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. 2018 Mar 5;7:e31989. doi: 10.7554/eLife.31989

Figure 4. CUT60 prevents ATP16 repression by Transcriptional Interference.

(A) ChIP-qPCR experiments for histone H3K36 trimethylation (H3K36me3) relative to the histone H3 levels at the promoter and gene body of ATP16 in wild type (black), cut60Δ::URA3 (white) and cut60Δ::URA3++ (grey) strains. Error bars are s.e.m. of three biological replicates. * indicates p values of p<0.05 comparing %IP of H3K36/H3 in cut60Δ::URA3 to the %IP of H3K36me3/H3 in wild type. Position of ChIP-qPCR probes indicated in top panel. (B) Schematic representation of synthetic circuits. mCherry replaces MED2, YFP replaces ATP16. Circuit I represents wild type circuit containing CUT60, circuit II represents the cut60Δ::SUT129 mutant circuit. (C) Relative fluorescence of mCherry and YFP in circuit I (white) and circuit II (black). Error bars are s.e.m. of nine biological replicates, the mean was derived from multiple thousand individual measurements. *** indicates p values of p<0.0001. (D) Relative YFP fluorescence of circuit I in wild type (white) and set2Δ (light grey) background, and of circuit II in wild type (black) and set2Δ (light grey) background. YFP fluorescence levels are normalized to the corresponding wild-type values. Error bars are s.e.m. of six biological replicates. ** indicates p values p < 0.01 comparing YFP fluorescence in set2Δ to the respective wild type. (E) Relative YFP fluorescence in circuit I in wild type (white), spt6-1004 (dark grey) and spt16-197 (light grey) background, and in circuit II in wild type (black), spt6-1004 (dark grey) and spt16-197 (light grey) background. YFP fluorescence is normalized to the corresponding wild-type values. Error bars are s.e.m. of six biological replicates. * and ** indicate p values of p<0.05 and p<0.01 respectively comparing YFP fluorescence in spt6-1004 and spt16-194 to the respective wild type.

Figure 4.

Figure 4—figure supplement 1. Set2p, Spt6p and Spt16p participate in Transcriptional Interference of ATP16.

Figure 4—figure supplement 1.

(A) Enrichment of DNA corresponding to the Actin control gene for H3 (white) and H3K36me3 (grey) antibodies relative to no antibody control (black, values indicated) in wild type, cut60Δ::URA3 and cut60Δ::URA3 ++ strains. Error bars are s.e.m. of 3 biological replicates. *and *** indicate p values of p<0.05 and p<0.001 comparing H3K36me3 to no antibody control. (B) ChIP-qPCR experiments for histone H3 at the promoter and gene body of ATP16 in wild type (black), cut60Δ::URA3 (white), cut60Δ::URA3++ (grey) strains. Enrichment is normalized to ACT1. Error bars are s.e.m. of three biological replicates. (C) ChIP-qPCR for H3K36me3 levels at the promoter and gene body of ATP16 in wild type (black), cut60Δ::URA3 (white), cut60Δ::URA3++ (grey) strains. Enrichment is normalized to ACT1 locus. Error bars are s.e.m. of three biological replicates. * indicates p values of p<0.05 comparing H3K36me3 at the promoter of ATP16 in cut60Δ::URA3 to wild type. (D) Histograms showing the frequency of YFP fluorescence values of individual cells of cell populations containing circuit I (top panel, n = 13.000 cells) or circuit II (bottom panel, n = 12.000 cells). The distributions suggest variation around one distinct fluorescence state rather than a bi-modal distribution. (E) Relative mCherry fluorescence of circuit I in wild type (white) and set2Δ (light grey) background, and of circuit II in wild type (black) and set2Δ (light grey) background. mCherry fluorescence levels are normalized to the circuit I or circuit II wild type data. Error bars are s.e.m. of six biological replicates. (F) Relative mCherry fluorescence of circuit I in wild type (white), spt6-1004 (dark grey) and spt16-197 (light grey), and of circuit II in wild type (black), spt6-1004 (dark grey) and spt16-197 (light grey). mCherry fluorescence levels are normalized to the circuit I or circuit II wild type data. Error bars are s.e.m. of six biological replicates. ** and *** indicate p values of p<0.01 and respectively p<0.001 comparing mCherry fluorescence in spt6-1004 and spt16-194 to the respective wild types.