Elsevier

Experimental Cell Research

Volume 315, Issue 19, 15 November 2009, Pages 3233-3241
Experimental Cell Research

Review
Epigenetic specification of centromeres by CENP-A

https://doi.org/10.1016/j.yexcr.2009.07.023Get rights and content

Abstract

Centromeres are the chromosomal loci that direct the formation of the kinetochores. These macromolecular assemblies mediate the interaction between chromosomes and spindle microtubules and thereby power chromosome movement during cell division. They are also the sites of extensive regulation of the chromosome segregation process. Except in the case of budding yeast, centromere identity does not rely on DNA sequence but on the presence of a special nucleosome that contains a histone H3 variant known as CenH3 or CENP-A (Centromere Protein A). It has been therefore proposed that CENP-A is the epigenetic mark of the centromere. Upon DNA replication the mark is diluted two-fold and must be replenished to maintain centromere identity. What distinguishes CENP-A nucleosomes from those containing histone H3, how CENP-A nucleosomes are incorporated specifically into centromeric chromatin, and how this incorporation is coordinated with other cell cycle events are key issues that have been the focus of intensive research over the last decade. Here we review some of the highlights of this research.

Introduction

The centromere/kinetochore complex orchestrates chromosome segregation and, along with telomeres and origins of replication, constitutes a key element to ensure faithful propagation of the genome. After the cloning of the first centromere sequence from Saccharomyces cerevisiae by Clarke and Carbon [1], many researchers tried to identify a similar “magic sequence” in other organisms. However, the complexity of centromere sequences became clear upon their discovery in Schizosaccharomyces pombe [2], [3]. Fission yeast centromeres are 50–100 kb long, in contrast to 125 bp in budding yeast, and contain a complex arrangement of repeated sequences. This difference in sequence organization led Pluta et al. to distinguish between point centromeres (those of S. cerevisiae) and regional centromeres (those of S. pombe) [4]. The presence of repeated sequences appears to be a characteristic of most centromeres analyzed to date from plants, flies and humans [5], [6], [7]. Importantly, centromeres form at a subset of these repetitive DNA arrays and thus the site of kinetochore assembly appears to be determined by factors other than DNA sequence alone. Indeed, early studies revealed the epigenetic nature of centromeres. In dicentric chromosomes (i.e., carrying two centromeres) only one of them is active for recruitment of kinetochore proteins while the other is inactivated [8], [9]. Steiner and Clarke [10] showed that a nonfunctional centromere present on a circular minichromosome could be converted to a functional one without changes in the content, structural arrangement or chemical modification state of the DNA. Strong evidence supporting that a particular DNA sequence is neither necessary nor sufficient for centromere specification comes from the existence of neocentromeres that arise at otherwise acentric fragments resulting from a chromosome rearrangement. These neocentromeres are devoid of the repeated sequences found at canonical centromeres but recruit most of the centromeric and pericentromeric proteins required for proper chromosome segregation [11].

What, if not DNA sequence, determines the site of kinetochore assembly? Increasing experimental evidence suggests that chromatin composition and organization play a major role in centromere specification and propagation [12], [13], [14]. Centromere Protein A, CENP-A, is a histone H3 variant that replaces canonical histone H3 in the centromeric nucleosomes of all eukaryotes [15], [16], [17], [18], [19]. Inactivation or down regulation of CENP-A in different experimental systems results in chromosome segregation defects and eventually cell death, and its presence is required for assembly of all other centromeric proteins [20], [21], [22], [23], [24], [25], [26]. It has been therefore proposed that CENP-A is the epigenetic mark of the centromere. Although we are far from understanding the molecular determinants of this mark and its inheritance, recent studies have provided exciting clues that we summarize in this review.

Section snippets

What makes CENP-A unique?

CENP-A localizes specifically at active centromeres, although its overexpression can lead to promiscuous localization all over the chromosomes [27], [28], [29]. This 17-kDa histone variant displays over 60% sequence identity to canonical H3 over the C-terminal domain that includes the “histone fold” domain (HFD) [15]. In contrast, the N-terminal region of CENP-A shares the flexible nature of histone tails but shows little amino acid sequence similarity to H3 or any other histone variant, and it

When does CENP-A deposition occur?

Maintenance of centromere identity requires incorporation of new CENP-A during or after replication of centromeric DNA. In S. cerevisiae, all pre-existing CENP-A is replaced by newly synthesized CENP-A during S phase [58] whereas in S. pombe, two pathways of CENP-A deposition exist at different times of the cell cycle, S phase and G2 [59], [60]. Experiments in Arabidopsis thaliana suggest that most CENP-A is loaded in G2 by a replication-independent mechanism [61]. Early studies in human cells

Histone chaperones and CENP-A deposition

Despite their sequence and structural similarities, histone H3 variants are deposited by distinct chaperones: histone H3.1 is deposited by the Chromatin Assembly Factor 1 (CAF-1) complex during replication and DNA damage processing, while histone H3.3 is deposited at active chromatin by a complex containing the HIRA protein in a process independent of DNA synthesis [78]. Some evidences suggested that these two complexes may contribute to CENP-A deposition in yeast, but neither of them could be

Conclusions/perspectives

Our knowledge on how centromere identity is propagated from one generation to the next has increased enormously over the last decade. Many factors have been identified that interact with CENP-A physically and/or genetically, and a number of them have been shown to affect the incorporation or stabilization of CENP-A at centromeres (Fig. 1). However, the exact role of most of these factors remains unknown. The development of an in vitro assay for CENP-A incorporation from purified components

Acknowledgments

We thank Juan Méndez (CNIO) and members of the lab for critically reading the manuscript, and colleagues in the field for helpful discussions. We apologize to those whose work is not cited due to space limitations. Research in our lab is supported by the Spanish Ministry of Science and Innovation (grants BFU2007-66627 and CSD2007-0015, and FPI predoctoral fellowship to P.S.), Fundación Caja Madrid, the Epigenome Network of Excellence (EU) and EMBO (postdoctoral fellowship ALTF 77-2007 to R.B.).

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