Sequence-specific physical properties of African green monkey alpha-satellite DNA contribute to centromeric heterochromatin formation

Centromeres of mammalian chromosomes are rich in repetitive DNAs that are packaged into specialized nucleoprotein structures called heterochromatin. In humans, the major centromeric repetitive DNA, alpha-satellite DNA, has been extensively sequenced and shown to contain binding sites for CENP-B, an 80-kDa centromeric autoantigen. The present report reveals that African green monkey (AGM) cells, which contain extensive alpha-satellite arrays at centromeres, appear to lack the well-characterized CENP-B binding site (the CENP-B box). We show that AGM cells express a functional CENP-B homolog that binds to the CENP-B box and is recognized by several independent anti-CENP-B antibodies. However, three independent assays fail to reveal CENP-B binding sites in AGM DNA. Methods used include a gel mobility shift competition assay using purified AGM alpha-satellite, a novel kinetic electrophoretic mobility shift assay competition protocol using bulk genomic DNA, and bulk sequencing of 76 AGM alpha-satellite monomers. Immunofluorescence studies reveal the presence of significant levels of CENP-B antigen dispersed diffusely throughout the nuclei of interphase cells. These experiments reveal a paradox. CENP-B is highly conserved among mammals, yet its DNA binding site is conserved in human and mouse genomes but not in the AGM genome. One interpretation of these findings is that the role of CENP-B may be in the maintenance and/or organization of centromeric satellite DNA arrays rather than a more direct involvement in centromere structure.

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Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization.

Molecular and Cellular Biology ◽

10.1128/mcb.16.9.5169 ◽

1996 ◽

Vol 16 (9) ◽

pp. 5169-5177 ◽

Cited By ~ 28

Author(s):

K Yoda ◽

T Nakamura ◽

H Masumoto ◽

N Suzuki ◽

K Kitagawa ◽

...

Keyword(s):

Dna Binding ◽

Gene Structure ◽

Satellite Dna ◽

African Green Monkey ◽

Alpha Satellite ◽

Alpha Satellite Dna ◽

Centromeric Satellite ◽

Centromere Protein ◽

Green Monkey ◽

Human And Mouse

Centromere protein B (CENP-B) is a centromeric DNA-binding protein which recognizes a 17-bp sequence (CENP-B box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of alpha-satellite DNA, but there has been no report of CENP-B boxes or CENP-B in the centromere domains of its chromosomes. To elucidate the AGM CENP-B-CENP-B box interaction, we have analyzed the gene structure, expression, biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was established to be highly homologous to that of human and mouse CENP-B. In particular, the DNA binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. Immunoblotting and DNA mobility shift analyses revealed CENP-B to be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhibited the same DNA binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antibodies, a centromere-specific localization of AGM CENP-B could be demonstrated. We also isolated AGM alpha-satellite DNA with a CENP-B box-like sequence with CENP-B affinity. These results not only prove that CENP-B functionally persists in AGM cells but also suggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B boxes with the core recognition sequence or CENP-B box variants) in centromeric satellite DNA.

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Analysis of the alpha-Satellite DNA from African Green Monkey Cells by Restriction Nucleases

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1977.tb11399.x ◽

1977 ◽

Vol 74 (2) ◽

pp. 343-352 ◽

Cited By ~ 26

Author(s):

Friedrich FITTLER

Keyword(s):

Satellite Dna ◽

African Green Monkey ◽

Alpha Satellite ◽

Alpha Satellite Dna ◽

Green Monkey ◽

African Green Monkey Cells

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Identification of a subdomain of CENP-B that is necessary and sufficient for localization to the human centromere.

The Journal of Cell Biology ◽

10.1083/jcb.116.5.1081 ◽

1992 ◽

Vol 116 (5) ◽

pp. 1081-1093 ◽

Cited By ~ 73

Author(s):

A F Pluta ◽

N Saitoh ◽

I Goldberg ◽

W C Earnshaw

Keyword(s):

Satellite Dna ◽

Centromeric Heterochromatin ◽

Amino Terminus ◽

Major Protein ◽

Alpha Satellite ◽

Alpha Satellite Dna ◽

Human Centromere ◽

Bacterial Enzyme

We have combined in vivo and in vitro approaches to investigate the function of CENP-B, a major protein of human centromeric heterochromatin. Expression of epitope-tagged deletion derivatives of CENP-B in HeLa cells revealed that a single domain less than 158 residues from the amino terminus of the protein is sufficient to localize CENP-B to centromeres. Centromere localization was abolished if as few as 28 amino acids were removed from the amino terminus of CENP-B. The centromere localization signal of CENP-B can function in an autonomous fashion, relocating a fused bacterial enzyme to centromeres. The centromere localization domain of CENP-B specifically binds in vitro to a subset of alpha-satellite DNA monomers. These results suggest that the primary mechanism for localization of CENP-B to centromeres involves the recognition of a DNA sequence found at centromeres. Analysis of the distribution of this sequence in alpha-satellite DNA suggests that CENP-B binding may have profound effects on chromatin structure at centromeres.

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CHARACTERIZATION OF HUMAN CHROMOSOMAL CONSTITUTIVE HETEROCHROMATIN

Gulustan-Black Sea Scientific Journal of Academic Research ◽

10.36962/gbssjar5302202008 ◽

2020 ◽

Vol 53 (02) ◽

pp. 08-11

Author(s):

Aytakin Hasanova

Keyword(s):

Satellite Dna ◽

Constitutive Heterochromatin ◽

Chromosome Breakage ◽

Centromeric Heterochromatin ◽

Crossing Over ◽

Alpha Satellite ◽

Alpha Satellite Dna ◽

Chromosome Regions ◽

Chromosome Disjunction

Heterochromatin of centromeric chromosome regions contains late replicating, largely repetitive DNA. It is suggested that heterochromatin participates in chromosome pairing, crossing-over and in chromosome disjunction control (1,3). Centromeric heterochromatin, a variety of heterochromatin, is a tightly packed form of DNA.Centromeric heterochromatin is a constituent in the formation ofactive centromeres in most higher-order organisms; the domain exists on both mitotic and interphase chromosomes. (4,5,6,8) Centromeric heterochromatin is usually formed on alpha satellite DNA in humans; however, there have been cases where centric heterochromatin and centromeres have formed on originally euchromatin domains lacking alpha satellite DNA; this usually happens as a result of a chromosome breakage event and the formed centromere is called a neocentromere.

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