Robust CENP-A incorporation in human cells is independent of transcription and cohesin components.

Centromeres are essential chromosomal components that ensure proper cell division by serving as assembly sites for kinetochores, which connect chromosomes to spindle microtubules. Centromeres are marked by the evolutionarily conserved centromere-specific histone H3 variant, CENP-A, which is deposited into centromere nucleosomes during G1 in human cells. Centromeres retain cohesin, a ring-like protein complex, during mitosis, protecting sister chromatid cohesion and centromere transcription to prevent chromosome missegregation. Previous work in Drosophila has suggested that centromere transcription and centromeric RNAs are important for CENP-A deposition in chromatin. During mitosis, centromeric cohesin is critical for centromere transcription. However, it is not clear how or whether centromeric transcription and cohesin contribute to CENP-A deposition in G1 in human cells. To address these questions, we combined a cell synchronization strategy with the Auxin Inducible Degron technology and transcription inhibition in human cells. In contrast with Drosophila cells, our results demonstrated that neither centromeric transcription nor cohesin is required for CENP-A deposition in human cells. Our data demonstrate clear differences in the CENP-A deposition mechanism between human and Drosophila cells. These findings provide deeper insights into the plasticity underlying centromere maintenance and highlight evolutionary divergence in centromere maintenance systems across species.