Centromere targeting element within the histone fold domain of Cid.
Centromeres require specialized nucleosomes; however, the mechanism of localization is unknown. Drosophila sp. centromeric nucleosomes contain the Cid H3-like protein. We have devised a strategy for identifying elements within Cid responsible for its localization to centromeres. By expressing Cid from divergent Drosophila species fused to green fluorescent protein in Drosophila melanogaster cells, we found that D. bipectinata Cid fails to localize to centromeres. Cid chimeras consisting of the D. bipectinata histone fold domain (HFD) replaced with segments from D. melanogaster identified loop I of the HFD as being critical for targeting to centromeres. Conversely, substitution of D. bipectinata loop I into D. melanogaster abolished centromeric targeting. In either case, loop I was the only segment capable of conferring targeting. Within loop I, we identified residues that are critical for targeting. Most mutations of conserved residues abolished targeting, and length reductions were deleterious. Taken together with the fact that H3 loop I makes numerous contacts with DNA and with the adaptive evolution of Cid, our results point to the importance of DNA specificity for targeting. We suggest that the process of deposition of (Cid.H4)2 tetramers allows for discriminating contacts to be made between loop I and DNA, providing the specificity needed for targeting.
Vermaak
Danielle
D
Howard Hughes Medical Institute, Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Hayden
Hillary S
HS
Henikoff
Steven
S
D000595:Amino Acid Sequence; D000818:Animals; D002503:Centromere / Q000378:metabolism*; D000076247:Centromere Protein A; D017124:Conserved Sequence; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D049452:Green Fluorescent Proteins; D006657:Histones / Q000737:chemistry* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008938:Mitosis; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000076247:Centromere Protein A; C427237:Cid protein, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins