Figure 5.
Figure 5. HSE and HSAS sequences are necessary to anchor hsp-16.2 at the nuclear rim. (A) Nascent (left) and spliced (right) RNA levels of hsp-16.2 (◇) and mCherry (▪) before and at six time points after HS for the indicated loci. Values are normalized to its-1, a transcribed rDNA spacer. (B) Scheme of the hsp-16.2/41 promoter, containing two HSF-1 binding sites, HSE1 and HSE2, on either side of the HSAS sequence, with hsp-16.2 or mCherry transcription driven toward the right. Changes in mutated versions of HSE1, HSAS, or HSE2 are shown below the sketch. (C) Quantitation of subnuclear positions of the GFP-lacI signal for the MosSCI insertion of WT or mutated hsp-16.2 promoters; color-coded as in B. Zone 1 values, bar labels, and asterisks are defined as in Fig. 2 (B and C). Cell numbers were (left to right) n = 117, 309, 367, 339, 306, 95, 346, 329, 347, and 249. P-values versus random = 0.17, 0.02, 0.80, 0.73, 0.33, 0.31, 3 × 10−5, 1 × 10−6, 1 × 10−6, and 0.59, respectively. (D) Nascent and spliced RNA levels of mCherry driven from WT or mutated hsp-16.2 promoters at MosSCI insertion, normalized to its-1. Values are shown for HSE1/2mt, HSASmt, and WT, color-coded as in B. (E) HSF-1 is essential for peripheral targeting of the hsp-16.2 tg #1 both before and after HS. Quantification of positions of the GFP-lacI signal for progeny from control RNAi or hsf-1 RNAi fed adult progeny as in Fig. 2 B. Zone 1 scoring, asterisks, and hatching are defined the same as in Fig. 2 (B and C). Numbers counted were (left to right) n = 681, 704, 281, and 244. P-values versus random = 3 × 10−4, 3 × 10−7, 0.16, and 0.07, respectively. (F) Quantitation of GFP-lacI focus position for WT or mutated hsp-16.2 promoter MosSCI inserts (coded as in B) and the lacO-tagged insert tel V in progeny of hsf-1 RNAi fed adults. Scoring, asterisks, and hatching are as in Fig. 2 (B and C). Loci counted were (left to right) n = 309, 209, 367, 209, 339, 227, 95, 198, 346, 210, 329, 242, 347, 204, 91, and 209. P-values versus random = 0.02, 0.16, 0.80, 0.54, 0.73, 0.92, <0.01, <0.01, 3 × 10−5, 0.56, 1 × 10−6, 0.88, 1 × 10−6, 0.86, <0.01, and <0.01, respectively.

HSE and HSAS sequences are necessary to anchor hsp-16.2 at the nuclear rim. (A) Nascent (left) and spliced (right) RNA levels of hsp-16.2 (◇) and mCherry (▪) before and at six time points after HS for the indicated loci. Values are normalized to its-1, a transcribed rDNA spacer. (B) Scheme of the hsp-16.2/41 promoter, containing two HSF-1 binding sites, HSE1 and HSE2, on either side of the HSAS sequence, with hsp-16.2 or mCherry transcription driven toward the right. Changes in mutated versions of HSE1, HSAS, or HSE2 are shown below the sketch. (C) Quantitation of subnuclear positions of the GFP-lacI signal for the MosSCI insertion of WT or mutated hsp-16.2 promoters; color-coded as in B. Zone 1 values, bar labels, and asterisks are defined as in Fig. 2 (B and C). Cell numbers were (left to right) n = 117, 309, 367, 339, 306, 95, 346, 329, 347, and 249. P-values versus random = 0.17, 0.02, 0.80, 0.73, 0.33, 0.31, 3 × 10−5, 1 × 10−6, 1 × 10−6, and 0.59, respectively. (D) Nascent and spliced RNA levels of mCherry driven from WT or mutated hsp-16.2 promoters at MosSCI insertion, normalized to its-1. Values are shown for HSE1/2mt, HSASmt, and WT, color-coded as in B. (E) HSF-1 is essential for peripheral targeting of the hsp-16.2 tg #1 both before and after HS. Quantification of positions of the GFP-lacI signal for progeny from control RNAi or hsf-1 RNAi fed adult progeny as in Fig. 2 B. Zone 1 scoring, asterisks, and hatching are defined the same as in Fig. 2 (B and C). Numbers counted were (left to right) n = 681, 704, 281, and 244. P-values versus random = 3 × 10−4, 3 × 10−7, 0.16, and 0.07, respectively. (F) Quantitation of GFP-lacI focus position for WT or mutated hsp-16.2 promoter MosSCI inserts (coded as in B) and the lacO-tagged insert tel V in progeny of hsf-1 RNAi fed adults. Scoring, asterisks, and hatching are as in Fig. 2 (B and C). Loci counted were (left to right) n = 309, 209, 367, 209, 339, 227, 95, 198, 346, 210, 329, 242, 347, 204, 91, and 209. P-values versus random = 0.02, 0.16, 0.80, 0.54, 0.73, 0.92, <0.01, <0.01, 3 × 10−5, 0.56, 1 × 10−6, 0.88, 1 × 10−6, 0.86, <0.01, and <0.01, respectively.

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