ABSTRACT
Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling.
Subject(s)
Centromere Protein A/metabolism , Drosophila Proteins/metabolism , Peptide Elongation Factors/metabolism , Transcription Factors/metabolism , Animals , Blotting, Western , Cell Cycle/genetics , Cell Cycle/physiology , Cell Line , Centromere Protein A/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Drosophila , Drosophila Proteins/genetics , Flow Cytometry , Fluorescent Antibody Technique , HeLa Cells , Humans , Immunoprecipitation , Mitosis/genetics , Mitosis/physiology , Peptide Elongation Factors/genetics , Transcription Factors/geneticsABSTRACT
The centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, centromere protein (CENP)-A, yet how its self-propagation is achieved remains poorly understood. Here, we develop a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C, and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify CAL1 to be required for dCENP-C loading onto chromatin in cooperation with dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of Drosophila centromere identity.
Subject(s)
Centromere/metabolism , Drosophila melanogaster/metabolism , Amino Acid Sequence , Animals , Cell Line , Centromere Protein A/chemistry , Centromere Protein A/metabolism , Chromatin/metabolism , Chromosomes, Human/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Female , Humans , Nucleosomes/metabolism , Protein Binding , Protein Domains , Protein Multimerization , Protein Structure, SecondaryABSTRACT
The evolutionarily conserved nucleoplasmin family of histone chaperones has two paralogues in Drosophila, named Nucleoplasmin-Like Protein (NLP) and Nucleophosmin (NPH). NLP localizes to the centromere, yet molecular underpinnings of this localization are unknown. Moreover, similar to homologues in other organisms, NLP forms a pentamer in vitro, but the biological significance of its oligomerization has not been explored. Here, we characterize the oligomers formed by NLP and NPH in vivo and find that oligomerization of NLP is required for its localization at the centromere. We can further show that oligomerization-deficient NLP is unable to bind the centromeric protein Hybrid Male Rescue (HMR), which in turn is required for targeting the NLP oligomer to the centromere. Finally, using super-resolution microscopy we find that NLP and HMR largely co-localize in domains that are immediately adjacent to, yet distinct from centromere domains defined by the centromeric histone dCENP-A.
Subject(s)
Centromere Protein A/chemistry , Centromere/chemistry , Drosophila Proteins/chemistry , Drosophila melanogaster/genetics , Nuclear Proteins/chemistry , Nucleoplasmins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Cells, Cultured , Centromere/metabolism , Centromere Protein A/genetics , Centromere Protein A/metabolism , Chromatin/chemistry , Chromatin/metabolism , Cloning, Molecular , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Gene Expression , Models, Molecular , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleophosmin , Nucleoplasmins/genetics , Nucleoplasmins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, but the mechanism underlying its recruitment to mammalian centrioles is not understood. In flies, Plk4 recruitment depends on Asterless, whereas nematodes rely on a distinct protein, Spd-2. Here, we have explored the roles of two homologous mammalian proteins, Cep152 and Cep192, in the centriole recruitment of human Plk4. We demonstrate that Cep192 plays a key role in centrosome recruitment of both Cep152 and Plk4. Double-depletion of Cep192 and Cep152 completely abolishes Plk4 binding to centrioles as well as centriole duplication, indicating that the two proteins cooperate. Most importantly, we show that Cep192 binds Plk4 through an N-terminal extension that is specific to the largest isoform. The Plk4 binding regions of Cep192 and Cep152 (residues 190-240 and 1-46, respectively) are rich in negatively charged amino acids, suggesting that Plk4 localization to centrioles depends on electrostatic interactions with the positively charged polo-box domain. We conclude that cooperation between Cep192 and Cep152 is crucial for centriole recruitment of Plk4 and centriole duplication during the cell cycle.