ABSTRACT
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.
Subject(s)
Autoantigens , Centromere/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA, Satellite , DNA-Binding Proteins , Regulatory Sequences, Nucleic Acid , Base Sequence , Cell Division , Cell Line, Transformed , Cells, Cultured , Centromere/chemistry , Centromere Protein B , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Artificial, Mammalian , Chromosomes, Human, Pair 21/chemistry , Chromosomes, Human, Pair 21/genetics , Chromosomes, Human, Pair 21/metabolism , DNA, Satellite/chemical synthesis , DNA, Satellite/genetics , DNA, Satellite/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , In Situ Hybridization, Fluorescence , Mitosis , Point Mutation , Repetitive Sequences, Nucleic AcidABSTRACT
Degenerate probe DNA, homologous to part of the 234-bp repeated mouse gamma (major) satellite DNA, was generated by primer-directed in vitro DNA amplification using the polymerase chain reaction with oligonucleotide primers that anneal in the most conserved parts of the repeat. Probe labeling with biotin was performed during DNA polymerization. In situ hybridization of probe DNA with metaphase chromosome preparations showed exclusive binding of probe molecules to the centromeric region of mouse chromosomes. We applied the probe DNA for labeling of mouse heterochromatin in metaphase chromosomes, as well as interphase cell nuclei, and compared results of probe visualization using avidin tagged with either fluorescein or alkaline phosphatase in combination with a chromogenic substrate.
