Wash interacts with Lamin and affects nuclear organization. Jeffrey M. Verboon, Hector Rincon, Tim Werwie, Tobias Ragoczy, Dave Scalzo, Steven Erikson, Jeff Delrow, Mark Groudine, Susan Parkhurst. Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N., Seattle, WA 98109.

   The Wiskott-Aldrich Syndrome (WAS) family proteins have been shown to promote the formation of branched actin in the cytoplasm by activating the Arp2/3 complex. However, there is a growing body of work suggesting that actin and actin nucleation factors may also have a role in the nucleus. We find that Wash, a WAS family member, is present in the nucleus and associates with specific chromosome regions. Futhermore, wash mutants have an altered nuclear morphology, where the normally smooth, spherical nuclear envelope is puckered and amorphous. Interestingly, we find that Wash directly binds to B-type Lamin, a nuclear intermediate filament that lines the inside of the nuclear envelope. Recently, Lamin has been shown to play a role in gene repression as specific chromosomal regions associate with Lamin at the nuclear envelope and these Lamin Associated Domains (LADs) correspond with transcriptionally inactive genome regions. We performed chromatin profiling for Wash and Lamin in Kc cells and find that ~85% of the chromosome regions that Wash associates with overlap with LADs. Lamin chromatin profiling in wash knockdown cells results in a significant loss of LADs indicating that Wash is necessary for the proper formation of LADs. We also find that general nuclear architecture is impaired in wash knockdown cells as we see a loss of the repressive marker HP1, nucleolar staining by fibrillarin, and Cajal bodies by coilin, as well as disruption of chromosome territories by FISH. Our results suggest that the proper tethering of genomic regions to the nuclear envelope by Wash and Lamin may not only be important for maintaining repressive LAD domains but may also function to help organize the nucleus. Currently, we are purifying Wash nuclear complexes to gain a better understanding of how Wash may be performing its nuclear functions.