UBC Theses and Dissertations
Identifying gene regulatory networks controlled by bone morphogenetic protein-signaling in Drosophila and murine genomes Othonos, Katerina Maria
Bone morphogenetic proteins (BMPs) are a group of phylogenetically conserved signaling proteins, first identified to play important roles in bone formation. Since their discovery, they have been recognized to contribute to embryonic development and adult homeostasis in a multitude of tissues, by regulating cellular lineage commitment, morphogenesis, differentiation, proliferation, and apoptosis. BMPs transduce their signals through intracellular downstream effectors, primarily the Smad transcription factors, many of which bind to genomic BMP-responsive cis-regulatory elements (BMP-CREs) to direct gene expression. Despite their importance in cellular processes and maintenance, BMP-CREs remain largely unidentified at a genomic level for most BMP-dependent cellular processes. The overall objectives of this thesis were to experimentally characterize the widespread function of a novel low-affinity BMP-CRE motif in the Drosophila nervous system and to identify the BMP-driven regulatory network underlying mammalian chondrogenesis. To address the first goal, we used computational methods to identify this novel BMP-CRE through the Drosophila genome and used in vivo transgenic reporters to determine their function in the Drosophila nervous system. Our results show that this BMP-CRE is used within multiple enhancers to mediate their BMP-dependent activity. For our second goal, we used poly-A transcriptome sequencing (RNA-seq) to characterize differentially expressed genes (DEGs) during chondrogenesis in primary murine cells. Amongst these DEGs, we identified transcription factors/cofactors with previously unknown roles in chondrogenesis that are of interest for further study. Further, we used histone modification ChIP-seq to identify more than 2000 candidate regulatory regions in the vicinity of BMP-responsive DEGs. Using computational tools, we examined these candidate regulatory regions for Smad-binding sites using BMP-CRE motifs identified in Drosophila. We then applied multiple selection criteria to prioritize likely BMP-responsive regulatory regions and assessed four novel regions for BMP-responsive reporter expression, using mouse primary limb mesenchymal (PLM) cells. Among these, we identified two BMP-responsive regulatory regions, including one within 50kb of the transcription factor Jdp2, a gene with previously unknown roles in chondrogenesis. The genomic mapping of BMP-CREs remains incomplete. Mutations in these cis-regulatory sites and BMP-regulated genes could potentially result in disease, and therefore their identification is of critical importance to help further our understanding of disorders in various human tissues.
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