Gene looping persists following a cycle of GAL10 activation and repression. (A) Schematic depiction of the GAL locus. The positions of the divergent P1 and T1 primer pairs used in 3C analysis and the positions of the PCR products generated in RT–PCR and ChIP analyses are indicated. (B) Time course of GAL10 activation and repression. Cells were grown in 2% glucose (Glc) to mid-log phase, shifted to 2% galactose (Gal) for 2.5 h, then shifted back to 2% glucose. The numbers in parentheses above the line correspond to time points below the line and to lane numbers in C–E. (C) GAL10 and ACT1 (control) mRNA levels were assayed by RT–PCR. (Lane 1) GAL10 transcript levels were quantified by normalizing to the preinduction level, set at 1. (D) RNAP II occupancy of the GAL10 promoter was determined by ChIP using an antibody (8WG16) to the unphosphorylated form of RNAP II. (Lane 1) The data were quantified by dividing the immunoprecipitation:input ratio for GAL10 to the immunoprecipitation:input ratio for an intergenic region on chromosome V (Komarnitsky et al. 2000; Singh and Hampsey 2007; data not shown) and were normalized to the preinduction sample. (E) Gene looping between the GAL10 promoter and terminator regions was determined by 3C using the P1 and T1 primer pairs as described previously (Singh and Hampsey 2007). Control PCR represents the intergenic region of chromosome V, generated using convergent primers, confirming that equal amounts of template DNA were present in all reactions. (Lane 1) The data were quantified by dividing the P1–T1 PCR signals by the control PCR signals for each sample and were normalized to the ratio for the preinduction sample. Results are presented as fold increase below each lane in C–E.

GAL1p-SEN1 undergoes rapid reactivation and correlates with the persistence of looping. (A) Schematic depiction of the GAL1p-SEN1 gene, depicting HindIII sites (H) and positions of the primer pairs (P1 and T1) used in 3C analysis, and the region amplified by RT–PCR analysis of mRNA levels. (B) Time course of GAL1p-SEN1 activation, corresponding to the data presented in C and D. (C,D) SEN1 transcript levels (RT–PCR) and GAL1p-SEN1 looping (3C) were determined as described in Figure 1. (E) Schematic depiction of the time course of GAL10 activation, repression, and reactivation, corresponding to the data presented in F and G. (F,G) Analogous to C and D, extended to include a cycle of glucose repression and subsequent reactivation. (H,I) Quantification of the RT–PCR and 3C data for GAL1p-SEN1 reactivation. In B and E, the numbers in parentheses above the line correspond to time points below the line and to lane numbers in C, D, F, and G.

Gal4 persists at the GAL10 promoter and is associated with looping. (A) Schematic depiction of the time course of GAL10 activation and reactivation, corresponding to the data presented in B and C. The numbers in parentheses above the line correspond to time points below the line and to the lane numbers in B and C. (B) ChIP analysis of RNAP II occupancy of the GAL10 regulatory region in isogenic wild-type and sua7-1 strains using an antibody to the Rpb3 subunit of RNAP II. (C) ChIP analysis of Gal4 occupancy of the GAL10 regulatory region. All data were quantified as described in the legend for Figure 1D.

The rapid reactivation kinetics of GAL10 is associated with gene looping. (A) Schematic depiction of the time course of GAL10 activation, corresponding to the data presented in B and C. (B,C) Transcript levels (RT–PCR) and GAL10 looping (3C) were determined as described in Figure 1. The sua7-1 mutant encodes the TFIIB E62K replacement and is isogenic to the wild-type (WT) strain. (D) Schematic depiction of the time course of GAL10 activation, repression, and reactivation, corresponding to the data presented in E and F. (E,F) Analogous to B and C, extended to include a cycle of glucose repression and subsequent reactivation. (G,H) Quantification of the RT–PCR and 3C data for GAL10 activation and reactivation. In A and D, the numbers in parentheses above the line correspond to time points below the line and to lane numbers in B, C, E, and F. The data in G and H summarize the data shown in B, C, E, and F.

Effects of Set1 and Snf2 on GAL10 looping and memory. (A) Schematic depiction of the time course of GAL10 activation and reactivation, corresponding to the data presented in B and C. (B,C) Transcript levels (RT–PCR) and GAL10 looping (3C), as described in Figure 1, were assayed using isogenic SUA7 (WT), sua7-1; SET1 (WT), set1Δ; and SNF2 (WT), snf2Δ strain pairs. (D) Schematic depiction of the time course of GAL10 activation and repression for the data presented in E. (E) GAL10 looping (3C) using the same wild-type and snf2Δ strain pair as in B. All data were quantified as described in Figure 2 and results are presented as fold change below each lane in B, C, and E.