Doctor of Philosophy, Case Western Reserve University, 2018, Genetics

The ability to culture stem cells in vitro has provided access to a wide variety of cellular states that are inaccessible or too rare to study otherwise. Here we demonstrate the use of stem cells to model two distinct developmental transitions, and examine the role that epigenetic regulation of transcription plays in controlling stem cell fate and function.First, we examine the transition from mouse embryonic stem cells (mESCs), representative of the pre-implantation epiblast, to mouse epiblast stem cells (mEpiSCs), representative of the post-implantation epiblast. These two states, while maintained by distinct signaling pathways, both retain the ability to re-integrate into their respective tissue of origin and contribute to the development of the entire organism. While the transcriptome of the two cell types is largely similar, they rely on dramatically different sets of enhancers to regulate that expression. 97% of the genes that are shared between the two states undergo a change in enhancer usage in the transition. The enhancers that arise in the mEpiSC state are present, but inactive, in the preceding mESC state and become enhancer clusters downstream in development.Second, we examine the development of oligodendrocytes from oligodendrocyte progenitors (OPCs). Oligodendrocytes wrap neural axons in a lipid-rich sheath known as myelin. This myelin is required for proper signal conduction, but is subject to immune attack in multiple sclerosis (MS). OPCs replace damaged oligodendrocytes early in the course of disease, but eventually fail. A chemical genetics screen reveals that BET bromodomain proteins are required for oligodendrocyte development. These proteins are epigenetic readers that integrate chromatin state and other signals to allow transcription elongation at many genes. Other inhibitors of elongation show similar effects on development, and treatment with BET bromodomain inhibitor (S)-JQ1 blocks activation of oligodendrocyte genes. Elongation genes are e (open full item for complete abstract)

Committee: Paul Tesar PhD (Advisor); Peter Scacheri PhD (Committee Chair); Helen Salz PhD (Committee Member); Ahmad Khalil PhD (Committee Member) Subjects: Bioinformatics; Biology; Developmental Biology; Genetics

Master of Science, Miami University, 2024, Biology

The acquisition of wings is often regarded as the main driving force for the evolutionary success of insects. Although the exact tissues that contributed to the evolution of wings are still hotly debated, the wing is generally considered to have evolved from a part of the body wall and/or associated branch structures. But the detailed molecular mechanisms underlying wing evolution are still elusive. Decades of studies in Drosophila have revealed a large network of genes (wing gene network, WGN) that governs the development of wings. The vestigial gene (vg) is a key gene in the WGN. Interestingly, a network of several wing genes, including vg, also operates in the body wall of various insects and even non-insect arthropods, suggesting that a network like the WGN (preWGN) in the body wall precedes the emergence of insect wings. Here we provide evidence that wing enhancers of vg evolved through modification of ancestral body wall enhancers. We demonstrated that (i) wing and body wall enhancers of vg share the location at the vg locus, (ii) wing and body wall activities of vg boundary enhancer are inseparable, and (iii) STREs within the enhancers are essential for wing activity but dispensable from body wall activity.

Committee: Yoshinori Tomoyasu (Advisor); Katia Del Rio-Tsonis (Committee Member); Jennifer Schumacher (Committee Member) Subjects: Biology; Evolution and Development

Ph.D., Antioch University, 2023, Antioch New England: Marriage and Family Therapy

