Interdisciplinary Graduate Program
Biochemistry and Molecular Pharmacology
First Thesis Advisor
Job Dekker, Ph.D.
Gene Expression Regulation, Chromosomes, Nuclear Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic
Long-range chromosomal interactions have been discovered in a number of organisms, suggesting that gene regulation through direct physical association with regulatory elements and/or other genes is a common and conserved phenomenon. This thesis investigates the relationship between direct physical contact of genomic loci and how these interactions may play a role in gene regulation. Analysis of such levels of chromosomal organization has been made possible in part by the emergence of Chromosome Conformation Capture (3C). This technique makes use of formaldehyde crosslinking to trap interacting chromosomal fragments, which can be detected after a number of manipulations. By adapting the 3C protocol for use in intact Saccharomyces cerevisiaecells, one can determine the structure of a chromosome or chromosomal region without introducing artifacts due to the harsh isolation of nuclei.
A number of 3C-based technologies, such as 4C (Circular 3C or 3C-on-Chip) and 5C (3C Carbon Copy) have added to the knowledge of physical association of genes with regulatory elements and/or other genes. Here, we present a new non-biased technology that allows for determination of chromosomal interactions between all fragments throughout a genome. We present two-dimensional heatmaps of chromosomal interactions for all 16 chromosomes in yeast. These techniques promise to shed light onto the biochemical process by which clustering of genes and elements can result in up- or down-gene expression, which is still poorly understood.
To understand how chromosomal interactions play a role in gene regulation, we study clustering of heterochromatic loci. Clustering of heterochromatic loci in silenced nuclear compartments is a phenomenon that has been observed throughout evolution. These clusters are thought to represent nuclear sub-compartments that are enriched in silencing proteins, while the rest of the nucleus is depleted in such factors. Chromosome III in Saccharomyces cerevisiae contains four heterochromatic regions: the two telomeres and the silent mating type loci, HML and HMR, located on either end of the chromosome. Our work describes a long-range interaction between the heterochromatic regions on chromosome III. We analyze the mechanism that drive these interactions and reveal roles for silencing proteins and proper nucleosome assembly in mediating heterochromatic clustering. In addition we identify a novel step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.
Miele, A. Analysis of Long-Range Chromosomal Interactions in Saccharomyces cerevisiae: A Dissertation. (2009). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 413. DOI: 10.13028/bcde-vk98. https://escholarship.umassmed.edu/gsbs_diss/413
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