CENP-A and CENP-B collaborate to create an open centromeric chromatin state

Centromeres, the sites within chromosomes responsible for accurate genome repartitioning, are epigenetically defined via replacement of canonical histone H3 by the histone variant CENP-A forming specific nucleosomes with increased DNA flexibility. In human cells, CENP-A nucleosomes and thus centromeres localize to genomic regions containing extended tandem repeats of alpha-satellite DNA. There, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. CENP-A provides a major recruitment point for many CCAN member proteins. One factor, CENP-B, binds to a specific DNA sequence contained in about half of alpha-satellite repeats. CENP-B is a dimer and is involved in maintaining centromere stability and, together with CENP-A, shapes the basic layer of the centromeric chromatin state. While recent studies have revealed the structure of large parts of the CCAN complexes, the nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence resonance energy transfer (FRET) and colocalization imaging as well as dynamic experiments in cells to show that CENP-A incorporation establishes a far more dynamic and open chromatin state compared to canonical H3. We investigate whether CENP-A marks a landing spot for CENP-B, and find that on the single nucleosome level, CENP-B does not prefer H3 over CENP-A nucleosomes. However, in a chromatin fiber context, CENP-B binding is suppressed by higher-order chromatin structure. The increased dynamics of CENP-A chromatin create an opening, allowing CENP-B access and binding. In turn, bound CENP-B further opens the chromatin fiber structure, potentially via bending the bound DNA. Finally, transient knockdown of CENP-A expression in cells increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.