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Genomic size of CENP-A domain is proportional to total alpha satellite array size at human centromeres and expands in cancer cells

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Abstract

Human centromeres contain multi-megabase-sized arrays of alpha satellite DNA, a family of satellite DNA repeats based on a tandemly arranged 171 bp monomer. The centromere-specific histone protein CENP-A is assembled on alpha satellite DNA within the primary constriction, but does not extend along its entire length. CENP-A domains have been estimated to extend over 2,500 kb of alpha satellite DNA. However, these estimates do not take into account inter-individual variation in alpha satellite array sizes on homologous chromosomes and among different chromosomes. We defined the genomic distance of CENP-A chromatin on human chromosomes X and Y from different individuals. CENP-A chromatin occupied different genomic intervals on different chromosomes, but despite inter-chromosomal and inter-individual array size variation, the ratio of CENP-A to total alpha satellite DNA size remained consistent. Changes in the ratio of alpha satellite array size to CENP-A domain size were observed when CENP-A was overexpressed and when primary cells were transformed by disrupting interactions between the tumor suppressor protein Rb and chromatin. Our data support a model for centromeric domain organization in which the genomic limits of CENP-A chromatin varies on different human chromosomes, and imply that alpha satellite array size may be a more prominent predictor of CENP-A incorporation than chromosome size. In addition, our results also suggest that cancer transformation and amounts of centromeric heterochromatin have notable effects on the amount of alpha satellite that is associated with CENP-A chromatin.

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Abbreviations

CENP-A:

Centromere protein A

CCD:

Charged coupled device

CHEF:

Contour-clamped homogenous electric field

ChIP:

Chromatin immunoprecipitation

CSK buffer:

Cytoskeleton buffer

DNA:

Deoxyribonucleic acid

E7:

E7 protein subunit of HPV

FISH:

Fluorescence in situ hybridization

FITC:

Fluorescein isothiocyanate

FBS:

Fetal bovine serum

HAT:

Hypoxanthine/aminopterin/thymidine

HPV:

Human papillomavirus

HOR:

Higher order repeat

HSAX:

Homo sapiens chromosome X

HSAY:

Homo sapiens chromosome Y

Kb:

Kilobase

MEM:

Minimal essential medium

Mb:

Megabase

PCR:

Polymerase chain reaction

PVDF:

Polyvinylidene fluoride

Rb:

Retinoblastoma protein

RPMI:

Roswell Park Memorial Institute medium

PFGE:

Pulsed field gel electrophoresis

SDS:

Sodium dodecyl sulfate

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Acknowledgments

We thank Cyrus Vaziri (University of North Carolina, Chapel Hill) for providing cell lines DIP3 and DIP3-E7 and Chris Shaw for technical assistance. This work was supported in part by grants from the American Cancer Society (ACS IRG-72-001-29-IRG), March of Dimes Foundation (6-FY06-377 and 6-FY10-294), and NIH NIGMS (R01 GM069514) to BAS.

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Author notes

  1. Brankica Mravinac

    Present address: Division of Molecular Biology, Ruder Boskovic Institute, Zagreb, Croatia

Authors and Affiliations

  1. Duke Institute for Genome Sciences & Policy, Duke University, 101 Science Drive, Box 3382, Durham, NC, 27708, USA

    Lori L. Sullivan, Brankica Mravinac & Beth A. Sullivan

  2. Department of Genetics and Genomics, Boston University School of Medicine, 715 Albany Street, E645, Boston, MA, 02118, USA

    Christopher D. Boivin & Ihn Young Song

  3. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, 27710, USA

    Beth A. Sullivan

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  1. Lori L. Sullivan
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  2. Christopher D. Boivin
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Correspondence to Beth A. Sullivan.

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Responsible Editor: Conly Rieder

Lori L. Sullivan and Christopher D. Boivin contributed equally to this work.

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Sullivan, L.L., Boivin, C.D., Mravinac, B. et al. Genomic size of CENP-A domain is proportional to total alpha satellite array size at human centromeres and expands in cancer cells. Chromosome Res 19, 457–470 (2011). https://doi.org/10.1007/s10577-011-9208-5

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