Active RNA pol II is necessary for peripheral anchoring of the hsp-16.2 promoter. (A) MosSCI insertion at ttTi5605 of the hsp-16.2 promoter driving either one or two mCherry genes as indicated. The top shows control lacking the hsp-16.2 promoter. (B) Quantitation of the GFP-lacI position for lacO-only control and uni- or bidirectional hsp-16.2 promoter constructs (A). Scoring, zone 1 plotting, asterisks, and hatching are the same as in Fig. 2 (B and C). Loci counted were (left to right) n = 117, 95, 309, 346, 232, and 246. P-values versus random = 0.17, 0.31, 0.02, 3 × 10−5, 7 × 10−7, and 3 × 10−11, respectively; for 1× versus 2× genes, both with and without HS, P < 0.05. (C) A thermosensitive (ts) mutation in RNA pol II AMA-1 impairs anchoring of the hsp-16.2 tg #1. GFP-lacI signal positions in WT or ama-1(m218m251) embryos were scored and zone 1 values were plotted before and 20 min after HS (hatched), as in Fig. 2 (B and C). The lacO-tagged tel V insert in the same ama-1ts background shows the opposite effect upon AMA-1 inactivation (HS). Loci counted were (left to right) n = 309, 346, 179, 96, 155, and 155. P-values versus random = 0.02, 3 × 10−5, 6 × 10−5, 0.39, 2 × 10−9, and <10−10, respectively. (D) Scheme of HS kinetics (red indicates samples in B and C) showing gradual temperature increase (E) testing ama-1ts mutants for the hsp-16.2 tg1 position. At × and ○, images were taken and quantified. (E) Progressive temperature increase in the indicated ama-1 mutants releases hsp-16.2 tg #1 without HS. Locus scoring, zone 1, and asterisks are the same as in Fig. 2 (B and C). Loci counted were (left to right) n = 186, 217, 208, 167, 199, 210, 256, 274, 85, 123, 76, and 39. P-values versus random = 6 × 10−6, 1 × 10−8, 0.02, 0.70, 4 × 10−6, 3 × 10−7, 0.01, 0.93, 2 × 10−5, 1 × 10−12, 3 × 10−8, and 2 × 10−4, respectively.
