Maize Centromere Structure and Evolution: Sequence Analysis of Centromeres 2 and 5 Reveals Dynamic Loci Shaped Primarily by Retrotransposons

Abstract

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.

Author Summary

Centromeres tend to be the last regions to be assembled in genome projects, as their mapping is hampered by their characteristically high repeat DNA content and lack of genetic recombination. Using unique markers derived from these repeat-rich regions, we were able to generate and annotate physical maps of two maize centromeres. Functional centromeres are defined not so much by their primary DNA sequence as by the presence of CENH3, a special histone that replaces canonical histone H3 in centromeric nucleosomes. Little is known about how deposition of CENH3 is regulated, or about the interplay between centromeric repeats and CENH3. By graphing the density of CENH3 nucleosomes onto the physical map, we delineated the functional centromeres in today's maize genome. We then used the large number of LTR retrotransposon insertions, for which the corn genome is well known, as “archeological evidence” to reconstruct the historic centromere boundaries. This was possible because i) some retrotransposon families of maize (CRM) appear to possess a unique ability to preferentially target centromeres during integration and ii) insertion times of individual retrotransposons can be calculated. Here we show that the centromere boundaries in maize have changed over time and are heavily influenced by centromeric and non-centromeric repeats.