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M.S., Rutgers University - 2003
Thesis Advisor: Marc Gartenberg, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology
School of Public Health Building
2nd Floor Conference Room
Monday, April 23, 2012
Faithful segregation of genomes is arguably one of the most important goals of cell division, essential for viability and development of every life form. Chromosome segregation in eukaryotes requires that sister chromatids are held together from S-phase when they are generated until anaphase onset when two identical sets of chromosomes separate. This adhesive process, termed sister chromatid cohesion, is mediated by an evolutionarily conserved protein complex aptly named cohesin. This ring-shaped complex binds near centromeres to assure bi-orientation on spindle microtubules. Cohesin binds elsewhere on chromosomes and recent evidence suggests that such binding influences other chromosome-centered events like gene regulation and the repair of DNA damage. Genes encoding cohesin subunits or factors that regulate the subunits have also been implicated in human diseases, now collectively known as cohesinopathies. These include Warsaw Breakage Syndrome, Cornelia de Lange Syndrome and Roberts Syndrome. Most is known about Cornelia de Lange Syndrome where the disease pathology is thought to result from dysregulation of critical developmental genes. Less is known about Roberts syndrome where the most striking cellular distinction is abnormal loss of cohesion in heterochromatic domains. The disease is caused by mutation of the Esco2, a protein acetyltransferase that activates cohesin by acetylating specific subunits.
I used yeast Saccharomyces cerevisiae to explore two aspects of sister chromatid cohesion. In the first area of focus, I characterized the impact of a Roberts Syndrome patient derived mutation on sister chromatid cohesion in yeast. In general, I found that cohesion of chromosomal arms persisted. However, I discovered that cohesion of localized domains of heterochromatin as well as euchromatin was disrupted. In the case of heterochromatin where most of my studies were centered, loss of cohesion activity was not accompanied by loss of the cohesin complex from DNA. In addition, heterochromatic genes remained transcriptionally silenced, in agreement with other Gartenberg lab studies that have found that cohesion was dispensable for steady-state repression of the loci studied. I concluded that Roberts Syndrome mutations can cause localized cohesion defects without changes in gene silencing, and that these cohesion defects can be masked in assays for global chromatid cohesion.
In budding yeast most of the cohesin on chromosomal arms binds intriguing positions relative to euchromatic genes. In my second area of focus, I investigated the factors that contribute to cohesin binding and function at a model euchromatic gene, URA3. Unexpectedly, I found that the DNA bound transcriptional activator, Ppr1, was necessary and sufficient for cohesion of the gene, even when the gene was not induced. The protein supports cohesion by enriching cohesin on the gene. I also studied the influence of transcriptional induction on cohesion of the gene. Remarkably, the topology of the DNA template influenced the fate of cohesion, which did not strictly correlate with the fate of cohesin binding. The results are discussed in terms of how the ring shaped cohesin complex binds euchromatic regions and how this complex responds to RNA polymerase movement.