Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Shannon Fisher

Abstract

The vertebrate skeleton forms via two distinct modes of ossification, membranous and endochondral. Osteoblasts are also heterogeneous in embryonic origin; bone formed by either mode can be derived from neural crest cells or mesoderm. In contrast, all bone develops via a common genetic pathway regulated by the transcription factor Runx2. Runx2 is required for bone formation, and haploinsufficiency in humans causes the skeletal syndrome cleidocranial dysplasia, demonstrating the importance of gene dosage. Despite the central role of Runx2 in directing bone formation, little is understood about how its expression is regulated in development. We took an unbiased approach to identify direct regulatory inputs into Runx2 transcription by identifying cis-regulatory elements associated with the human gene. We assayed conserved non-coding elements in a 1 Mb interval surrounding the gene for their ability to direct osteoblast expression in transgenic zebrafish. We identified three enhancers spaced out across the interval. Within each we identified conserved transcription factor binding sites required for their activity, and further showed distinct and specific regulation of each. The enhancer in the last intron of RUNX2 itself is positively regulated by the FGF signaling pathway, an enhancer in the last intron of the adjacent gene, SUPT3H, is regulated by canonical Wnt signaling, and a distant downstream enhancer requires a conserved Dlx binding site for its activity. While all of these pathways and factors have been previously implicated in bone formation, our results provide the first direct links to the common genetic pathway regulating osteogenesis, transcription of Runx2. These findings further illustrate the integration of multiple regulatory inputs at the level of transcription of a key developmental gene, and highlight the role of Runx2 as the gatekeeper for changes in skeletal morphology achieved through alterations in gene expression.

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