The current study is quantitative research that used a web-based survey from multiple religious denominations in the United States to examine the relationships among perceptions of stress, work-family spillover, marital satisfaction, and family functioning of pastors. Emotional exhaustion, hours worked, personal accomplishment, and social support were analyzed as predictors from the work domain. Two measures of spillover were used to evaluate family stressors and enhancers. The stressors and enhancers were tested as mediators between work domain and personal/family life. Life satisfaction, marital satisfaction and family dysfunction were analyzed as outcome variables. The sample included 83 pastors from a convenience sample including 62 males and 21 females, with backgrounds diverse in culture, education, and denomination. Analyses were conducted using IBM SPSS (Version 26), including Pearson's correlations along with regression analysis using PROCESS macro to test for mediation. It was found that social support does increase family enhancers and work-family positive spillover but contrary to predictions, personal accomplishment appeared to impact family life by increasing emotional exhaustion, stressors, and family dysfunctions. Greater incidents of emotional exhaustion and hours worked were found to reduce the incident of enhancers and work-family positive spillover and increase family stressors and work-family negative spillover. Greater incidents of emotional exhaustion were found to reduce marital satisfaction and increase family dysfunctions. This research has practical and clinical implications for pastors, spouses, and children of pastors, educators, family scientists, therapists, and organizations who employ pastors.

Committee: Kevin Lyness PhD (Committee Chair); Lucille Byno PhD (Committee Member); Janet Robertson PhD (Committee Member) Subjects: Families and Family Life; Individual and Family Studies; Mental Health; Occupational Health; Psychotherapy; Therapy

PhD, University of Cincinnati, 2023, Medicine: Molecular and Developmental Biology

DNA of the human genome contains sequences which encode for functional gene products, but also contains sequences which regulate gene expression. Cis-regulatory elements (CRE) are critical for coordinating gene expression programs that dictate cell-specific differentiation and homeostasis. Recently developed self-transcribing active regulatory region sequencing (STARR-Seq) has allowed for genome-wide annotation of functional CREs. Despite this, STARR-Seq assays are only employed in cell lines, in part, due to difficulties in delivering reporter constructs. Herein, we implemented and validated a STARR-Seq–based screen in human CD4+ T cells using a non-integrating lentiviral transduction system. Lenti-STARR-Seq is the first example of a genome-wide assay of CRE function in human primary cells, identifying thousands of functional enhancers and negative regulatory elements (NREs) in human CD4+ T cells. We find an unexpected difference in nucleosome organization between enhancers and NRE: enhancers are located between nucleosomes, whereas NRE are occupied by nucleosomes in their endogenous locations. We also describe chromatin modification, eRNA production, and transcription factor binding at both enhancers and NREs. Our findings support the idea of silencer repurposing as enhancers in alternate cell types. Collectively, our study suggests that Lenti-STARR-Seq is a successful approach for CRE screening in primary human cell types and provides an atlas of functional CREs in human CD4+ T cells.

Committee: Artem Barski Ph.D. (Committee Chair); Raphael Kopan Ph.D. (Committee Member); Matthew Weirauch Ph.D. (Committee Member); Ralph Vatner (Committee Member); Louis Muglia M.D. Ph.D. (Committee Member) Subjects: Biology

PhD, University of Cincinnati, 2023, Medicine: Molecular Genetics, Biochemistry, & Microbiology

An important cell-to-cell signaling pathway, Notch signaling, is involved in the development of nearly every organ in metazoans. The Notch signaling pathway is important in both development and homeostasis and is critical for viability. Canonical Notch signaling occurs when a signal sending cell with a ligand on the cell surface interacts with a receptor on the cell surface of a signal receiving cell. Upon this interaction, two cleavage events take place, releasing the intracellular domain of Notch (NICD). NICD translocates into the nucleus and binds to the transcription factor CSL along with a coactivator named Mastermind. The formation of this complex allows for activation of target genes. Regulation of the Notch signaling pathway is very important and changes to Notch signal strength can have profound effects on Notch-dependent processes. Decreases in Notch signal strength have been associated with developmental syndromes such as Adams-Oliver syndrome and Alagille syndrome. Increases in Notch signal strength have been linked to a number of cancers, including T-cell acute lymphoblastic leukemia (T-ALL). However, we still have much to learn about how changes in Notch signal strength affect cell-specific responses. We unexpectedly found that simply inserting enhancers with multiple dimeric Notch binding sites (SPSs) next to binding sites for the Grainyhead pioneer transcription factor into Drosophila melanogaster flies resulted in wing nicks, a classic Notch phenotype in flies. To better understand this wing nicking phenomenon, we sought to define the parameters or rules that contribute to this Notch-like wing nicking phenotype. To do so, we designed and generated synthetic reporter assays, used a combination of bioinformatic and biochemical approaches, performed Drosophila genetics, and carried out chromatin accessibility experiments. Surprisingly, we found that only some, but not all, endogenous SPSs can replace the synthetic SPSs and still produce wing nicks in t (open full item for complete abstract)

