A Multiplicity of Cis-Regulatory Elements Drive Regulatory Gene Expression for a Drosophila Pigmentation Trait

Date of Award

5-1-2025

Degree Name

M.S. in Biology

Department

Department of Biology

Advisor/Chair

Amit Singh

Abstract

An open question of biological inquiry is what are the genetic and molecular changes responsible for the diversification of animal form. While these changes remain largely unresolved, case studies suggest that alterations to how conserved genes are expressed will be often encountered. Hence, of high interest are the DNA sequences, known as cis-regulatory elements or CREs, which often act as enhancers of gene expression. Some of the best studied animal CREs function as enhancers that drive a specific aspect of a gene’s expression in “modular” manner, while additional modular enhancers are responsible for the other aspects of the regulated gene’s expression. This modularity is often given as an explanation as to why gene expression evolution plays such a large role in trait evolution. In recent years, a growing list of genes in multiple animal species has been shown to be regulated by redundant, non-modular, enhancers. In this thesis, we used the evolution of the male-specific, dimorphic, pattern of abdomen pigmentation of the Drosophila (D.) melanogaster fruit fly as a model trait to study the CREs controlling the expression of several key genes. 29 DNA sequences were evaluated from the D. melanogaster genome that were previously predicted by the SCRMshaw bioinformatic tool as potential pupal abdomen enhancers. Among these were four predicted CREs for both the grainy head and homothorax gene loci, and ten in the Eip74EF gene locus. RNA-interference for these three transcription factor genes revealed that each is needed for normal pigmentation. 22 of the 29 sequences were validated as pupal epidermis enhancers through transgenic Green Fluorescent Protein reporter transgene assays. This included two of the homothorax predications, and all of the grainy head and Eip74EF predictions. The two homothorax CREs were removed from the genome by a CRISPR/Cas9 homology dependent repair approach. This resulted in subtle, incompletely penetrant and variably expressive pigmentation phenotypes, consistent with a partially-redundant role for these CREs in homothorax expression regulation. Homothorax protein expression and the enhancer activities of the two CREs were shown to be conserved among fruit fly species with the derived dimorphic pigmentation and the ancestral monomorphic pigmentation phenotype. Similarly, the comparable sequences from the Eip74EF gene of the monomorphic D. willistoni had melanogaster-like enhancer activities in reporter transgene assays. Thus, this gene’s extensive CRE redundancy predated the origin of the dimorphic trait and has been conserved for more than 30 million years. The origin and diversification of this dimorphic fruit fly pigmentation trait has been connected in other studies to changes to modular CREs for genes that are part of the trait’s gene regulatory network. The work here demonstrates that this gene regulatory network also includes several important genes that are regulated by non-modular redundant CREs. Together these studies suggest that gene regulatory networks can include genes with modular regulation and genes with redundant regulation. When these traits evolve, changes can be anticipated for the CREs of modularly regulated genes, while the expression of the redundantly regulated genes will remain conserved.

Keywords

Biology, Evolution and Development, Genetics, Molecular Biology, Morphology

Rights Statement

Copyright 2025, author.

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