CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA.
Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.
Ohzeki
Jun-ichirou
J
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Nakano
Megumi
M
Okada
Teruaki
T
Masumoto
Hiroshi
H
D001324:Autoantigens; D001483:Base Sequence; D002455:Cell Division; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002503:Centromere / Q000737:chemistry / Q000378:metabolism*; D051936:Centromere Protein B; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D022221:Chromosomes, Artificial, Mammalian; D002891:Chromosomes, Human, Pair 21 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004276:DNA, Satellite / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008938:Mitosis; D017354:Point Mutation; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid
D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004276:DNA, Satellite; D004268:DNA-Binding Proteins