Transcriptional regulation at the nuclear pore complex
By: by Christopher Richard Brown
Published: 2008
Language: eng
Number of pages: 205
Description: Chromosomes are non-randomly localized within the eukaryotic nucleus. While this sub-nuclear organization has been observed for nearly a century, the functional benefits of such positioning remain elusive. However, recent evidence from the budding yeast, Saccharomyces cerevisiae, has implicated the nuclear transport machinery as a key element in both the spatial organization of the genome and transcriptional regulation. We examined interactions between the nuclear transport machinery and the S. cerevisiae genome by performing genomic location analysis on several nucleoporins, β-karyopherins, and the Ran guanine nucleotide exchange factor (RanGEF). We found that the majority of our factors associated with transcriptionally active genes, while the RanGEF associated with inactive genes. In addition, genes associated with the nuclear transport machinery were enriched for Rap1 binding sites in their promoter regions, regardless of their transcriptional state. We also demonstrated the relocalization of the GAL locus to the nuclear periphery upon transcriptional induction, concomitant with a loss of binding to the RanGEF. Further studies examined the effects of a developmental stimulus, treatment with the mating pheromone alpha factor, on the global positioning of the yeast genome. We showed that upon alpha factor-induction, dozens of activated genes became associated with the nuclear transport machinery. Next, we focused our studies on the nuclear pore-associated myosin-like protein, Mlp1, a component of the nuclear pore that has a role in mRNA export. We showed that Mlp1 associated in a RNA-dependent manner with the majority of alpha factor-induced genes. In addition, we probed high-resolution, tiled microarrays of yeast chromosome III and found that Mlp1 associated with origins of replication, intergenic sites, and the 3'-ends of transcriptionally active genes.

To examine this process in higher eukaryotes, we performed genomic location analysis of Nup93 in HeLa S3 cells. We generated high-resolution, chromosome-wide binding maps of Nup93 in the presence and absence of a histone deacetylase inhibitor (HDACI). We found that Nup93-associated sites were enriched in promoter regions, euchromatin, and differentially expressed genes following HDACI treatment. In addition, we observed the physical relocalization of several genomic loci with respect to the nuclear periphery. In summary, this work has advanced our understanding of nuclear organization and transcriptional regulation. We show that the spatial positioning of active genes at the nuclear pore complex is a functionally important, global mechanism of transcriptional control. The demonstration of such differential positioning in human cells suggests that the nuclear periphery is a complex, evolutionarily conserved domain of transcriptional regulation in eukaryotic nuclei.
Notes: "December 2007."

Thesis (Ph.D., Division of Medical Sciences (Biological Chemistry and Molecular Pharmacology)--Harvard University, 2008.

Includes bibliographical references.