Testing the functional consequences of rapid centromeric DNA and protein evolution

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PROJECT SUMMARY/ABSTRACT The centromere is a network of proteins rooted to centromeric DNA by nucleosomes containing a centromere-specific histone H3 paralog, CENP-A. Despite the centromere’s conserved role in chromosome segregation, a subset of its proteins that are essential for its function exhibit molecular signatures of rapid adaptive evolution. Repetitive centromeric satellite DNA also rapidly evolves, drastically diverging in sequence between species. The “centromere drive hypothesis” proposes that a genetic conflict between centromeric DNA and proteins causes their rapid evolution. Specifically, centromeric DNAs behave selfishly and increase their inheritance through female meiosis (drive) by increasing binding affinity for centromeric proteins. Selfish centromeres are also detrimental to the organism, which selects for novel centromeric protein variants with a lower affinity for the selfish DNA, thereby suppressing drive. The centromere drive hypothesis further predicts that distinct evolutionary lineages undergo unique bouts of centromeric DNA-protein co-evolution, leading to deleterious centromeric DNA-protein incompatibilities in hybrids that promote reproductive isolation. I have developed a novel experimental system to test three key propositions of the centromere drive hypothesis. The first is that centromeric DNA repeat variants differentially recruit centromere proteins. By fertilizing the eggs of M. musculus with the sperm of divergent Murinae species, I test whether the two species centromeric DNA repeats differ in their ability to recruit centromeric proteins from the hybrid zygote cytoplasm. My preliminary data indicate that Mus pahari CENP-A nucleosomes have a higher binding affinity for a key centromeric protein scaffold, CENP-C, than M. musculus CENP-A nucleosomes. For my first aim, I will biochemically reconstitute the M. pahari CENP-A nucleosome and test whether the centromeric DNA that wraps it imparts this increase in CENP-C binding. For my second aim, I will test whether rapidly evolving centromere protein orthologs (variants) differentially bind to centromere DNA. I will transiently express various centromere protein orthologs in hybrid zygotes and determine whether their binding preferences for the two species’ centromeres differ. For my third aim, I will test whether divergent centromere DNAs result in deleterious incompatibilities in hybrids. I found that in M. musculus / M. pahari hybrid zygotes, a subset of M. pahari centromeres are mispackaged. I will generate a more contiguous M. pahari genome assembly to determine if unique M. pahari centromeric DNA sequences underly this mispackaging, and test whether mispackaging causes deleterious chromosome missegregation. The hybrid embryo system that I developed serves as a powerful tool to interrogate not only functional divergence of centromeric DNA and proteins, but also genetic conflict more broadly by uncoupling selfish DNAs from their species-specific suppressors. This system and my training will position me to establish a future research program around diverse mammalian selfish genetic elements (see Career Goals).