Abstract 3471: Replication of megabase-size alpha-satellite DNA arrays in human centromere

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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Repetitive Fragile Sites: Centromere Satellite DNA As a Source of Genome Instability in Human Diseases

Genes ◽

10.3390/genes9120615 ◽

2018 ◽

Vol 9 (12) ◽

pp. 615 ◽

Cited By ~ 25

Author(s):

Elizabeth M. Black ◽

Simona Giunta

Keyword(s):

Satellite Dna ◽

Genome Instability ◽

Fragile Sites ◽

Special Focus ◽

Alpha Satellite ◽

Chromosomal Locus ◽

Alpha Satellite Dna ◽

Human Centromere ◽

Repetitive Nature ◽

Human Genome Assembly

Maintenance of an intact genome is essential for cellular and organismal homeostasis. The centromere is a specialized chromosomal locus required for faithful genome inheritance at each round of cell division. Human centromeres are composed of large tandem arrays of repetitive alpha-satellite DNA, which are often sites of aberrant rearrangements that may lead to chromosome fusions and genetic abnormalities. While the centromere has an essential role in chromosome segregation during mitosis, the long and repetitive nature of the highly identical repeats has greatly hindered in-depth genetic studies, and complete annotation of all human centromeres is still lacking. Here, we review our current understanding of human centromere genetics and epigenetics as well as recent investigations into the role of centromere DNA in disease, with a special focus on cancer, aging, and human immunodeficiency–centromeric instability–facial anomalies (ICF) syndrome. We also highlight the causes and consequences of genomic instability at these large repetitive arrays and describe the possible sources of centromere fragility. The novel connection between alpha-satellite DNA instability and human pathological conditions emphasizes the importance of obtaining a truly complete human genome assembly and accelerating our understanding of centromere repeats’ role in physiology and beyond.

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Surprising deficiency of CENP-B binding sites in African green monkey alpha-satellite DNA: implications for CENP-B function at centromeres.

Molecular and Cellular Biology ◽

10.1128/mcb.16.9.5156 ◽

1996 ◽

Vol 16 (9) ◽

pp. 5156-5168 ◽

Cited By ~ 37

Author(s):

I G Goldberg ◽

H Sawhney ◽

A F Pluta ◽

P E Warburton ◽

W C Earnshaw

Keyword(s):

Binding Site ◽

Satellite Dna ◽

Binding Sites ◽

Present Report ◽

African Green Monkey ◽

Dna Arrays ◽

Alpha Satellite ◽

Mobility Shift ◽

Alpha Satellite Dna ◽

Green Monkey

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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A functional neo-centromere formed through activation of a latent human centromere and consisting of non-alpha-satellite DNA

Nature Genetics ◽

10.1038/ng0697-144 ◽

1997 ◽

Vol 16 (2) ◽

pp. 144-153 ◽

Cited By ~ 195

Author(s):

Desirée du Sart ◽

Michael R. Cancilla ◽

Elizabeth Earle ◽

Jen-i Mao ◽

Richard Saffery ◽

...

Keyword(s):

Satellite Dna ◽

Alpha Satellite ◽

Alpha Satellite Dna ◽

Human Centromere

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CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains

Journal of Cell Science ◽

10.1242/jcs.115.11.2317 ◽

2002 ◽

Vol 115 (11) ◽

pp. 2317-2327 ◽

Cited By ~ 1

Author(s):

Valeria Politi ◽

Giovanni Perini ◽

Stefania Trazzi ◽

Artem Pliss ◽

Ivan Raska ◽

...

Keyword(s):

Dna Sequences ◽

Satellite Dna ◽

Alpha Satellite ◽

Alpha Satellite Dna ◽

Human Centromere ◽

Ultrastructural Characterization ◽

First Time

CENP-C is a fundamental component of the centromere, highly conserved among species and necessary for the proper assembly of the kinetochore structure and for the metaphase-anaphase transition. Although CENP-C can bind DNA in vitro,the identification of the DNA sequences associated with it in vivo and the significance of such an interaction have been, until now, elusive. To address this problem we took advantage of a chromatin-immunoprecipitation procedure and applied this technique to human HeLa cells. Through this approach we could establish that: (1) CENP-C binds the alpha-satellite DNA selectively; (2) the CENP-C region between amino acids 410 and 537, previously supposed to contain a DNA-binding domain, is indeed required to perform such a function in vivo;and (3) the profile of the alpha-satellite DNA associated with CENP-C is essentially identical to that recognized by CENP-B. However, further biochemical and ultrastructural characterization of CENP-B/DNA and CENP-C/DNA complexes, relative to their DNA components and specific spatial distribution in interphase nuclei, surprisingly reveals that CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains. Our results, besides extending previous observations on the role of CENP-C in the formation of active centromeres, show, for the first time, that CENP-C can associate with the centromeric DNA sequences in vivo and, together with CENP-B, defines a highly structured organization of the alpha-satellite DNA within the human centromere.

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