Year: 2023
Type: article
Abstract: Watching documentaries as a child, I became fascinated by how genomes encode the instructions to make all the cells of an organism. I studied Biochemistry at Oxford University as the subject seemed to provide a mechanistic understanding of living systems. During my time at Oxford, I completed my part II thesis (similar to a master's project) in Prof. Doug Higgs' lab. I learned about the regulation of gene expression during development of blood cells and the disease ATRX which causes alpha thalassemia and neurological defects in patients via misregulation of gene expression. While we were studying the effects of this disease on gene expression within the blood system, I wondered if studying both the blood and the brain may help find generalizable principles and mechanisms driving the disease. For this reason, I wanted to do my Ph.D. in a system where I could study many different types of cells. I decided to work with stem cells and transcription factors involved in the specification of cell fate. I did my Ph.D. at Imperial College London at the MRC London Medical Sciences Center with Dr. Meng Li. I studied how midbrain dopaminergic neurons are made in developing mouse and chick brains and applied this knowledge to stem cells to create dopaminergic neurons in a dish. The hope was that these stem cell-derived dopaminergic neurons would serve as a platform for drug screening and therapeutic approaches for patients with Parkinson's disease. While I value the stem cell system, at the time, it was not the ideal system to explore how genomes encode gene expression in time and space. My stem cell cultures were often heterogenous; a mixture of neural-like cells and other cells, most commonly cardiac cells beating in the dish. And one could never truly know if the cells in the dish recapitulated the endogenous dopaminergic neurons. Through my research experiences, I thought that experimental approaches in whole developing embryos would be better suited for understanding how our genomes encode the instructions for making an organism. I set about looking for a system in which I could study enhancers in high-throughput within whole developing organisms. Prof. Mike Levine spoke about Ciona at a British Society of Developmental Biology meeting, and I was hooked. I realized that Ciona, with its close relation to vertebrates and the power of electroporation to incorporate plasmids into millions of embryos, would be an ideal organism for whole embryo high-throughput reporter assays to study enhancers. Thus, Ciona is an ideal system to decipher how the instructions for development are encoded in our genomes. I started my postdoc with Prof. Mike Levine in 2012. I developed a synthetic enhancer library screen (SEL-seq) to test many millions of enhancers for activity in developing Ciona (Figure 1a). I used SEL-Seq to test 2.5 million variants of a neural Otx-a enhancer to examine how this enhancer activated by two pleiotropic factors (ETS and GATA) encodes neural-specific expression within the anterior sensory vesicle and dorsal nerve cord (Farley et al., 2015) (Figure 1b). From these screens, we discovered that enhancers need low or suboptimal affinity transcription factor binding sites to correctly encode tissue-specific expression. If these low-affinity sites are replaced with high-affinity sites, then the enhancer is no longer restricted to the neural lineages but is also active in many other tissues where FGF signaling or GATA are present. These studies illustrate that the use of suboptimal affinity sites is critical to ensure that the enhancer remains under combinatorial control of ETS and GATA and is only active where the concentration of these two factors is just right. Similar results showing that low-affinity Hox sites were important for specificity in flies were also reported (Crocker et al., 2015). We also found that the organization of sites (order, spacing, and orientation) in the endogenous Otx-a sequence is not optimal for the highest level of transcription (Farley et al., 2015). Changing the spacing between the sites within the enhancer could increase the levels of transcription, giving stronger neural expression. Indeed, optimizing the affinity and spacing within a version of the Otx-a enhancer results in a complete loss of tissue specificity; expression is no longer restricted to the neural tissue (a6.5 and b6.5 lineages) but is also seen in the notochord, endoderm, and posterior sensory vesicle (Figure 1b,c). The use of low-affinity sites that are non-optimally organized prevents aberrant activation of the enhancers by a single factor and means that combinatorial control is required to activate transcription (Farley et al., 2015). The realization that enhancers contain incredibly degenerate binding sites was alarming as it meant enhancers were even more complex than we originally thought. Luckily, we noticed a relationship between the affinity and organization of the binding sites. Low-affinity sites within native enhancers had an organization that led to higher levels of transcriptional output, while higher-affinity sites had less optimal spacing for transcriptional output. Thus, there appeared to be an interplay between affinity and organization of binding sites (enhancer grammar) (Farley et al., 2015; Farley et al., 2016; Jindal & Farley, 2021). As a postdoc and in my own lab, we have used these grammatical rules to find tissue-specific enhancers within the genome (Farley et al., 2016; Song et al., 2023). In my own lab at UC San Diego, I continue to harness Ciona for high-throughput enhancer screens to find rules governing enhancers. We have found signatures of enhancer grammar conserved across chordates—in Ciona, mice, and humans (Song et al., 2023). We've also expanded to look at how violations in regulatory principles governing enhancers can drive organismal-level phenotypes in Ciona and other species, such as mice and humans (Jindal et al., 2023; Lim et al., 2022). We've found that single nucleotide variants (SNVs) can increase binding site affinity driving ectopic expression and organismal phenotypes as severe as a second heart in Ciona and extra fingers in mice and humans (Jindal et al., 2023; Lim et al., 2022). Thus, the principle of suboptimization of developmental enhancers to encode tissue-specific expression, which we initially discovered in Ciona, applies to other organisms too. Furthermore, violating this principle leads to major phenotypes in tunicates and vertebrates. I am incredibly grateful to Prof. Mike Levine for supporting me while I pursued the high-throughput enhancer screens in Ciona. If it were not for him, I would not have been able to realize these experiments, which formed the foundation of the research conducted within my own lab. While occasionally challenging, the Levine lab was also fun. I'd never been around so many people who loved enhancers as much as I did. My time in the Levine lab was full of exciting conversations and brainstorming, which helped me develop into the scientist I am today. Now in my own lab, I enjoy being surrounded by enhancerophiles all the time. So far, two graduate students and one postdoc have graduated from my lab and joined industry. My lab now consists of two postdocs, three graduate students, and three undergraduates (Figure 2). I hope many of them remain in the Ciona community. To support my research, I've been fortunate to receive the NIH New Innovator Award, NSF CAREER Award, and NHGRI R01. I'd like to thank everyone who has worked with me during the course of my research. Special thanks to all members of the Farley lab, past and present, for contributing to our discoveries. I'd also like to thank all Levine lab alumni, but especially members of the Levine lab from 2012-2016, for helpful discussions and support. Thanks to Doug Higgs for introducing me to gene regulation and his continued support and advice. Thank you Meng Li and Emily Gale for mentoring me through my PhD. Finally, thank you, Mike Levine, for introducing me to the amazing model organism and proto-vertebrate – Ciona, and for supporting me as I developed high-throughput enhancers screens in Ciona.
Source: genesis
DOI: 10.1002/dvg.23577
Author: Emma Kirsten Farley
Institution: University of California San Diego
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,450
OpenAlex ID: W4389037979