Project Summary/Abstract This proposal outlines an investigation into the role of chromatin-tethering proteins in maintenance of peripheral heterochromatin and the integrity of nuclear lamina. It is well established that heterochromatin association with the nuclear lamina plays an important role in development, and contributes to maintenance of the adopted cell fate. Genetic mutations in lamina proteins, such as is seen in Hutchinson-Gilford progeria syndrome (HGPS), is associated with alterations in the morphology of the nuclear lamina and disruption of peripheral heterochromatin. However, the precise link between nuclear lamina mutations and heterochromatin positioning remains unclear. I propose to test if the chromatin-tethering protein PRR14 plays a critical role in this process and therefore functions in the premature aging pathway. Our data demonstrate that PRR14 organizes a significant fraction of heterochromatin at the nuclear lamina. Lack of PRR14 results in detachment of the peripheral heterochromatin from the nuclear lamina, while PRR14 overexpression substantially increases the fraction of heterochromatin located at the nuclear periphery. Previous findings implicate PRR14 as an epigenetic repressor and its loss can release transcriptional gene silencing. Furthermore, our results demonstrate that PRR14 loss results in nuclear lamina defects similar to those observed in diseases associated with nuclear lamina mutations. Here we propose to study the role of PRR14 in maintenance of chromatin organization at the nuclear lamina and assess how nuclear lamina mutations act through PRR14 to cause dysregulation of lamina-associated chromatin. We will determine the effect of PRR14 loss on heterochromatin organization at the nuclear lamina and define epigenetic features that mark genome regions for nuclear lamina localization via PRR14. Further, we will determine how nuclear lamina mutations affect the PRR14 function in organizing chromatin at the nuclear lamina by testing how Progerin (a mutant form of Lamin A protein) affects the PRR14-nuclear lamina interaction and triggers nuclear lamina and peripheral heterochromatin defects. Finally, to determine if PRR14-nuclear lamina association is affected by farnesyl group modification on Progerin, we will test whether farnesyltransferase inhibitors restore PRR14 function in tethering heterochromatin in HGPS cells. Our work will provide critical insights into the fundamental knowledge of chromatin association with the nuclear lamina. This would significantly advance our understanding of molecular mechanism behind nuclear lamina and genome organization defects observed in laminopathy diseases.