CENP-A (1, 2) is a centromere specific homologue of the core nucleosomal protein histone H3. Biochemical evidence obtained by purification of mammalian CENP-A suggests that CENP-A coassembles with the other core histones in a particle that co-purifies with nucleosome core particles. A potential homologue of CENP-A has been found in budding yeast as a gene required for stable chromosome inheritance and as a high copy suppressor of a novel histone H4 mutation with a mitotic arrest phenotype. These data point to the idea that the centromere is differentiated from the chromosome arms at the most fundamental level of chromosomal organization - that of the nucleosome.
My laboratory is interested in the assembly and function of CENP-A at human centromeres. One question we have focused on is: how is CENP-A targeted for specific assembly at centromeres? By constructing a series of chimeric genes in which segments of CENP-A were systematically exchanged with the corresponding regions of histone H3 we have learned that A) the histone fold domain of CENP-A, rather than its unique N-terminal tail, is required for assembly at centromeres and B) regions predicted to correspond with DNA contact sites of histone H3 within a nucleosome are necessary for efficient assembly at centromeres. These data suggest that DNA binding may contribute to targeting CENP-A for assembly at centromeres.
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| Epitope tagged CENP-A derivatives were prepared by substituting defined structural domains with corresponding histone H3 sequences. Subcellular distribution was assayed after transfection. | Mapping the elements essential for centromeric targeting onto the structure of the nucleosome reveals that predicted DNA contact sites play a targeting role. | Identification of alpha satellite DNA as the primary sequence associated with CENP-A will allow us to determine whether CENP-A nucleosomes have unique DNA recognition properties. |
The possibility that CENP-A assembles into a nucleosome-like particle with unique DNA recognition properties is currently being investigated in the laboratory. An essential step was to identify the DNA sequences bound to CENP-A in chromosomes. We developed a chromatin immunoprecipitation procedure to specifically isolate CENP-A chromatin and clone the associated DNA, showing that the predominant sequence class associated with CENP-A is alpha-satellite DNA, the major DNA constituent of human centromeres. Simultaneously, the demonstration that CENP-A is localized at the inner kinetochore plate, where it is associated specifically with active centromeres, allowed us for the first time to identify bona fide kinetochore-associated DNA of human centromeres.
Currently we are purifying CENP-A to develop an in vitro assembly system for CENP-A nucleosomes and chromatin. One experimental priority is to determine whether a CENP-A nucleosome has unique DNA recognition properties.
Regulation of CENP-A expression also appears to play an important role in centromere targeting.
CENP-A vital statistics:
Entrez DNA | Entrez Protein | Swissprot | Chromosomal location | Pubmed
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