Genetics, epigenetics and back again: Lessons learned from neocentromeres

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Abstract

The duplication and segregation of the genome during cell division is crucial to maintain cell identity, development of organisms and tissue maintenance. Centromeres are at the basis of accurate chromosome segregation as they define the site of assembly of the kinetochore, a large complex of proteins that attaches to spindle microtubules driving chromosome movement during cell division. Here we summarize nearly 40 years of research focussed on centromere specification and the role of local cis elements in creating a stable centromere. Initial discoveries in budding yeast in the 1980s opened up the field and revealed essential DNA sequence elements that define centromere position and function. Further work in humans discovered a centromeric DNA sequence-specific binding protein and centromeric α-satellite DNA was found to have the capacity to seed centromeres de novo. Despite the early indication of genetic elements as drivers of centromere specification, the discovery in the nineties of neocentromeres that form on unrelated DNA sequences, shifted the focus to epigenetic mechanisms. While specific sequence elements appeared non-essential, the histone H3 variant CENP-A was identified as a crucial component in centromere specification. Neocentromeres, occurring naturally or induced experimentally, have become an insightful tool to understand the mechanisms for centromere specification and will be the focus of this review. They have helped to define the strong epigenetic chromatin-based component underlying centromere inheritance but also provide new opportunities to understand the enigmatic, yet crucial role that DNA sequence elements play in centromere function and inheritance.

Section snippets

Genetic elements instructing centromere formation

Initial studies were performed in the budding yeast Saccharomyces cerevisiae, where the site of microtubule attachment became known as a ‘‘point centromere’’. Pioneering studies showed a mere 125 bp DNA sequence containing three conserved DNA elements (CDE I, II and III) to be key for active centromere formation [[1], [2], [3]] (Fig. 1A). The finding that the centromeric DNA sequence placed onto a plasmid carrying a yeast replication origin is all that is required for the plasmid to behave as a

CENP-A, an epigenetic centromere mark

Contemporaneous with the discovery of neocentromeres, it became apparent that centromeres feature an unusual chromatin structure. One of the initial set of centromere proteins discovered along with CENP-B and CENP-C was CENP-A, a histone-like component tightly associated with chromatin at centromeres, suggesting that it may function as a centromere-specific core histone [13,[20], [21], [22]], a notion confirmed by its cloning that revealed it to be a novel histone H3 variant [23].

CENP-A-based

Neocentromeres, origins of a centromere paradigm shift

The first clear indication for the epigenetic regulation of the centromere came from the discovery of human neocentromeres, the first of which was described in 1993. This neocentromere was derived from a rearrangement of chromosome 10, resulting in a centromere-containing ring chromosome and an acentric linear chromosome lacking any centromeric DNA. The latter acquired centromere proteins at a novel location, constituting a functional centromere that rescued mitotic maintenance of the

Artificial systems for neocentromere generation

In the pathogenic yeast Candida albicans, regional centromeres can expand from 4 to 15 kb and CENP-A is assembled into unique sequences that cover around 3 kb on each chromosome which are surrounded by direct or inverted repeats lacking classical pericentric heterochromatin [84]. By taking advantage of the higher rates of homologous recombination in Candida, a selectable marker was used to replace the centromere of chromosome 5. Surviving cells maintained the chromosome by neocentromere

Common elements driving the formation of neocentromeres

The accumulated collection of different neocentromeres, as well as the artificial generation of neocentromeres in different species allows us to define shared features that are required for centromere specification. All neocentromeres described up to date are universally marked by CENP-A [17,[91], [92], [93]] (Fig. 3A). While perhaps not surprising, it underscores the critical role of the centromere-specific histone variant in building the structural core of the kinetochore [94]. In addition to

Centromere size

Mapping neocentromere positions along the genome has allowed for a comparison of centromere domains and specific DNA sequences supporting centromere formation. One relevant parameter is centromere size. In S.pombe, the CENP-ACnp1 domain expanded to around 20 kb in the artificially generated neocentromeres, a size comparable to the endogenous centromeres (15 kb) [87]. A similar picture emerges from chicken DT40 cells whose endogenous non-repetitive centromeres expand to ~35 kb while

General features of centromeric chromatin

At the primary DNA sequence level, the most conserved feature across neocentromeres is the tendency to form preferentially on AT rich sequences [81,90], although the preference is slight and by no means sufficient to explain centromere formation. Moreover, some human neocentromeres are enriched in repetitive LINE elements and a potential role of the L1 retrotransposons in the regulation of neocentromere activity has been suggested [102]. At the chromatin level, more similarities arise. The

The role of higher order chromatin features in centromere function

From a more recent perspective, analysis performed in Candida, revealed that neocentromeres generated at different loci along the chromosome resulted in kinetochores that cluster with active endogenous centromeres within the nucleus [86]. This result suggested that the higher order chromatin organization may be another important feature in defining sites that can support centromere formation. Indeed, 4C analysis performed in chicken DT40 cells revealed that following neocentromere formation,

Centromere drifting of evolutionary new centromeres

As we have seen above, there is no universal agreement about what local features are responsible for neocentromere specification, and it is quite likely that random chance plays a major role. It is generally clear that chromatin-based epigenetic identity plays a dominant role. If indeed centromere inheritance is largely uncoupled form the underlying DNA sequence, this would make a clear prediction: Pure chromatin-based replication of centromere identity would be subject to stochastic

From genetic centromere specification to epigenetics and back again

The local drift of CENP-A domains is expected from a self-templating epigenetically defined mechanism for centromere specification. In fact, due to the existence of ENCs in repetitive DNA tracks, it has been hypothesized that a possible role for the repetitive centromeric DNA is to generate a safe space for CENP-A drifting to occur, avoiding migration towards genomic regions containing essential genes [135] (Fig. 4). Thus, the presence of alpha-satellite DNA may promote centromere maintenance

Future perspectives

Through the study of neocentromeres we have discovered several salient features of centromeric chromatin structure that specifies centromere positions. While there is no broad consensus on exactly what are the local DNA sequence and chromatin features favourable for centromeres to form, many advances have been made regarding the elements required for centromere specification.

Artificial systems for neocentromere formation, developed in different model organisms have been particularly powerful

Acknowledgements

Salary and research support to MMP and LETJ is provided by an ERC-consolidator grant ERC-2013-CoG-615638 and a Senior Research Fellowship in Basic Biomedical Science.

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