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Genomic analysis of ribosomal DNA and its application to the investigation of disease pathogenesis

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2011, Doctor of Philosophy, Case Western Reserve University, Genetics.
The synthesis of rRNA is critical to all growing organisms, accounting for well over half of all cellular transcription. Highlighting its central function in cellular life, dysregulation of rRNA biogenesis and subsequent ribosome assembly has been implicated in human genetic diseases and cancer. While the transcription of rRNA is highly regulated at the chromatin level, it has not been analyzed by genomic methods due to the exclusion of rDNA from current genome assemblies. This work describes a novel method of analysis that enables the alignment of high-throughput sequencing data to a single copy of rDNA in the context of the full human genome. Integrated analysis of genomic datasets reveals that the coding region of rDNA is contained within nucleosome-poor open chromatin with high transcriptional activity. We find that histone modifications are enriched not only at the rDNA promoter but also at novel sites within the noncoding intergenic spacer. The distribution of active modifications is more similar within and between cell types than that of repressive modifications. Using ChIP-seq, we show that the nucleolar protein UBF is bound to sites throughout the genome and may play a role in regulating the transcription of nucleoplasmic genes. Lastly, the insulator-binding protein CTCF is bound to a site proximal to the junction between adjacent rDNA repeats, potentially indicating a role for transcriptional insulation in the regulation of rRNA transcription. We apply this method to a disease-relevant protein, CHD7, a chromatin-remodeling enzyme mutated in the developmental disorder CHARGE syndrome. CHARGE syndrome shares several clinical features with known disorders of ribosome biogenesis. ChIP-seq analysis reveals robust association of CHD7 across the transcribed region of rDNA. Immunofluorescence and subcellular fractionation confirm the nucleolar localization of a substantial fraction of CHD7. Knockdown experiments show that CHD7 functions to positively regulate rRNA biogenesis by counteracting DNA methylation at the rDNA promoter. Lastly, CHARGE-relevant tissues from Chd7-mutant mouse embryos display reduced levels of precursor rRNA. Taken together, these studies provide a novel means for assessing protein occupancy at rDNA that can be applied to disease-relevant chromatin-associated proteins in order gain novel insights into disease pathogenesis. Additionally, the work presented herein defines a novel role for CHD7 as a positive regulator of rRNA synthesis and suggests that dysregulated rRNA synthesis is involved in the pathogenesis of CHARGE syndrome.
Peter Scacheri, PhD (Advisor)
Guangbin Luo, PhD (Committee Chair)
Helen Salz, PhD (Committee Member)
Derek Abbott, MD/PhD (Committee Member)
224 p.

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Citations

  • Zentner, G. E. (2011). Genomic analysis of ribosomal DNA and its application to the investigation of disease pathogenesis [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1321478238

    APA Style (7th edition)

  • Zentner, Gabriel. Genomic analysis of ribosomal DNA and its application to the investigation of disease pathogenesis. 2011. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1321478238.

    MLA Style (8th edition)

  • Zentner, Gabriel. "Genomic analysis of ribosomal DNA and its application to the investigation of disease pathogenesis." Doctoral dissertation, Case Western Reserve University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1321478238

    Chicago Manual of Style (17th edition)