Defining the Minimal Trigger for Human Centromere Formation

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Human centromeres are large chromosomal loci specified by the presence of the histone H3 variant CENP-A, typically found at an array of alpha-satellite repeat sequences. As centromeres are omnipresent and constitutively inherited, little is known about their initial formation. Yet, witnessing the birth of a centromere allows us to discover the design principles of a centromere assembly. Human neocentromere formation is a rare event in nature and most studies to date have focused on seeding centromeres either on ectopic extrachromosomal arrays of alpha-satellite DNA or by nucleating CENP-A chromatin artificially. While insightful, these approaches do not inform us about the underlying chromatin and genomic characteristics required for neocentromere specification. Yet, understanding how to build a centromere is key to basic chromosome biology and will instruct the engineering of synthetic mammalian chromosomes.Recently, the host laboratory developed a CRISPR-Cas9-driven centromere deletion technology to isolate a spontaneously-formed epigenetically seeded human neocentromere (Neo4p13). I will capitalize on this methodology to discover what are the key elements needed for efficient neocentromere seeding on an intact human chromosome. Specifically, as spontaneous neocentromere formation is rare, I will determine the minimal copy number of two key centromere ingredients, CENP-A nucleosomes and alphoid DNA, that is sufficient to efficiently generate neocentromeres. Moreover, I will compare the role of active and silenced chromatin in the nucleation of these neocentromeres. Further, I will define the contribution of alphoid DNA to existing neocentromeres through the insertion of an alphoid DNA array adjacent to our current neocentromere, and at a distal site. At the interface of fundamental centromere biology and advanced chromosome engineering, this proposal will improve our ability to reliably build stable centromeres in the context of functional human chromosomes.