This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Actin and actin-related proteins (ARPs) are conserved members of several chromatin-modifying complexes including yeast remodelers (which harness the energy released from ATP hydrolysis to move nucleosomes) RSC, SWI/SNF, SWR1 and INO80 as well as HAT complex NuA4 (Bao and Shen, 2007). ARP7 and ARP9 are obligate heterodimers (Szerlong et al., 2003) and members of both RSC and SWI/SNF. Recently our lab identified the HSA (helicase and SANT associated), a conserved domain, as responsible for actin/ARP-association with these complexes forming an HSA/Arp7/Arp9 module (unpublished results). The HSA domain in RSC is located on catalytic subunit Sth1, and we identified a genetic and regulatory relationship between the ARP7/9-HSA and the ATPase domain of Sth1 (unpublished). As ARP2/3 function to seed actin-filament formation only by physically dimerizing on the helical portion of the VCA domain of WASP (Hitchcock-DeGregori, 2003), we believe ARP7 and ARP9 must dimerize on the HSA domain to function properly in RSC. We further believe that this module regulates the ATPase function of RSC, but need to identify the proteins outside this module that influence regulation. We would like to identify other proteins that bind the ARP7/9 dimer in RSC to further elucidate the mechanism of ARP regulation of RSC ATPase and remodeling activity. We propose performing a yeast two-hybrid screen with the Sth1-HSA domain (fused to the Gal4-DNA binding domain). The HSA domain will bind an ARP7/ARP9 dimer in vivo (unpublished), forming the module, a trimer. HSA/ARP module-binding partners, and thus, RSC ATPase-regulatory factors, should be identified in this screen. References:Bao, Y., and Shen, X. (2007). SnapShot: Chromatin Remodeling Complexes. Cell 129, 632.Hitchcock-DeGregori, S. E. (2003). Now, swing your partner! 3D-domain switching of WASP activates Arp2/3 complex. Nat Struct Biol 10, 583-584.Szerlong, H., Saha, A., and Cairns, B. R. (2003). The nuclear actin-related proteins Arp7 and Arp9: a dimeric module that cooperates with architectural proteins for chromatin remodeling. Embo J 22, 3175-3187.
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