UC Berkeley

Regardless of size, shape, or function, all cells must rapidly respond to a changing environment, especially in adverse conditions. Various environmental stresses including heat shock, osmotic stress, and nutrient starvation consequently induce dramatic changes in the molecular composition of cellular machinery. Importantly, the cell's adjustment to a new homeostasis is accomplished through a variety of mechanisms, but most predominantly through the regulation of gene expression which is normally thought to function through the interplay of specific transcriptional repressors and activators. Recent evidence suggests, however, that more novel forms of transcriptional regulation may exist, including the repositioning of specific genes to distinct subdomains within the nucleus and through global changes in genome organization. Moreover, it has been hypothesized that such changes may function to supplement more traditional models of transcriptional regulation. Thus, upon environmental stress, cells may dramatically modify global transcriptional programs through a reorganization of their genome, facilitating a more rapid response to an adverse environment. The budding yeast Saccharomyces cerevisiae represents a remarkable model for elucidating how the organization of the genome and the repositioning of specific genes may function to regulate gene expression. In yeast, certain cellular stresses are known to stimulate the repositioning of genes within the nucleus and alter the global organization of the genome. Despite considerable research into the dynamic nature of genome organization, however, many questions remain as to the true function of gene positioning and the mechanism(s) by which cells establish specific organizational conformations of the genome.

In the following dissertation, I first provide evidence on the function of repositioning a specific gene to the nuclear periphery by examining the consequences of improperly localizing the GAL locus of budding yeast. Next, I describe my attempts to discern the coordinated diffusion of co-regulated genes as well as my findings on the independent movement of loci positioned various distances apart on the same chromosome. Finally, I describe novel findings on the mechanism of chromatin mobility through experiments focused on understanding the biological determinants of intracellular diffusion. Together, my results suggest that chromatin mobility is, in part, actively driven, which may function to facilitate more rapid alterations in genome organization and thus more rapid regulation of gene expression.

I first set out to understand the function of repositioning specific genes to distinct locations within the nucleus. In yeast, the GAL locus, comprised of GAL1, GAL7, and GAL10 and necessary for the metabolism of galactose, re-localizes from a central position of the nucleus when repressed to the nuclear periphery when transcriptionally active. To understand the function of this specific repositioning, I took advantage of mutants in different macromolecular complexes, namely the nuclear pore complex and the SAGA transcriptional complex, which both fail to properly localize the GAL locus. I demonstrate that the GAL locus is negatively regulated at the nuclear periphery as both mutants, when compared to wild- type, displayed massively upregulated transcription of GAL1 upon activation and a distinct lag in repression. Importantly, the increased induction kinetics of GAL1 mRNA is mirrored by increased expression levels of GAL1 protein, suggesting that the over-expressed GAL1 mRNA is functional and that export of the mRNA is not perturbed despite the mislocalization of the GAL locus. In my model, the GAL locus is positioned at the nuclear periphery to facilitate more rapid transcriptional repression when cells encounter their preferred carbon source, glucose.

Co-regulated genes may localize to transcriptional complexes known as transcription factories, hypothesized to facilitate more rapid and efficient transcription through colocalization and sequestration of specific transcriptional machinery. Because the GAL locus is known to re-localize at or near nuclear pore complexes upon activation, which may provide a scaffold for such transcriptional machinery, I next sought to observe transcription-induced coordinated diffusion of the two GAL loci within diploid yeast. To do so, I utilized a microscope capable of tracking both uniquely-labeled loci simultaneously using a double-helical point spread function. Although I did not discern evidence of potential transcription factories, I did reveal insights into the flexibility of chromatin by analyzing intrachromosomal loci separated by known distances. Overall, my results suggest that coordinated mobility of distinct loci correlates directly with their distance apart in space, regardless if they are intra or interchromosomal.

Finally, I explored the mechanism of chromatin mobility and, thereby, the driving force behind the re-organization of the genome by comparing the diffusion rates of chromatin and other macromolecules in distinct conditions. I began my investigation by starving cells of glucose and determining the effect on macromolecular mobility. Interestingly, both chromatin and mRNPs (messenger ribonucleoprotein particles) display a massive confinement in their mobility in such conditions. The reduction in mobility cannot be explained by a decrease in ATP, but can be replicated through a reduction of intracellular pH. I then show that both glucose starvation and reduction of intracellular pH induce a decrease in cell volume, increasing molecular crowding and reducing macromolecular mobility. Furthermore, I demonstrate a dependence of chromatin mobility on the cytoskeleton as simultaneous treatment with actin and microtubule depolymerizers specifically confines chromatin mobility. Lastly, by uncoupling metabolism and macromolecular mobility I propose that the preservation of an optimal cell volume is signaled independently from the energy status of the cell and discuss implications and future directions of this work.