| Mitosis is the cell cycle stage during which sister chromatids are segregated to form two new daughter cells. Accurate chromosome segregation ensures that each daughter cell inherits an identical genome. Chromosome segregation requires chromosome attachment to mitotic spindle microtubules through a multi-protein complex called the kinetochore. Kinetochores monitor proper microtubule attachment to chromosomes and couple microtubule dynamics to chromosome segregation. Errors in chromosome segregation lead to chromosome abnormalities that alter gene expression and genome stability. Changes in gene expression and genome stability are proposed to contribute to the development of numerous diseases such as cancer.;During mitosis, kinetochores assemble at a specialized region on each chromosome called the centromere. In higher eukaryotes, the centromere is visualized as the primary constriction point on a mitotic chromosome. At the molecular level, the centromere is a unique chromatin domain composed of alpha-satellite DNA repeats and nucleosomes containing the histone H3.1 variant centromere protein-A (CENP-A). CENP-A directs the assembly of most other centromere proteins and is thought to be the epigenetic mark that establishes centromere position. CENP-A is essential in all eukaryotes, and CENP-A mutations result in chromosome missegregation and cell death.;CENP-A is required for proper centromere and kinetochore function, but the molecular mechanism of how CENP-A is specifically assembled at centromeres is not well understood. In general, nucleosome assembly requires histone chaperones and ATP-dependent chromatin remodeling factors. For example, during DNA replication the canonical histones including H3.1, H4, H2A, and H2B are assembled onto DNA by the CAF-1 complex. In contrast, the histone variant H3.3 is assembled onto DNA by HIRA, and the SWR1 complex exchanges histone H2A for the histone variant H2AZ. The histone chaperones and ATP-dependent chromatin remodeling complexes that act on histone variants selectively recognize histone variants over canonical histones, so we reasoned that factors involved in CENP-A assembly would bind to CENP-A and not H3.1.;To identify and study potential factors involved in CENP-A assembly, we first reconstituted centromeric nucleosomes. We developed methods for the purification of recombinant CENP-A/H4 tetramers and the assembly of CENP-A/H4 mononucleosomes on a variety of DNA templates. Using our reconstituted centromeric nucleosomes as a tool, we investigated the molecular mechanism of CENP-A assembly and found that histone acetyltransferase-1 (Hat-1) and retinoblastoma associated protein-46 (RbAp46) selectively bound to the CENP-A N-terminal tail and not the H3.1 N-terminal tail. In vitro the Hat-1/RbAp46 complex acetylated CENP-A/H4 tetramers, but not nucleosomes, predominantly on H4 lysine 12 (K12) and to a lesser extent on H4 lysine 5 (K5).;To study the in vivo role of H4 K5 and K12 acetylation in CENP-A chromatin assembly, we developed a histone tetramer microinjection assay. In human tissue culture cells, we microinjected recombinant CENP-A/H4 K5Q, K12Q mutant tetramers that mimic constitutive acetylation, CENP-A/H4 K5R, K12R mutant tetramers that mimic constitutive deacetylation, or wild type CENP-A/H4 tetramers. When CENP-A/H4, CENP-A/H4 K5Q, K12Q, or CENP-A/H4 K5R, K12R tetramers were microinjected in HeLa cells during G2, we found that the microinjected CENP-A localized to centromeres at a similar frequency for all three types of tetramers. In contrast, when CENP-A/H4 tetramers were microinjected at G1/S phase, CENP-A associated with the H4 lysine mutants preferentially assembled at centromeres compared to CENP-A associated with wild type H4. Our results suggest that the post-translational modification of H4 K5 and K12 is important for promoting CENP-A assembly at centromeres in a cell cycle dependent manner. |
