Abstract
Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (N)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in Saccharomyces cerevisiae CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of Schizosaccharomyces pombe CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (Δclr4), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover.
Highlights
Precise partitioning of chromosomes during mitosis is critical for genomic stability, and the loss of faithfulness during the segregation of genetic and epigenetic information is associated with many human diseases, including cancers and birth defects [1,2]
We found that the SpCENP-Acnp1-4PA mutant was almost as stable as the wild-type protein in a protein turnover assay [28]
These observations suggested that the ‘GRANT-proline’ residues have no effect on the proteasome-associated turnover of SpCENP-A protein
Summary
Precise partitioning of chromosomes during mitosis is critical for genomic stability, and the loss of faithfulness during the segregation of genetic and epigenetic information is associated with many human diseases, including cancers and birth defects [1,2]. Chromosome segregation is brought about by the attachment of mitotic spindle microtubules that emanate from centrosomes (or spindle pole bodies) at cell poles to the kinetochores, which are specialized mega-protein complexes assembled onto chromosomal loci called centromeres [3,4]. The centromere consists of a higher order chromatin architecture that performs many critical mitotic functions, such as inculcating mechanical strength, to safeguard the structural integrity of the chromosomes; checkpoint functions, to ensure precise timing of chromosomal separation; and bi-directional orientation of the kinetochore-microtubule attachment [5,6,7]. CENP-A epigenetically marks the position of the centromere by assembling a specialized chromatin architecture, on which other centromere proteins of the constitutive centromere-associated network (CCAN) and the KNL-1/Mis complex/Ndc complex network (KMN) can be mounted for assembly of the kinetochore [6,8]. The level of overexpressed CENP-A, along with that of other kinetochore proteins, may be used as a prognostic marker to predict patient survival and therapeutic response [21]
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