Decoding the Non-coding Genome: Novel Technologies for the Characterization of Non-coding Elements and Variation

One of the key frontiers in genomics research is decoding the function of non-coding sequence and variation. Non-coding sequence, once thought to be junk DNA, is now known to regulate gene expression in a tissue-specific manner, and is frequently found to be mutated in cases of complex human disease. Despite their importance in human disease, non-coding regions are vastly understudied compared to protein coding regions. This is in part due to the abundance of non-coding sequences currently predicted to comprise 98.8% of the genome compared to protein coding regions, which make up only 1.2%. To complicate things further, most of this sequence is non-functional. A non-coding mutation may lead to a change in gene expression or a difference in human phenotype, yet it could show no change in gene expression at all. Therefore, there is considerable demand for novel computational and experimental tools focused on identifying functional non-coding sequences, and prioritizing variation associated with gene expression regulation and human disease. The focus of the work in this dissertation is the development of novel tools to identify functional non-coding regulatory sequences, and to prioritize the variation that falls within these sequences. I will introduce the following computational tools, the SNP Effect Matrix Pipeline (SEMpl) and the SNP Effect Matrix Pipeline with Methylation (SEMplMe). These methods integrate data from genome-wide annotations of functional elements, such as sites of transcription factor protein binding (ChIP-seq), open chromatin (DNase-seq), and DNA methylation (WGBS), to generate predictions of the consequences of nucleotide and methylation changes to binding affinity in transcription factor binding sites. As transcription factor binding sites are the building blocks of larger regulatory sequences, such as regulatory elements, functional alterations caused by the introduction of a variant or DNA methylation may lead to aberrant gene expression. SEMpl and SEMplMe are easy to use tools to help researchers prioritize the hundreds of putative regulatory variants that emerge from high-throughput studies, such as genome-wide association studies. This will greatly increase the rate at which non-coding variation can be experimentally validated. I will also introduce experimental tools focused on identifying larger blocks of regulatory non-coding sequence: cis-regulatory elements. Cis-regulatory elements are sequences that are able to alter or drive gene expression. Currently, a large body of in- formation exists for regulatory elements that are associated with an increase in gene expression, known as positive regulatory elements. However, regulatory elements associated with a decrease in gene expression, also known as negative regulatory elements, are comparatively understudied. To help fill this gap in knowledge between positive and negative regulatory elements, I helped develop two novel methodologies that are able to invert negative regulation into a positive reporter signal. The observed positive output allows negative regulatory elements to be characterized in a spatio-temporal manner in vivo in whole animals. This advancement will allow negative regulatory elements to be studied in a manner similar to what has already been achieved for positive regulatory elements for the first time. Together, the studies in this dissertation investigate non-coding regulatory sequence genome-wide through the development of novel tools which prioritize regulatory variation and identify and characterize regulatory elements.PHDGenetics and Genomics PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/166151/1/ssnishi_1.pd

The Inducible lac Operator-Repressor System Is Functional in Zebrafish Cells

BackgroundZebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, an E. coli lac operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish.ResultsHere we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish.ConclusionOur results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities

SEMplMe: a tool for integrating DNA methylation effects in transcription factor binding affinity predictions

Abstract Motivation Aberrant DNA methylation in transcription factor binding sites has been shown to lead to anomalous gene regulation that is strongly associated with human disease. However, the majority of methylation-sensitive positions within transcription factor binding sites remain unknown. Here we introduce SEMplMe, a computational tool to generate predictions of the effect of methylation on transcription factor binding strength in every position within a transcription factor’s motif. Results SEMplMe uses ChIP-seq and whole genome bisulfite sequencing to predict effects of methylation within binding sites. SEMplMe validates known methylation sensitive and insensitive positions within a binding motif, identifies cell type specific transcription factor binding driven by methylation, and outperforms SELEX-based predictions for CTCF. These predictions can be used to identify aberrant sites of DNA methylation contributing to human disease. Availability and Implementation SEMplMe is available from https://github.com/Boyle-Lab/SEMplMe .http://deepblue.lib.umich.edu/bitstream/2027.42/173166/1/12859_2022_Article_4865.pd

Integrating climate adaptation and transboundary management: Guidelines for designing climate-smart marine protected areas

Climate change poses an urgent threat to biodiversity that demands societal responses. The magnitude of this challenge is reflected in recent international commitments to protect 30% of the planet by 2030 while adapting to climate change. However, because climate change is global, interventions must transcend political boundaries. Here, using the California Bight as a case study, we provide 21 biophysical guidelines for designing climate-smart transboundary marine protected area (MPA) networks and conduct analyses to inform their application. We found that future climates and marine heatwaves could decrease ecological connectivity by 50% and hinder the recovery of vulnerable species in MPAs. To buffer the impacts of climate change, MPA coverage should be expanded, focusing on protecting critical nodes for the network and climate refugia, where impacts might be less severe. For shared ecoregions, these actions require international coordination. Our work provides the first comprehensive framework for integrating climate resilience for MPAs in transboundary ecoregions, which will support other nations’ aspirations. © 2023 The AuthorsOpen access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Integrating climate adaptation and transboundary management:Guidelines for designing climate-smart marine protected areas

Climate change poses an urgent threat to biodiversity that demands societal responses. The magnitude of this challenge is reflected in recent international commitments to protect 30% of the planet by 2030 while adapting to climate change. However, because climate change is global, interventions must transcend political boundaries. Here, using the California Bight as a case study, we provide 21 biophysical guidelines for designing climate-smart transboundary marine protected area (MPA) networks and conduct analyses to inform their application. We found that future climates and marine heatwaves could decrease ecological connectivity by 50% and hinder the recovery of vulnerable species in MPAs. To buffer the impacts of climate change, MPA coverage should be expanded, focusing on protecting critical nodes for the network and climate refugia, where impacts might be less severe. For shared ecoregions, these actions require international coordination. Our work provides the first comprehensive framework for integrating climate resilience for MPAs in transboundary ecoregions, which will support other nations’ aspirations.</p