Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone.

Abstract

CENP-A, a centromere-specific 17-kDa protein, has histone-like properties. However, in contrast to the common somatic histones, CENP-A is quantitatively retained in bull spermatozoa, and we have exploited this fact to purify CENP-A to apparent homogeneity. Partial sequence analysis of the purified protein indicates that CENP-A is a distinctive gene product. Some CENP-A sequences are highly similar to regions of histone H3. Other segments of CENP-A are not related to H3 or any other histone. These unrelated segments are presumably involved in loaliing CENP-A to centromeric DNA or in centromere-specific functions of CENP-A..The centromeric region of the chromosome is responsible for its integration into the mitotic spindle and for its proper segregation poleward during anaphase (1)(2)(3)(4). Several proteins have been identified that are associated with the centromere (5-10). For the most part, their functions are presently unknown. Recently, however, Bischoff et al. (11) reported that a 47-kDa centromere-specific autoantigen is highly homologous to the translation product ofRCCI, a gene involved in regulating mammalian chromosome condensation. This antigen may be identical to CENP-D, a 50-to 60-kDa centromeric protein reported to be present in a variety of species (7, 9, 10).CENP-A, a centromere-specific protein of 17 kDa, appears to be associated with kinetochore chromatin or with chromatin closely apposed to the outermost domain of the kinetochore, as judged by indirect immunofluorescence (9) and immunoelectron microscopy (12). This localization suggests it may play a direct role in kinetochore function during mitosis.We have extensively analyzed CENP-A and have shown that it appears to be a core histone by the following criteria: (i) it is extracted with histones from chromatin by dilute mineral acids, (ii) it elutes with histones H3 and H4 in apparent tetrameric complexes during ion exchange (13) or sizing-column chromatography (D.K.P., K.O., and R.L.M., unpublished observations), and, most importantly, (iii) it is a component of highly purified nucleosome core particles (13). CENP-A is exceptional among somatic histones not only because it is centromere specific but also because it is quantitatively retained in chromatin during spermatogenesis in mammals, even in species where other histones are quantitatively replaced by protamines (14).The selective retention of this protein in bull sperm, where other histones are absent, has now allowed us to extract and purify CENP-A to homogeneity. We have also subjected the purified CENP-A from bull sperm to partial sequence analysis and report here that it is a distinctive histone, with sequences similar to those of H3, as well as segments that are not related to histones or homologous to any other known mammalian centromeric protein sequences (11, 15). MATERIALS AND METHODSPurification and Acid Extraction of Nuclei ad ReversePhase Chromatography of Acid-Extracted Proteins. Calf thymus nuclei and bull sperm nuclei were purified and extracted with 1 M NaCl/0.25...

Hierarchical Inactivation of a Synthetic Human Kinetochore by a Chromatin Modifier

“…In model 1 (Figure 6B), the kinetochore is built on a chromatin foundation of CENP-A nucleosomes (Palmer and Margolis, 1985;Palmer et al, 1991;Yoda et al, 2000;Black et al, 2007;Dalal et al, 2007;reviewed in Carroll and Straight, 2006;Cheeseman and Desai, 2008;Vagnarelli et al, 2008). In this model, eviction of CENP-A or prevention of its targeting caused by chromatin modifiers would disrupt the foundation for the kinetochore as a primary effect, and the structure would subsequently fall apart.…”

Dynamic distribution of TTK in HeLa cells: insights from an ultrastructural study

“…Structurally, the kinetochore is composed of four layers: an innermost plate that apparently consists of a specialized layer of chromatin, an interzone, an outer plate that has been argued to consist of tightly packed fibers [4,5], and an outermost fuzzy, fibrous corona that is most clearly seen after microtubule disassembly [6,7]. Although kinetochore morphology has been documented in numerous ultrastructural studies [8][9][10][11][12], there is little information about molecular composition and the respective localization of known kinetochore proteins or protein complexes, except for a handful kinetochore proteins such as CENP-A (attached to to centromeric heterochromatin) [13], CENP-B (underneath the inner plate) [14], CENP-C (a component of the inner plate [1], CENP-E [a component of the corona fiber) [ 7], CENP-F (a component of outer plate) [15], Bub1 and BubR1 [components of the corona fiber) [16].…”

Somatic Histones Are Components of the Perinuclear Theca in Bovine Spermatozoa

“…Human sperm nuclei retain up to 15% of somatic core histone content (23)(24)(25). In contrast, somatic histones from bovine sperm nuclei represent less than 1% of calf thymus nuclei content (26). Somatic histones have also been isolated in the mature sperm of mouse (27,28) and rat (29), although it remained questionable whether their source was exclusively nuclear (27).…”