Figure 7. Summary on the role of factors that promote the integrity of H3 chromatin during transcription in preventing promiscuous CENP-A^Cnp1 deposition.

(A) In wild-type cells, a limited amount of free CENP-A^Cnp1 is available to accumulate outside endogenous centromeres at which kinetochore proteins act to attract CENP-A^Cnp1. This limited pool of free CENP-A^Cnp1 can be preferentially deposited to specific sites such as centromeric central domain (CC) and subtelomeric regions (ST). Intensity of red color on the chromosome and the plasmid represents relative “receptiveness” of the locus for CENP-A^Cnp1 incorporation. Bold arrow indicates regions where CENP-A^Cnp1 incorporation normally occurs without overexpression (i.e. centromeres). Dashed arrows indicate regions where de novo assembly of CENP-A^Cnp1 is expected under conditions where CENP-A^Cnp1 deposition is stimulated (e.g. when CENP-A^Cnp1 is overexpressed or flanking heterochromatin is provided). (B) In cells with defective FACT, non-centromeric regions become permissive to CENP-A^Cnp1 (indicated by red color all over the chromosome and the plasmid). Endogenous centromeres drive CENP-A^Cnp1 assembly at proximal euchromatic regions when they become permissive to CENP-A^Cnp1 assembly (indicated by dark red color at centromere-proximal regions). CENP-A^Cnp1 incorporation when overexpressed is not significantly elevated at CC and ST in FACT mutants compared to wild-type cells, suggesting that FACT action may be already limited at these sites. Without CENP-A^Cnp1 overexpression, the limited pool of free CENP-A^Cnp1 is distributed to non-centromeric regions and cannot accumulate at normally preferred sites such as CC and ST. (C) In cells with defective Clr6-CII, only specific regions such as centromere-proximal euchromatic regions, CC and ST become permissive to CENP-A^Cnp1. Clr6-CII has a weaker impact on H3 chromatin compared to FACT and thus loss of Clr6-CII function promotes CENP-A^Cnp1 incorporation only at regions where CENP-A^Cnp1 assembly is predisposed (centromere-proximal regions) or FACT action is limited (CC and ST). In Clr6-CII mutants, limited pool of free CENP-A^Cnp1 is not distributed and can accumulate at preferred sites, allowing de novo assembly of CENP-A^Cnp1 chromatin on a plasmid bearing CC in the absence of flanking heterochromatin.