Figure 1.

The FPC is essential for the maintenance of heterochromatic gene silencing

(A) Illustration of the FPC (Mrc1, Swi1/Tof1, Swi3/Csm3) at the replisome, showing the CMG helicase (MCM2-6, Cdc45), Mcl1/Ctf4, DNA primase (Polα), and polymerase epsilon (Polε).

(B) Heterochromatic mating-type region between the IR-L and IR-R boundaries depicting the silent mating-type cassettes mat2-P and mat3-M, Kint2::YFP and (EcoRV)::mCherry fluorescent reporter genes, RNAi-dependent nucleation center cenH, Atf1 transcription factor binding sites, and REII and REIII silencing elements.

(C) Establishment of mCherry silencing following clr4⁺ reintroduction in FPC mutants.

(D) Cells with derepressed reporters in clonal cultures of FPC mutants (n = 6). Data are represented as mean ± SD.

(E) Micrographs of FPC mutants expressing mCherry. Scale bar: 10 μm.

(F) Histograms of mCherry fluorescence (mean, black; replicates n = 6, red).

(G) Interpretation of loss-of-silencing events in (H). The mCherry locus asymmetrically loses heterochromatic silencing in S phase, and the protein production starts in G2 (top cell). The expressed (mCherry ON) and repressed (mCherry OFF) chromatids segregate to sister cells. In subsequent cell divisions, mCherry protein is produced in the ON lineage (blue arrows) but is diluted in the OFF lineage (yellow arrows).

(H) Loss of silencing in the mrc1Δ mutant followed by time-lapse microscopy. White arrowheads point to cells experiencing a loss-of-silencing event. Blue arrowheads point to ON cell lineages and yellow arrows point to OFF lineages. Yellow arrows are omitted at 10 h for clarity. Scale bar: 10 μm.