(484.3) Investigating the Transition of the Core Centromeric Nucleosome Complex from Interphase to Mitosis using Chemical Biology Tools

Poster Board Number: A15

Praveen Kumar Allu (Biochemistry and Biophysics, Penn Center for Genome Integrity, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania), Rachel Lackner (University of Pennsylvania), David Chenoweth (University of Pennsylvania), Ben Black (University of Pennsylvania)

Centromeres are the location on each chromosome where kinetochores form in mitosis to connect to spindle microtubules that physically separate sister chromatids to the daughter cells. A 16-subunit complex termed the constitutive centromere associated network (CCAN) is bound to nucleosomes harboring the histone variant, CENP-A, and is essential for assembling the mitotic kinetochore. The core centromere nucleosome complex (CCNC) consists of CENP-A nucleosomes and CCAN subunits, CENP-C and CENP-N. In prior studies, we proposed that the CCNC undergoes an alteration at mitotic onset where one of two copies of CENP-N is lost. This model indicates that there would remain only one CENP-A nucleosome surface with both CENP-N and CENP-C that is competent to recruit the CCAN and, in turn, assemble the kinetochore. We hypothesize that the asymmetric surfaces on CENP-A nucleosomes provide a level of organization of centromeric chromatin that is important for chromosome segregation. We are developing a system to study these functional relationships in cells using chemical biology approaches. We have created a cell line with complementary SNAP and HALO tags on the CCNC components and employed the SNAP-HALO cross linker, HaXS8, to tether CCNC components (CENP-A with CENP-N) irreversibly on nucleosomes. Initial experiments indicate that this system is able to retain CENP-N at high levels on mitotic centromeres in an HaXS8 concentration-dependent fashion. This provides additional support for the model of a centromeric chromatin transition as cells enter mitosis, and our system holds promise to test predictions that arise from it.