Committee: Brian Gebelein Ph.D. (Committee Chair); Kenneth Campbell Ph.D. (Committee Member); William Miller Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Makiko Iwafuchi-Doi Ph.D. (Committee Member) Subjects: Genetics

Doctor of Philosophy, Miami University, 2022, Biology

Scientists have long been fascinated by morphological novelties, which at times seem to spring out of the ancestral form from no pre-existing structure (or structures, i.e. a complex novel trait). With molecular biology, it is relatively straightforward to work out the genes and regulatory interactions responsible for the proper development of these structures in extant species. However, what is more important from an evolutionary perspective (and more difficult to determine) is how a developmental gene regulatory network (GRN) is first pieced-together to create a novel structure. An intriguing possibility is that two or more ancestral GRNs may become cross-wired to drive the formation of a complex novel structure that is radically different from its origin tissues. The objective of this dissertation is to better understand how pre-existing GRNs from more than one origin tissue may combine to spark the origin of a complex morphological novelty, focusing on the origin of the insect wing. This chapter will provide background knowledge on the evolutionary impact of wing origin on the insects (Section 1.2), as well as relevant information on the development and morphology of the wings and proposed origin tissues (Section 1.3). This is followed by a brief history of the wing origin debate utilizing traditional comparative morphology (Section 1.4), a review of the major components of the wing GRN in the fruit fly (Section 1.5), and finally, a discussion on what evo-devo studies have discovered regarding the origin of the wing GRN (Section 1.6).

Committee: Yoshinori Tomoyasu (Advisor); Xin Wang (Committee Member); Michael Robinson (Committee Member); Jennifer Schumacher (Committee Member); Paul James (Committee Member) Subjects: Biology; Developmental Biology; Entomology; Evolution and Development

Doctor of Philosophy, Case Western Reserve University, 2020, Pathology

Meningiomas are the most common primary intracranial tumor with current classification offering limited therapeutic guidance. Here, we interrogated meningioma enhancer landscapes from 33 tumors to stratify patients based upon prognosis and identify novel meningioma-specific dependencies. Enhancers robustly stratified meningiomas into three biologically distinct groups (adipogenesis/cholesterol, mesodermal and neural crest) distinguished by distinct hormonal lineage transcriptional regulators. Meningioma landscapes clustered with intrinsic brain tumors and hormonally-responsive systemic cancers with meningioma subgroups reflecting progesterone or androgen hormonal signaling. Enhancer classification identified a subset of tumors with poor prognosis, irrespective of histological grading. Super enhancer signatures predicted drug dependencies with superior in vitro efficacy to treatment based upon the NF2 genomic profile. Inhibition of DUSP1, a novel and druggable meningioma target, impaired tumor growth in vivo. Collectively, epigenetic landscapes empower meningioma classification and identification of novel therapies.

