Nucleocytoplasmic Communication in Progeria

Hutchinson Gilford Progeria Syndrome (HGPS) is a rare and devastating syndrome that affects children and is characterized by premature aging. It is caused by a mutation in the gene encoding for lamin A. Lamin A is an intermediate filament protein that forms polymers and associates with other proteins to form a network that sits on the nuclear side of the inner nuclear envelope, called the lamina. Lamin A is farnesylated after translation and its farnesyl group is inserted into the nuclear membrane. During its maturation, lamin A is cleaved from the nuclear envelope by the metalloproteinase Zmpste24. The mutant form of lamin A, termed progerin, lacks the Zmpste24 cleavage site. Thus, while mature wild-type lamin A is not directly attached to the nuclear envelope, progerin retains its attachment, altering the lamina structure. This disruption of the nuclear lamina results in nuclear morphology changes, a global reduction in heterochromatin marks, and gene expression changes. Children who have this disease display aging symptoms that include atrophy of subcutaneous muscle and fat, alopecia, and atherosclerotic disease. Their life expectancy is approximately 13 years. Since the progerin mutation was discovered in 2003, work has focused on identifying cellular phenotypes in Progeria. However, the pathways that connect progerin to HGPS cellular phenotypes remain unclear. A major driving hypothesis in the Progeria field is that the reduction in heterochromatin at the nuclear periphery exhibited by patient cells results in the misexpression of age-related genes. In this study, we present an alternative mechanism by which changes in chromatin affect downstream cellular phenotypes. RCC1 is a chromatin-associated protein and is also a regulator of the RanGTPase. These two proteins are essential for nuclear transport. Here, we hypothesize that changes in the nuclear lamina lead to perturbation of chromatin, which affects the functions of RCC1 and Ran. As these are vital players in nuclear transport, their reduced function is predicted to affect many cellular processes. In chapter 3 of this work we show that the Ran, which is normally highly concentrated in the nucleus, is mislocalized in HGPS patient fibroblasts. We explore the link between the mislocalization of Ran and the reduction of heterochromatin methyl marks in Progeria. Treatment of normal fibroblasts with BIX01294, a compound that inhibits the methyltransferases responsible for dimethylation of lysine 9 on histone 3, is sufficient to disrupt Ran. These data suggest a possible pathway whereby chromatin perturbation can result in Ran system changes. In chapter 4, we explore the consequences that result from Ran mislocalization. One major function of Ran is the dissociation of import complexes entering the nucleus. Surprisingly, we find that the disruption of Ran localization does not inhibit bulk nuclear import. Instead, it specifically affects the import of large cargo. One affected cargo, the nucleoporin Tpr, imports into the nucleus as a dimer at 543 kD. We found…