FIG. 7.

Spt2 is required for nucleosome reassembly in the wake of RNAP II at the SRG1 intergenic region. (A) Diagram explaining the experimental procedure designed to analyze histone H3 recovery upon repression of GAL1-FMP27. WT, wild type. (B) spt2Δ mutation affects significantly histone H3 redeposition associated with the last wave of transcription at the GAL1-FMP27 transcribed region. Yeast cells from wild-type (YAN1047) or spt2Δ (YAN1048) strains were grown in galactose medium to mid-log phase. Glucose was then added to the medium, and the cells were cross-linked and harvested at the indicated time points. Histone H3 level was analyzed by ChIP assays using chromatin extracted from wild-type or spt2Δ strains. For each strain, the value of the ratio IP/input calculated for the time zero min was arbitrarily set at 1. (C) Diagram explaining the experimental procedure designed to analyze histone H3 recovery at the SRG1 3′ end upon repression of pGAL1-SRG1. (D) spt2Δ mutation affects significantly histone H3 redeposition associated with the last wave of transcription at the SRG1 region corresponding to the SER3 promoter. The experiment was conducted as described for panel B with chromatin extracted from wild-type (YAN1040) or spt2Δ cells (YAN1039). The ratio IP/input calculated for time zero in the wild type was arbitrarily set at 1. (E) Spt2 delays maximal SER3 induction. The SER3 mRNA and SCR1 levels were analyzed by RT-qPCR using total RNA extracted from cells treated as described for panel B. For each strain, the value of the ratio SER3 mRNA/SCR1 calculated for time zero was arbitrarily set at 1. The values represent the average of two to three independent experiments. Note that at time zero, as expected, the absolute levels in the spt2Δ strain are higher than those observed in wild-type cells.