Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres.
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 alphaPSer(10)H3 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.
Moreno-Moreno
Olga
O
Institute of Molecular Biology of Barcelona (IBMB), CSIC, Parc Científic de Barcelona, 08028 Barcelona, Spain.
Torras-Llort
Mònica
M
Azorín
Fernando
F
D000818:Animals; D002453:Cell Cycle; D002478:Cells, Cultured; D002503:Centromere / Q000737:chemistry*; D000076247:Centromere Protein A; D002843:Chromatin / Q000737:chemistry; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism*; D029721:Drosophila Proteins / Q000032:analysis* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D006657:Histones / Q000032:analysis* / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors
D000076247:Centromere Protein A; D002843:Chromatin; C427237:Cid protein, Drosophila; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006657:Histones; D007976:Leupeptins; D061988:Proteasome Inhibitors; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde