriptional activity. I used genome-wide methods to assay chromatin accessibility in anterior and posterior halves of Drosophila embryos. I found that genome-wide, chromatin accessibility is remarkably similar in the anterior and posterior half but that enhancers show greater accessibility in the half of their activity. These data are consistent with a model where a uniform chromatin landscape is established first and is later refined by spatially-patterned transcriptional activity./nZelda is a transcription factor that is necessary for enhancer activation in the early Drosophila embryo. Zelda has been observed to form clusters of high concentration in the nucleus. In chapter 3, I seek to address whether Zelda’s clustering activity is related to its regulatory functions by asking whether these clusters are near transcriptional loci. To answer this question, I developed a method to measure Zelda protein abundance at twenty-three Zelda targets and twenty-five non-targets in the same nucleus. I found that Zelda protein is modestly enriched at targets compared to non-targets. This enrichment is most likely driven by a small percentage of loci that reside in regions of high intensity Zelda protein./nThroughout my PhD, I have sought a method to create large numbers of site-specific mutants in vivo. This is challenging in Drosophila and other organisms where transgenic lines are generated by injection with low transformation rates. Mutating and rearranging binding sites has been largely successful at identifying important enhancer elements, like transcription factor binding sites, in the past. Therefore, a method to create large numbers of site-specific mutants would greatly enhance our ability to comprehensively dissect regions of the genome at greater scale. In chapter 4, I describe a method for generating diverse populations of site-specific enhancer variants where each fly laid in a cross possesses at most one copy of the mutant enhancer. I then show preliminary evidence that this method can efficiently generate diverse mutations. Though this method is not yet fully developed, it is the beginning of a new approach for characterizing enhancers that can be applied to any genomic region.

Advisor: Eisen, Michael B