FIGURE 8.

And-1 overexpression enhances the intensity of CENP-A at centromeres. A, And-1 interacts with CENP-A and H4 but not H3.1 or H3.3. 293T cells co-expressing FLAG-And-1 and either YFP-CENP-A, H3.1-YFP, H3.3-GFP, or H4-GFP were harvested and chromatin-free fractions prepared for co-immunoprecipitation (IP) assays. FLAG-And-1 immunoprecipitates were resolved by SDS-PAGE and immunoblotted for the indicated proteins. B, CENP-A intensity is increased in U2OS (FLAG-And-1) cells compared with that in U2OS (FLAG) cells. Asynchronous cells were harvested and immunostained for And-1 and CENP-A proteins. The intensity of CENP-A at centromeres is shown in the right panel. C, model shows the role of And-1 in the HJURP-mediated deposition of CENP-A at centromeres. Replication of sister chromatids during S phase dilutes the amount of CENP-A at the centromeres, which is interspersed with H3-containing nucleosomes. During late anaphase/telophase centromeric chromatin is primed for the deposition of new CENP-A by the Mis18 complex and other factors possibly through changing the acetylation status of the chromatin. At late mitosis or early G₁ phase, And-1 interacts with HJURP-CENP-A-H4 complexes and facilitates their assembly at centromeres in a process involving multiple proteins such as RbAp48/46 and Npm1 (data not shown).