LAUSR

Centromeres are essential for accurate chromosome segregation and are marked by centromere protein A (CENP-A) nucleosomes. Mis-targeted CENP-A chromatin has been shown to seed centromeres at non-centromeric DNA. However, the requirements for such de novo centromere formation and transmission in vivo remain unknown. Here, we employ Drosophila melanogaster and the LacI/lacO system to investigate the ability of targeted de novo centromeres to assemble and be inherited through development. De novo centromeres form efficiently at six distinct genomic locations, which include actively transcribed chromatin and heterochromatin, and cause widespread chromosomal instability. During tethering, de novo centromeres sometimes prevail, causing the loss of the endogenous centromere via DNA breaks and HP1-dependent epigenetic inactivation. Transient induction of de novo centromeres and chromosome healing in early embryogenesis show that, once established, these centromeres can be maintained through development. Our results underpin the ability of CENP-A chromatin to establish and sustain mitotic centromere function in Drosophila.