Committee: Jeremy Rich M.D., M.B.A., M.H.Sc (Advisor); Mark Jackson Ph.D. (Committee Chair); Paul Tesar Ph.D. (Committee Member); Drew Adams Ph.D. (Committee Member); Clive Hamlin Ph.D. (Committee Member); Nicholas Ziats Ph.D. (Committee Member); Marvin Natowicz M.D.,Ph.D. (Committee Member) Subjects: Bioinformatics; Biomedical Research; Cellular Biology; Oncology

Doctor of Philosophy, The Ohio State University, 2020, Molecular Genetics

Gene regulation is controlled by a variety of factors including cis-regulatory sequences, chromatin modifications, and trans-acting proteins. In some cases, the regulatory state of an allele can be transmitted to its homolog and maintained through meiosis through a process known as paramutation. I present my work in identifying and characterizing two genes whose function maintains the repressed state of the Pl1-Rhoades (Pl1-Rh) allele which is subject to paramutation. required to maintain repression3 (rmr3) mutants have a defect in a gene encoding an RNA polymerase (RNAP) subunit (RPB/D/E3) which participates in RNAP II and plant specific RNAP IV and V complexes as well as growth defects and a necrotic leaf phenotype. rmr12 encodes a chromodomain helicase DNA-binding 3 (CHD3) protein which in addition to its role in regulating Pl1-Rh has roles in development, pollen function, and gene regulation. I compare the genes differentially expressed in rmr12 mutants to those differentially expressed in mutants for other rmr factors which are primarily associated with RNAP IV function and 24 nucleotide (nt) RNA biogenesis. I highlight that RNAP IV has a much larger role in gene regulation than other factors involved in 24nt RNA biogenesis contributing to the model that RNAP IV has a 24nt RNA-independent role in regulating RNAP II transcription. I also demonstrate that RNAP IV and rmr12 have a moderate but significant overlap in targets suggesting a relationship between these factors not explained by current models of either RNAP IV or CHD3 function. I also characterize the molecular features of a tandem repeat in the Pl1-Rh allele which may serve as an enhancer and demonstrate that 24nt RNA production differs at this sequence between active and repressed allele states. Together my work investigates the relationships between repeat sequences, gene expression and gene silencing and the factors which mediate this repression including RNAP IV function, sRNA biogenesis, and ch (open full item for complete abstract)

Committee: Jay Hollick (Advisor); David Bisaro (Committee Member); Mark Parthun (Committee Member); Robin Wharton (Committee Member) Subjects: Genetics

Master of Science (MS), Wright State University, 2020, Anatomy

High-throughput genome-scale studies are becoming increasingly common as a means to discover genetic interactions. This methodology is particularly efficient when performed in the budding yeast Saccharomyces cerevisiae. Here, we (1) overexpress a large human gene library in yeast to assess how many of them are toxic, and (2) use the list of genes generated above to refine and analyze human genes previously identified to enhance toxicity of two ALS-associated proteins, FUS and TDP-43. By introducing each of 13,500 human genes into yeast, we demonstrated that the majority of these genes (about 97%) are not toxic to yeast when overexpressed. These results indicated that toxicity of human genes, such as FUS and TDP-43, are very likely due to their interactions with specific cellular pathways, rather than simply a non-specific effect of their overexpression in yeast. This is supported by our analysis showing that the toxic human genes are enriched in RNA metabolic processes and DNA transcription, both of which are conserved between yeast and human. Our lab previously identified a preliminary list of 685 human genes that enhance toxicity of FUS and TDP-43 in yeast. However, none of these genes were tested for their own toxicity, so they were possibly false positives, and never were removed from the list. Using the toxic genes identified above, we refined the enhancers list from 685 to 358 genes, out of which 138 genes enhanced TDP-43 toxicity, 335 genes enhanced FUS toxicity, and 115 genes enhanced both. Interestingly, functional classification of the genes that enhance toxicity from both FUS and TDP-43 revealed a group of cell cycle regulators that have been linked to the DNA damage response (DDR) and repair. Given that FUS and TDP-43 are RNA-binding proteins that have been implicated in DDR, these results suggest a possible mechanism of FUS and TDP-43 toxicity involving their abnormal activation of the cell cycle.

