Centromeric and ectopic assembly of CENP-A chromatin in health and cancer: old marks and new tracks

Abstract

The histone H3 variant CENP-A confers epigenetic identity to the centromere and plays crucial roles in the assembly and function of the kinetochore, thus ensuring proper segregation of our chromosomes. CENP-A containing nucleosomes exhibit unique structural specificities and lack the complex profile of gene expression-associated histone posttranslational modifications found in canonical histone H3 and the H3.3 variant. CENP-A mislocalization into noncentromeric regions resulting from its overexpression leads to chromosomal segregation aberrations and genome instability. Overexpression of CENP-A is a feature of many cancers and is associated with malignant progression and poor outcome. The recent years have seen impressive progress in our understanding of the mechanisms that orchestrate CENP-A deposition at native centromeres and ectopic loci. They have witnessed the description of novel, heterotypic CENP-A/H3.3 nucleosome particles and the exploration of the phenotypes associated with the deregulation of CENP-A and its chaperones in tumor cells. Here, we review the structural specificities of CENP-A nucleosomes, the epigenetic features that characterize the centrochromatin and the mechanisms and factors that orchestrate CENP-A deposition at centromeres. We then review our knowledge of CENP-A ectopic distribution, highlighting experimental strategies that have enabled key discoveries. Finally, we discuss the implications of deregulated CENP-A in cancer.

Figure 1.

(A) Schematic representations of the secondary structure of CENP-A and conventional histone H3, together with a depiction of the sites and nature of the major posttranslational modifications known to occur on their N-terminal tail (P, phosphorylation; Me, methylation; Ac, acetylation; Ub, ubiquitination). The 2 amino acid insertion within the L1 loop of CENP-A (R80, G81) that mediates specific interaction with CENP-N, as well as its C-terminal end involved in interaction with CENP-C, are indicated. Note that the αN helix of CENP-A is one helical turn shorter than that of H3. (B,C). Salient differences between the CENP-A and H3 nucleosome structures. (B). Side view of the CENP-A nucleosome (PDB entry 3AN2) together with superimposed conventional core histone octamer (PDB entry 1KX5). The small loop containing CENP-A amino-acids R80 and G81 that protrudes from the surface of the nucleosome is indicated (arrow). The right panel shows an enlargement of the L1 loop area, with a superimposition of the L1 loops from CENP-A (blue) and H3 (red); residues R80 and G81 of CENP-A are shown in green and yellow, respectively. The core histones H2A, H2B and H4 are in light brown in the H3 octamer, while those of the CENP-A octamer are in light blue. (C). Side views of the superimposed DNA from CENP-A (yellow) and conventional H3 (light blue) nucleosome core particle together with either CENP-A (dark blue, left) or H3 (red, middle); The right panel shows a superimposition of DNA from CENP-A- and H3 core particle together with CENP-A and H3. Note that the nucleosomal DNA ends are not visible in the crystal of the CENP-A nucleosome and that the αN-helix of CENP-A exhibits two turns only.

Figure 2.

Artificial and natural systems for the study of CENP-A ectopic deposition. (A). Illustration of the lacO/lacI tethering system developed in Drosophila. The upper panel depicts an array composed of multiple copies of the lac operator (lacO)(red triangles), allowing the tethering of centromeric/chromatin factors fused to the lac repressor (lacI). Introduction of plamids bearing such an array into flies leads to their stable ectopic integration (lower panel). Note that an inducible centromeric/chromatin factor-LacI fusion construction is often used, which allows the time windows of chromatin changes to be monitored. (B). Illustration of the synthetic, alphoid^tetO technology. The upper panel depicts an alphoid^tetO array derived from α satellite repeats (red triangles) by integration of tetO sequences (blue triangles), allowing tethering of chromatin factors expressed as tetR-fusion proteins. When inserted into a cloning vector and transfected into cells, such arrays can integrate ectopically into a chromosome arm or form a human artificial chromosome (HAC)(lower panel). Whereas ectopic alphoid^tetO array integration enables dissection of the chromatin factors and states that promote CENP-A deposition, the characterization of HACs also allows de novo centromere and kinetochore assembly to be explored. Beyond the scope of this review, it should be noted that de novo HAC formation requires the presence of CENP-B-box sequences in the α satellite DNA (not shown here) (reviewed by Molina et al. (112). Variations on the technology oulined here include the simultaneous tethering of factors with antagonist chromatin modifying activities Molina et al. (140).

Figure 3.

(A) Salient features of CENP-A deposition at the centrochromatin. Shown is a schematic summary of the CENP-A chaperone and deposition machinery including non-coding RNA originating from the underlying DNA. Maintenance of a boundary between centrochromatin and flanking pericentric heterochromatin domains (hereby illustrated by the SUV39H1/HP1-mediated deposition of H3K9me2/3) involves prevention of pericentric heterochromatin spreading into the centromere, which has been proposed to involve histone acetylation orchestrated by KAT7 and also histone turnover reactions mediated by the chromatin remodeler RSF1. KAT7-recruited RSF1 has also been suggested to promote CENP-A assembly directly. Not shown here are the histone epigenetic marks, turnover mechanisms and responsible chromatin factors that define the centrochromatin signature and impact its dynamics. These include a short window of H3K9 acetylation that accompanies Mis18/HJURP localization at the centromere, as well as histone modifications that contribute to a transcriptionaly-competent chromatin state. (B) Histone post-translational modifications that mark the centrochromatin and its flanking pericentric heterochromatin. Note that the H3K9me2 and H3K27me2 marks have been proposed to occur predominantly as a dual H3K9me2-K27me2 modification at the centromere.

Figure 4.

CENP-A deregulation and cancer. (A) Loss of p53 and oncogenic transformation lead to essential roles for CENP-A and its chaperone HJURP. In the model proposed by Filipescu et al. (181), the observed upregulation of CENP-A and HJURP in p53-null cells undergoing during malignant transformation allows cancer cells to sustain high proliferation rates by increasing the efficiency of CENP-A deposition and centromere propagation. Depletion of HJURP in such a model results in the rapid loss of CENP-A from the centromeres which, in cells lacking the cell-cycle arrest functions of p53, results in centromere dysfunction, chromosomal instability, aneuploidy and cell death. (B) Impact of CENP-A overexpression and ectopic localization on centromere dynamics and chromosome instability in cancer cells. Under normal conditions (upper panel), kinetochores assemble and function normally, providing sustained spindle microtubule attachment and enabling the orderly separation of sister chromatids. In CENP-A overexpressing cancer cells (lower panel), Shrestha et al. (8) propose that ectopic CENP-A molecules may titrate out and/or induce the loss of factors essential for proper kinetochore assembly and function at native centromeres, leading to chromosome segregation defects that are depicted here by aneuploidy and micronuclei formation.