Regulation of centromeres by the anaphase promoting complex [electronic resource]

The centromere is a specialized chromatin domain that is required for kinetochore formation and chromosomal attachment to the mitotic spindle. In higher eukaryotes, centromere identity is independent of the underlying DNA sequence and is thought to be propagated through epigenetic mechanisms. One of the defining features of centromeric chromatin is the presence of the histone H3 variant CENP-A, which is localized exclusively at centromeres. Many fields of evidence suggest that CENP-A incorporation serves as the epigenetic mark maintaining centromere identity. CENP-A loading is under strict temporal control, and unlike conventional histones is uncoupled from DNA replication; instead, CENP-A is loaded exclusively during late anaphase through earl G1 of the cell cycle. Uncovering the mechanisms that control the precise timing and exclusive localization of CENP-A incorporation will deepen our understanding of aspects fundamental to cell division and epigenetics. In order to take an unbiased approach to discovering genes involved in centromeric function, we undertook a whole genome RNAi screen in Drosophila looking for genes whose depletion resulted in a loss of CENP-A phenotype. In our screen, we discovered that the constitutive centromere protein CENP-C and newly discovered protein CAL1 were in a complex with CENP-A and required CENP-A loading. Additionally, we discovered that RNAi of the drosophila homologues of EMI1 and Cyclin A result in a loss of CENP-A through premature activation of the anaphase promoting complex (APC), which we theorized resulted in the destruction of an unidentified APC substrate required for CENP-A loading. Intrigued by the connection between the timing of CENP-A loading and APC activation, we adapted an in-vitro degradation assay to assess the possibility that Drosophila CENP-A, CENP-C, or CAL1 could be our theorized APC substrate. Our assay indicated that these Drosophila proteins are not APC substrates. This finding led us to screen a candidate pool of 20 human proteins implicated in centromere function. Our screen discovered that HsKNL2/M18BP1, an essential centromere protein required for CENP-A loading, is an in-vitro substrate of the anaphase promoting complex. KNL2 degradation is mediated through destruction box (D-Box) and KEN box motifs. Mutation of the KEN and D-Boxes confers stability to KNL2 both in-vitro and in-vivo. Additionally, we discovered that centromeric localization of KNL2 depends on conserved residues in its SANTA (SANT-Associated) domain. Expression of GFP-nondegradable-KNL2 is able to rescue the CENP-A loading defect of KNL2 siRNA, indicating that KNL2 removal by the APC is not required for CENP-A loading. Further experiments are ongoing to characterize the phenotypic effects of KNL2 stabilization, particularly whether KNL2 stabilization is sufficient to decouple CENP-A loading from the cell cycle. We are also testing models to explain KNL2's localization to centromeres during periods of high APC activity and their consequences to centromere function. The discovery that KNL2 is an APC substrate provides a mechanistic link to explain the carefully regulated timing of CENP-A loading. Through a multidisciplinary approach we have assigned novel roles to genes required for CENP-A assembly in Drosophila; we further extended those principles to human biology and have possibly uncovered a mechanistic explanation for CENP-A's unique loading dynamics.

Keywords: Biochemistry.
Advisor: Straight Aaron
Brown Patrick O'Reilly
Theriot Julie advisor. Thesis advisor