Committee: Shulin Ju Ph.D. (Advisor); Thomas Brown Ph.D. (Committee Member); Christopher Wyatt Ph.D. (Committee Member) Subjects: Cellular Biology; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2017, Chemical Engineering (Engineering and Technology)

Microbiologically influenced corrosion (MIC) has been a challenge in the oil and gas industry as well as other industries such as water treatment facilities, nuclear power plants and pulp and paper plants. Sulfate-reducing bacteria (SRB) are considered a major factor because sulfate is a widely available oxidant in anaerobic environments. This work studied the influence of flow effects on SRB biofilm growth in a glass cell bioreactor. Correlation between the cylindrical coupon rotation rate in the glass cell and the average flow velocity in the pipeline was theoretically derived with rough surface taken into account. Deadlegs attached to pipelines often are more prone to MIC. Because there is often no flow or low flow near the bottom of a deadleg, an open flow loop for MIC investigation in deadlegs using small flow rates to achieve similar mass transfer effects to fast flow pipeline was proposed. Mass transfer simulation using the computational fluid dynamics (CFD) software FLUENT was employed. Unlike regular recirculating flow loops used in most MIC studies, this setup was better at emulating real MIC in pipelines, avoiding the recycling of metabolic byproducts and damage of cells due to the high shear stress in the recycling pump. MIC treatment is usually about biocide treatment. Because of increasing environmental concerns and tightening regulations, a more environmentally benign treatment to mitigate MIC was evaluated in this work, utilizing biodegradable chelators such as ethylenediaminedisuccinate (EDDS) and N-(2-Hydroxyethyl)iminodiacetic acid disodium salts (HEIDA) to enhance biocide performance against planktonic and sessile SRB. It showed that biocide dosage to control biofilm growth could be reduced considerably when a chelator was used with the biocide. Furthermore, when 10% to 15% (v/v) methanol was added to the binary combination of biocide and EDDS treatment, mitigation of planktonic SRB growth was improved from an inhibiting effect to a kill (open full item for complete abstract)

Committee: Tingyue Gu (Advisor); Srdjan Nesic (Committee Member); Daniel Gulino (Committee Member); Peter Coschigano (Committee Member); Hao Chen (Committee Member) Subjects: Chemical Engineering; Engineering

MS, University of Cincinnati, 2017, Medicine: Genetic Counseling

Chromosomal microarrays have been used for over a decade to improve clinical diagnostic rates by identifying copy number variations (CNVs) across the genome. CNVs in noncoding regions of the genome are difficult to interpret because less is understood about the etiology of disease in these cases. Topologically-associating domains (TADs) describe the higher order chromatin structure that creates a landscape for enhancer-promoter interaction. The human genome has roughly two thousand such regions marked by high levels of chromatin interaction within these regions and separated by genomic boundaries, across which interaction occurs much less frequently. These domains and boundaries may provide the underlying genomic infrastructure for gene regulation and long range activation or repression of gene expression. Disruption of the boundary regions has been associated with human disease in two types of limb abnormalities and in other small disease specific populations. To date only one study has estimated the impact of TAD boundary deletions in an affected population and that study focused on large, known pathogenic deletions that were likely to disrupt multiple boundaries. In this study, a large scale, quantitative, comparative case-control study design was used to examine the presence of deletions across TAD boundaries by comparing patients with clinical indications for microarray testing to population-based controls. 5033 patients with a broad variety of clinical indications for microarray were compared with 312 controls to assess overall deletion burden, enrichment of topologically-associated domain boundary region disruption, and phenotypic trends among patients. A nonparametric t-test was used to assess the difference in the proportion of deletions which span an entire TAD boundary in cases compared to controls. No significant difference was found between cases and controls when both heterozygous and homozygous deletions were compared together. However, cases had sign (open full item for complete abstract)

Committee: Kristen Sund Ph.D. (Committee Chair); Iouri Chepelev Ph.D. (Committee Member); Lisa Dyer Ph.D. (Committee Member); Lisa Martin Ph.D. (Committee Member); Auvo Reponen Ph.D. (Committee Member); Teresa Smolarek Ph.D. (Committee Member) Subjects: Health Sciences

