Focus on the centre: the role of chromatin on the regulation of centromere identity and function

Abstract

The centromere is a specialised chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kinetochore, a critical structure that mediates binding of chromosomes to the spindle, monitors bipolar attachment and pulls chromosomes to the poles during anaphase. Identified more than a century ago as the primary constriction of condensed metaphase chromosomes, the centromere remained elusive to molecular characterisation for many years owed to its unusual enrichment in highly repetitive satellite DNA sequences, except in budding yeast. In the last decade, our understanding of centromere structure, organisation and function has increased tremendously. Nowadays, we know that centromere identity is determined epigenetically by the formation of a unique type of chromatin, which is characterised by the presence of the centromere-specific histone H3 variant CenH3, originally called CENP-A, which replaces canonical histone H3 at centromeres. CenH3-chromatin constitutes the physical and functional foundation for kinetochore assembly. This review explores recent studies addressing the structural and functional characterisation of CenH3-chromatin, its assembly and propagation during mitosis, and its contribution to kinetochore assembly.

Figure 1

Structural organisation of the different classes of eukaryotic centromeres. In holocentric organisms (C. elegans), centromeres form along the entire chromosome. Most eukaryotes, however, contain monocentric chromosomes, in which the centromere forms at a single, generally large, chromosomal region (C. albicans, S. pombe, Drosophila, H. sapiens). In S. cerevisiae, centromeric function resides in a small 125 bp long conserved DNA sequence. See text for details.

Figure 2

CenH3 is a highly divergent histone H3 variant that evolves very rapidly. Sequence comparison of the N-terminal domain (A) and the histone-fold domain (HFD) (B) of CenH3 proteins from different species, ranging from S. cerevisiae to humans, is shown. The sequence of canonical histone H3 is shown at the bottom for comparison. R-rich motives are indicated in A. Secondary structure of the HFD is indicated in B. The position of the CATD, which mediates centromeric targeting of CenH3 and confers distinct structural properties to CenH3-nucleosomes, is indicated.

Figure 3

Structural organisation of CenH3-chromatin. (A) CenH3-nucleosomes can be composed by (CenH3/H4/H2A/H2B)₂ octamers as are canonical nucleosomes. Alternatively, in Drosophila, the formation of ‘half-nucleosomes', composed by unusual (CenH3/H4/H2A/H2B) tetramers, has been proposed, and, in S. cerevisiae, it was reported that H2A/H2B dimers are replaced by Scm3 to form unusual (CenH3/H4/Scm3)₂ hexamers. (B) In centromeric chromatin, blocks of CenH3-nucleosomes are found interspersed with blocks containing canonical histone H3-nucleosomes. H3-blocks show a peculiar pattern of post-translational histone modifications being hypoacetylated and, at the same time, enriched in H3K4me2. During mitosis, these blocks can direct folding of centromeric chromatin into a specific higher-order structure, in which H3-blocks locate in the interface between sister chromatids and CenH3-blocks face out towards the kinetochore, an arrangement that could facilitate bipolar attachment.

Figure 4

Assembly and dynamic behaviour of CenH3-chromatin during cell cycle. Like other histone variants, CenH3 incorporates into chromatin independently of DNA replication. Deposition of newly synthesised CenH3 takes place during mitosis, at late telophase, or early G1. Specific CenH3 chaperones localise to the centromere coincidentally with deposition of new CenH3 and mediate assembly of CenH3-nucleosomes. During assembly, CenH3 might become resistant to proteolysis that, otherwise, degrades CenH3 and prevents deposition at non-centromeric sites. Before deposition, at late anaphase, specific complexes (Mis16/Mis18) seem to modify centromeric chromatin to allow assembly of new CenH3-nucleosomes. During DNA replication at S-phase, CenH3 concentration at centromeres is diluted and kinetochore assembly takes place before replenishment with new CenH3-nucleosomes. It is unclear whether ‘gaps' generated during DNA replication remain nucleosome-free or are filled by replicative H3-nucleosomes. It might also be possible that CenH3-nucleosomes are disassembled into ‘half-nucleosomes' to compensate for this deficit.

Figure 5

Kinetochores are large macromolecular entities. (A) Various protein complexes/networks are known to act at different stages of kinetochore assembly. These include the KNM network (KNL1, NDC80 and MIND), which is involved in microtubule binding, and the NAC/CAD network that directly associates to centromeric chromatin. (B) CenH3 is essential for kinetochore assembly. CenH3 is at the bottom of a complex network of interactions that, ultimately, leads to assembly of a fully functional kinetochore. Dependencies for centromeric/kinetochore localisation are indicated by solid arrows. Possible interactions, observed only in some species or not fully confirmed, are indicated by dotted arrows.