Neocentromeres: a place for everything and everything in its place

Abstract

Centromeres are essential for chromosome inheritance and genome stability. Centromeric proteins, including the centromeric histone centromere protein A (CENP-A), define the site of centromeric chromatin and kinetochore assembly. In many organisms, centromeres are located in or near regions of repetitive DNA. However, some atypical centromeres spontaneously form on unique sequences. These neocentromeres, or new centromeres, were first identified in humans, but have since been described in other organisms. Neocentromeres are functionally and structurally similar to endogenous centromeres, but lack the added complication of underlying repetitive sequences. Here, we discuss recent studies in chicken and fungal systems where genomic engineering can promote neocentromere formation. These studies reveal key genomic and epigenetic factors that support de novo centromere formation in eukaryotes.

Keywords: CENP-A; gene conversion; heterochromatin; histone; replication; transcription.

Figure 1. Engineered neocentromeres in DT40 chicken cells arise non-randomly near the original centromere

Endogenous chicken chromosomes Z and 5 contain CENP-A chromatin regions (black-filled curves; reddish-blue nucleosomes) that are ~35kb in size. (A) Removal of the 127kb of the centromere region of chromosome Z, including the 35kb CENP-A domain, and replacement with a bleomycin selectable marker cassette (blue box) using Cre-lox P genome engineereing led to neocentromere formation (yellow boxes) at various sites along chromosome Z. The location of neocentromere formation was preferentially skewed, with 76% of neocentromeres forming proximal to the original centromere. (B) Low levels of CENP-A were detected by ChIP-seq in a 2Mb region surrounding the endogenous centromere of chromosome 5. To test if these regions of more modest CENP-A incorporation are capable of nucleating a centromere in the absence of the adjacent, more enriched CENP-A domain, a smaller (67kb) region of centromere 5 was deleted. Nearly all neocentromeres (97%) formed adjacent to the original centromere, suggesting that in chicken cells, non-kinetochore CENP-A-enriched chromatin can seed neocentromere formation in the absence of the original centromere. Drawings are not drawn precisely to scale.

Figure 2. Induced neocentromeres in C. albicans form at high frequency near the original centromere

(A) Replacement of the 4.5kb C. albicans CEN7 with a URA3 marker (red box) resulted in neocentromere formation (yellow boxes) within 1–3kb on either side of the original centromere. The amount of CENP-A (reddish-blue nucleosomes) at the neocentromeres relative to the amount at the original CEN7 is denoted by the number of cartoon CENP-A nucleosomes (1 = reduced to 3 = normal amount at the endogenous centromere). (B, C) Larger deletions (6.5kb or 30kb) of the CEN7 region produced neocentromeres that were located 2–13kb from the original centromere. Notably, neocentromeres that formed farther from the original centromere contained lower amounts of CENP-A. (D) In a subset of neocentromere-containing strains, the neocentromeres disappeared and the endogenous CEN7 was restored by gene conversion. In these strains, the neocentromeres contained lower amounts of CENP-A (denoted schematically by the blue boxes and few CENP-A nucleosomes), suggesting that the amount of CENP-A may mark the completeness of a centromere, or its probability of being reverted by gene conversion. Drawings are not drawn precisely to scale.

Figure 3. The formation and fate of de novo centromeres arising at atypical genomic locations

Non-centromeric chromosomal loci contain low levels of CENP-A (red) and histone variants, including H2A.Z (yellow). Upon centromere loss, CENP-A is preferentially incorporated at existing CENP-A loci, whereas H2A.Z may guard against CENP-A incorporation. (A) Chromatin remodeling complexes and histone H3 chaperones monitor local chromatin structure and evict misincorporated CENP-A, resulting in centromere loss. (B) Alternatively, following centromere loss, CENP-A is incorporated at loci already containing a low level of CENP-A or other chromatin structures permissive to neocentromere formation, such as heterochromatin. HJURP association enables maturation of incomplete centromeres, followed by recruitment of centromere and kinetochore proteins necessary for neocentromere function. (C) Failure to recruit a sufficient amount of CENP-A in diploid organisms can result in incomplete neocentromere formation, which may be corrected by repositioning that results in CENP-A incorporation at a more favorable site or by homologous recombination (not shown). Neocentromeres can form, perhaps preferentially, within or adjacent to genes, resulting in reduced transcriptional activity adjacent to the mature neocentromere.