Abstract
CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.
Highlights
During mitotic cell division, microtubules are attached to kinetochores formed on each chromosome, to direct the segregation of sister chromatids into daughter cells [1,2,3]
No obvious difference in the CENP-B binding was detected between the CENP-A and H3.1 nucleosomes (Figure 1B)(46). This suggested that the CENP-B accessibility to the CENP-B box sequence located near the entry/exit sites may not be substantially different between the CENPA and H3.1 nucleosomes
To address the mechanism by which CENP-B binds to the CENP-A nucleosome, in the present study, we prepared the CENP-A and H3.1 nucleosomes complexed with the CENP-B DBD, and evaluated the CENP-B retention upon titration with a competitor DNA
Summary
Microtubules are attached to kinetochores formed on each chromosome, to direct the segregation of sister chromatids into daughter cells [1,2,3]. Genetic and cell biological studies revealed that CENP-A depletion induces chromosome missegregation, due to improper centromere formation [8,10,11,12,13,14]. The crystal structure revealed that CENP-A, H2A, H2B and H4 form the CENP-A nucleosome, with a histone octamer containing two each of CENP-A, H2A, H2B and H4 and the DNA left-handedly wrapped around it [15]. Biochemical and biophysical experiments confirmed the formation of the CENP-A nucleosome as the histone octamer [16,17,18,19,20]. The octameric nucleosomes containing CENP-A and its homologues were found in yeast, fly, and human cells [21,22,23,24,25]. The hemisome, consisting of one each of CENP-A, H2A, H2B and H4, has been proposed as another form [26,27,28], which may appear in a cell cycle-dependent manner [29,30]
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