Ciren, Danielle (February 2024) Decoding cis-regulatory control and evolution of conserved and divergent phenotypes in plants. PhD thesis, Cold Spring Harbor Laboratory.
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Abstract
Eukaryotic gene expression is regulated by specific genetic and physical interactions among cis-regulatory elements (CREs), which ensure gene expression is coordinated to a precise level, in a particular cell type, and at a specific time during development. This precision is facilitated by multiple epigenetic modifications, constantly remodeling the chromatin to exclude or encourage transcription factor binding at CREs. CREs are located in every genomic context, including upstream, downstream, within the gene itself (in the UTRs, introns, and exons), and even at distal sites that create DNA loops to contact promoters. It is the combination of multiple CREs, in multiple genomic locations, that come together to regulate genes in space and time. Throughout this thesis work, we have studied CREs and their interactions within an evolutionary framework, using a functional genetics approach. We sought to explore two main questions about the evolution of regulatory regions. The first, how do conserved genes in distantly related organisms maintain similar functions and spatiotemporal expression patterns, often amidst drastic cis-regulatory sequence divergence? And the second, how does variation in CREs contribute to phenotypic divergence? Using CRISPR-Cas9, we generated 89 unique mutations in cis-regulatory regions upstream and downstream of the dosagesensitive developmental genes CLAVATA3 (CLV3) and SELF PRUNING 5G (SP5G), and quantified the phenotypic effect. CLV3 is highly conserved among Arabidopsis and tomato, despite extremely diverged regulatory regions. We find evidence supporting a billboard model of CRE organization, in which the particular transcription factor bindings sites regulating CLV3 in both species are conserved, however their arrangement (spacing, order, orientation, etc.) is more flexible to change. In contrast, closely related species of wild and domesticated tomato have different flowering time responses to daylength, which has been attributed to differences in expression of SP5G. We found that multiple CREs are involved in the regulation of SP5G in the wild species, although none of our engineered cisregulatory alleles mimicked the domestication phenotype on its own. Further investigation into the genetic and physical interactions among SP5G CREs, as well as their molecular consequences, will 2 enhance our understanding of potential mechanisms of phenotypic divergence. Thus, we have found evidence for robustness and higher order complexity within cis-regulatory regions, in the context of both conserved and diverged traits. This work has explored fundamental principles of gene regulation using a functional genetics approach rarely applied in the field. Here we present a new perspective on cis-regulatory mechanisms of evolution, using in vivo mutagenesis experiments that have provided an improved understanding of the functional, phenotypic relevance of CREs and their interactions in the regulation of genes.
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