Surprising Deficiency of CENP-B Binding Sites in African Green Monkey α-Satellite DNA: Implications for CENP-B Function at Centromeres

Abstract

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, ␣-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 ␣-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 ␣-satellite, a novel kinetic electrophoretic mobility shift assay competition protocol using bulk genomic DNA, and bulk sequencing of 76 AGM ␣-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.

Organization and evolution of a novel cervid satellite DNA with yeast CDEI-like repeats

“…This hypothesis was supported by the following lines of evidence: (1) The sequence organization of centromeric domains on the primate X chromosomes is physically symmetrical (Schueler et al 2005), (2) organization of monomeric units in the pericentromeric regions is different from that of higher order repeat units in the centromeric region (Willard 1985;Alexandrov et al 1993;Puechberty et al 1999;Horvath et al 2000;Schueler et al 2001;Rudd and Willard 2004;Schueler et al 2005;Rudd et al 2006), (3) the genomes of lower primates show existing monomeric alpha satellites and no higher-order alpha satellites (Goldberg et al 1996;Alexandrov et al 2001), (4) human pericentromeric α-satellite monomers are frequently interrupted by LINE (long interspersed element), SINE (short interspersed element), and LTR (long terminal repeat) retrotransposons (Schueler et al 2005). In addition, the older L1P elements (primate-specific LINE interspersed repeats) exist in the more distal pericentromeric α-satellite monomers (Smit et al 1995;Schueler et al 2001).…”

In vitro assembly of the CENP‐B/α‐satellite DNA/core histone complex: CENP‐B causes nucleosome positioning

“…We have observed the similar nucleosome positioning around the CENP-B box regions using the isolated nuclei of HeLa cells (K. Yoda, S. Ando & T. Okazaki, Centromere specific nucleosomes are regularly punctuated by CENP-B/ CENP-B box interaction; submitted for publication). Nucleosome positioning in ␣-satellite DNA has previously been found in the African green monkey (Zhang et al 1983), but the positioning patterns on the African green monkey ␣-satellite were different from those in humans around the CENP-B box regions (Yoda et al, manuscript in preparation), which may reflect the difference in the organization of those repeats that the human ␣-satellite DNA containing CENP-B boxes predominantly consist of dimer subfamily (Yoda & Okazaki 1997) while the AGM ␣-satellite is a simple monomer repeat (Rosenberg et al 1978) with very little or no CENP-B boxes (Goldberg et al 1996;Yoda et al 1996). On the basis of the observation that the AGM CENP-B is actually localized at the centromere region with the monoclonal antibody (Yoda et al, unpublished observation) as well as with polyclonal antibody against CENP-B ( Yoda et al 1996), we speculate that CENP-B box-like sequences will probably exist in some unknown satellite sequence other than the ␣-satellite in the AGM centromere, as in the case of the Asian mouse Mus caroli (Kipling et al 1995).…”

Histone Modifications and Nuclear Architecture: A Review

“…In cycling cells, sites of methylated histone lysines were found dispersed throughout the nucleus during G1 but were mostly located at the nuclear periphery and around nucleoli in S phase. In S phase, the centromeres (which contain histone-like CENP proteins (Sullivan et al 1994;Goldberg et al 1996) were also associated with sites of highly methylated histone lysines, although some clusters of highly methylated lysines were observed outside the centromeric heterochromatin (Cremer et al 2004). Zinner et al (2006) further studied the threedimensional arrangement of specific histone lysine methylation sites (H3K4me3, H4K20me1, H3K9me1, H3K27me3, H4K20me3, and H3K9me3) in various human cell lines and described the spatial proximity of the methylated regions to centromeres or newly synthesized RNA.…”