Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres

Abstract

Centromere identity is determined by the formation of a specialized chromatin structure containing the centromere-specific histone H3 variant CENP-A. The precise molecular mechanism(s) accounting for the specific deposition of CENP-A at centromeres are still poorly understood. Centromeric deposition of CENP-A, which is independent of DNA replication, might involve specific chromatin assembly complexes and/or specific interactions with kinetochore components. However, transiently expressed CENP-A incorporates throughout chromatin indicating that CENP-A nucleosomes can also be promiscuously deposited during DNA replication. Therefore, additional mechanisms must exist to prevent deposition of CENP-A nucleosomes during replication and/or to remove them afterwards. Here, using transient expression experiments performed in Drosophila Kc cells, we show that proteasome-mediated degradation restricts localization of Drosophila CENP-A (CID) to centromeres by eliminating mislocalized CID as well as by regulating available CID levels. Regulating available CID levels appears essential to ensure centromeric deposition of transiently expressed CID as, when expression is increased in the presence of proteasome inhibitors, newly synthesized CID mislocalizes. Mislocalization of CID affects cell cycle progression as a high percentage of cells showing mislocalized CID are reactive against αPSer10H3 antibodies, enter mitosis at a very low frequency and show strong segregation defects. However, cells showing reduced amounts of mislocalized CID show normal cell cycle progression.

Detrimental incorporation of excess Cenp-A/Cid and Cenp-C intoDrosophilacentromeres is prevented by limiting amounts of the bridging factor Cal1

“…In principle, the interdependence of centromeric Cenp-A/Cid, Cenp-C and the low levels of Cal1 might provide robust control of centromeric Cenp-A/Cid amounts and could effectively protect cells against the consequences of accidental unbalanced Cenp-A/Cid excess. However, previous studies have demonstrated that overexpression of Cenp-A/Cid is sufficient to cause ectopic incorporation all along the chromosome and consequential mitotic defects (Van Hooser et al, 2001;Heun et al, 2006;Moreno-Moreno et al, 2006). The massive overexpression applied in these studies (70-fold) (Heun et al, 2006), which is rather unlikely to occur in physiological conditions, even accidentally, might have over-run negative regulation.…”

Ectopic Centromere Nucleation by CENP-A in Fission Yeast

“…Previous studies have shown that ubiquitin-mediated proteolysis of CENP-A is important for preventing CENP-A from euchromatin localization in budding yeast and Drosophila (Collins et al 2004;Moreno-Moreno et al 2006). To determine whether the N-terminal domain of CENP-A cnp1 plays a role in targeting the protein for degradation via the ubiquitin pathway in fission yeast, we first examined the stability of N-terminal-deleted CENP-A cnp1 -GFP compared to wildtype CENP-A cnp1 -GFP.…”

“…In Drosophila, overexpression of the CENP-A homolog, CID, can lead to ectopic formation of heritable functional kinetochores and significant chromosome missegregation (Heun Olszak et al 2011). In budding yeast and Drosophila, ubiquitin-mediated proteolysis of CENP-A has been shown to contribute to the removal of mislocalized CENP-A at noncentromeric regions (Collins et al 2004;Moreno-Moreno et al 2006;Hewawasam et al 2010;Ranjitkar et al 2010). However, the mechanisms that protect cells from erroneously assembling CENP-A at noncentromeric chromatin remain poorly understood.…”

Cse4 gets a kiss-of-death from Psh1

“…Overexpression of Cse4 in a psh1Δ strain is toxic and is associated with localization of Cse4 to ribosomal DNA and euchromatin, 4,5 Proteasomal degradation of CENP-A has been reported in human cells and flies. 11 A Psh1 ortholog in other eukaryotes is difficult to verify since the RING and Zinc fingers are highly conserved motifs in many genes from yeast to human. It will be interesting in the future to determine whether ubiquitin-dependent proteolytic control is an evolutionarily conserved mechanism to regulate Cen-H3.…”