FISH image showing maize chromosomes. Centromeres (CentC) are in green, knobs are in red.

While kinetochore proteins are reasonably well conserved, centromeric DNAs are among the most poorly conserved sequences known; the homology is so slim that it is nearly impossible to identify centromeres based on sequence alone. Our strategy for finding centromeres is to start with the kinetochores and work down, i.e., to use anti-kinetochore antisera as a way to pull down the associated centromeric DNA.

The two main components of maize centromeres are a 156 bp tandem repeat known as CentC, and a centromere-specific retroelement known as CRM (Centromeric Retroelement in Maize). By immunoprecipitating nucleosomes (by ChIP) containing CENH3, we were able to confirm that CentC and CRM are indeed major centromeric repeats. These results have now been amply confirmed by large-scale sequencing, fiber-fluorescent in situ hybridization, and extended chromatin assays (largely by our collaborator Jiming Jiang). Maize now has the best-characterized centromeres/kinetochores in plants, and as far as we know CentC and CRM are the only two viable candidates for bona fide centromeric repeats in this species.

Perhaps the most intriguing aspect of the centromeres in cereal grains are the CR elements, which are remarkably well conserved and entirely centromere-specific. We hypothesized that CR elements might promote transcription over the centromere. By combing the robust chromatin immunoprecipitation (ChIP) technique with RNA detection methods, we demonstrated that CRM as well as CentC are not only transcribed, but that much of the RNA remains tightly bound to the centromere/kinetochore complex. Our data and results from several other labs suggest that, contrary to common perception, centromeric chromatin is transcriptionally permissive.