Doctor of Philosophy, Case Western Reserve University, 2016, Pathology

Brain cancer is a devastating disease and a dreaded diagnosis for patients. Cancer within the brain, more than any other organ in the body, more than skin or colon or breast, is a difficult diagnosis to treat and live with because the brain is not just a part of the person, it is the person. Memories, personality, emotions, intelligence, speech and movement all originate in the brain. Therefore, when cancer, or the treatments used to fight the cancer, affect the brain by disrupting or damaging an area of the brain that controls these functions, it not only changes the body of the person, but changes the person. It is a particularly cruel disease. Glioblastoma is the most common and aggressive form of malignant brain cancer, which we currently have no way to effectively treat. The majority of patients die within 16 months of receiving a diagnosis, many times following aggressive surgery, radiation and chemotherapy, and then a sequelae of declining physical and cognitive function. Effective therapies are urgently needed for these patients. Many studies have identified potential therapeutic targets through in vitro screens or genomic sequencing, but this has not led to effective clinical therapies. Here we present a novel method of identifying potential drug targets in glioblastoma using advanced RNA interference screening in an in vivo orthotopic microenvironment. Using this system, we discover that screening within a functional microenvironment reveals novel targets that have been missed by traditional in silico or in vitro screening. We also demonstrate that our targets identified in vivo are more clinically relevant than targets we were able to identify in vitro using the same system. We focused our screening on epigenetic modifier genes, as many of these enzymes are sensitive to microenvironmental conditions and are potentially druggable. Within epigenetic modifier genes, we revealed that factors involved in transcription pause-release and elongation ar (open full item for complete abstract)

Committee: Jeremy Rich MD, MHSc, MBA (Advisor); Paul Tesar PhD (Advisor); Alan Levine PhD (Committee Chair); Peter Scacheri PhD (Committee Member); Bruce Trapp PhD (Committee Member); Steven Rosenfeld MD, PhD (Committee Member); Mark Jackson PhD (Committee Co-Chair); Clive Hamlin PhD (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Genetics; Medicine; Molecular Biology; Neurology; Pathology

Doctor of Philosophy, Case Western Reserve University, 2015, Genetics

Transcriptional enhancer elements are essential in the establishment and maintenance of gene expression programs that define cellular identity and function. DNA variants that predispose to common disease, identified by genome-wide association studies (GWAS), frequently lie in enhancer elements. Interpreting the impact of DNA variants on enhancer function and transcriptional output is key to determining the role of the disease-associated variants in the pathogenesis of disease. Here we present PreSTIGE (Predicting Specific Tissue Interactions of Genes and Enhancers), a method that identifies the specific gene targets of enhancers, and apply this methodology to delineate the impact of disease-associated variants on enhancer function. We demonstrate that for six autoimmune traits, GWAS association signals often arise from multiple polymorphisms in linkage disequilibrium (LD) that map to clusters of active enhancer elements. This result suggests a “multiple enhancer variant” hypothesis, in which several variants in LD alter enhancer function and cooperatively affect transcript levels. Multiple enhancer variants within a given locus are determined by PreSTIGE to target the same gene. To address the question of whether additional variants within a target gene's regulatory circuitry can alter the transcriptional effect of the locus, we identified variants that are inherited independently of known GWAS risk loci and interact with disease-associated variants. The interacting SNPs lie in enhancer elements that are constituents of the regulatory circuitry of the same gene. These variants, termed “outside variants” functionally modify the effect of the risk variants on target transcript levels and ultimately alter clinical risk. To further our understanding of the regulatory circuitry that dictates gene expression levels, we evaluated how transcriptional control is established and altered during early embryogenesis. We found that the regulatory circuitry of genes expressed (open full item for complete abstract)

Committee: Peter Scacheri (Advisor); Paul Tesar (Committee Chair); Thomas LaFramboise (Committee Member); Michael Harris (Committee Member) Subjects: Genetics

