Date of Award

Spring 5-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Photoreceptors are light-sensitive neurons that mediate vision, and they are the most commonly affected cell type in genetic forms of blindness. In mice, there are two basic types of photoreceptors, rods and cones, which mediate vision in dim and bright environments, respectively. The transcription factors (TFs) that control rod and cone development have been studied in detail, but the cis-regulatory elements (CREs) through which these TFs act are less well understood. To comprehensively identify photoreceptor CREs in mice and to understand their relationship with gene expression, we performed open chromatin (ATAC-seq) and transcriptome (RNA-seq) profiling of FACS-purified rods and cones. We find that rods have significantly fewer regions of open chromatin than cones (as well as >60 additional cell types and tissues), and we demonstrate that this uniquely closed chromatin architecture depends on the rod master regulator Nrl. Finally, we find that regions of rod- and cone-specific open chromatin are enriched for distinct sets of TF binding sites, providing insight into the cis-regulatory grammar of these cell types.

We also sought to understand how the regulatory activity of rod and cone open chromatin regions is encoded in DNA sequence. Cone-rod homeobox (CRX) is a paired-like homeodomain TF and master regulator of both rod and cone development, and CRX binding sites are by far the most enriched TF binding sites in photoreceptor CREs. The in vitro DNA binding preferences of CRX have been extensively characterized, but how well in vitro models of TF binding site affinity predict in vivo regulatory activity is not known. In addition, paired-class homeodomain TFs bind DNA as both monomers and dimers, but whether monomeric and dimeric CRX binding sites have distinct regulatory activities is not known. To address these questions, we used a massively parallel reporter assay to quantify the activity of thousands native and mutant CRX binding sites in explanted mouse retinas. These data reveal that dimeric CRX binding sites encode stronger enhancers than monomeric CRX binding sites. Moreover, the activity of half-sites within dimeric CRX binding sites is cooperative and spacing-dependent. In addition, saturating mutagenesis of 195 CRX binding sites reveals that, while TF binding site affinity and activity are moderately correlated across mutations within individual CREs, they are poorly correlated across mutations from distinct CREs. Accordingly, we show that accounting for baseline CRE activity improves the prediction of the effects of mutations in regulatory DNA from sequence-based models. Taken together, these data demonstrate that the activity of CRX binding sites depends on multiple layers of sequence context, providing insight into photoreceptor gene regulation and illustrating functional principles of homeodomain TF binding sites.

Language

English (en)

Chair and Committee

Joe C. Corbo

Committee Members

Shiming Chen, Donald F. Conrad, Kristen L. Kroll, Gary D. Stormo,

Comments

Permanent URL: https://doi.org/10.7936/0t56-bs28

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