PhD, University of Cincinnati, 2010, Pharmacy : Pharmaceutical Sciences

Transdermal pharmaceutical formulations, in most cases, contain chemical penetration enhancers. To properly select efficient chemical enhancers, an understanding of their mechanism(s) of action is essential. This dissertation evaluated the enhancement effects of known topical or cosmetic ingredients on human epidermal membrane (HEM) permeation. Permeation enhancement effect, Emax, was identified as enhancement effect induced by the enhancer on HEM permeation as the enhancer approaches the thermodynamic activity of its pure state in equilibrium with HEM. A structure enhancement relationship was established for the chemical enhancers studied. Emax was shown to be dependent of the lipophilicities. A guideline for the selection of chemical enhancers based on efficiency and duration of enhancement was determined based on octanol/water partition coefficient and calculated n-octanol solubility. This dissertation also studied topical formulations consisting of a volatile carrier system containing the chemical enhancers. To fully understand the enhancer induced permeation enhancement mechanism, the transport domain of skin i.e., the stratum corneum (SC) lipid domain was probed. This involved the determination of both enhancer and permeant uptake into the lipid domain of SC following enhancer treatment. It was concluded that the permeation enhancement mechanism is attributed to the enhancement of permeant partitioning into the transport rate limiting domain. Liposomes formulated from extracted human stratum corneum lipids (EHSCLL) were characterized and used to directly study this domain. The uptake of the chemical enhancers within the EHSCLL was determined and the results suggested a quantitative relationship between enhancer uptake in EHSCLL and enhancer efficiency. DSC and ATR-FTIR studies using enhancer treated intact SC supported that the mechanism of chemical enhancers is via their fluidization and perturbation of the SC lipid bilayer. This finding suggests that the enh (open full item for complete abstract)

Committee: Kevin Li PhD (Committee Chair); Gerald Kasting PhD (Committee Member); Giovanni Pauletti PhD (Committee Member); Apryll Stalcup PhD (Committee Member); R. Randall Wickett PhD (Committee Member); Abdel-Halim Ghanem PhD (Committee Member) Subjects: Pharmaceuticals

Master of Science (M.S.), University of Dayton, 2012, Biology

Phenotypes are the culmination of the spatial and temporal patterns of gene expression for numerous genes that comprise a gene regulatory network (GRN). These patterns are controlled by cis-regulatory elements (CREs). Genes are connected into GRNs when a CRE regulating its expression possess binding sites for network transcription factor proteins - so called regulatory linkages. Gains and losses of these linkages are candidates for a mechanistic path of CRE and GRN evolution; though linkage emergence remains poorly understood, as few case studies have revealed the ancestral and derived regulatory states in sufficient detail. The male-specific abdominal pigmentation of Drosophila melanogaster evolved from a sexually monomorphic ancestral state, with a key modification to the pigmentation GRN being the evolution of sexually dimorphic expression of the Bab transcription factor proteins. These proteins turn off expression of the yellow and tan genes that are required for melanic abdominal pigmentation. The research presented here addresses two questions. First, does Bab form direct regulatory linkages with the CRE(s) that control the male-specific expression of the Drosophila melanogaster yellow and tan genes? Second, when historically did these CREs and their regulatory linkages evolve? To answer these questions I: systematically mutated CRE sequences to expose motifs needed to integrate the repressive effects of Bab, and evaluated the regulatory activities of sequences related by common descent to the Drosophila melanogaster CREs. The results reveal the complex CRE encodings of putative Bab linkages and that these CREs and linkages evolved in the lineage of dimorphic species after it diverged from the lineage of monomorphic species. Future studies are needed to determine whether this divergence included the gain of Bab binding sites in the CREs of dimorphic species.

Committee: Thomas Williams PhD (Advisor); Mark Nielsen PhD (Committee Member); Amit Singh PhD (Committee Member) Subjects: Biology; Evolution and Development; Genetics; Molecular Biology