![Figure 2. The hsp-16.2 promoter is sufficient to anchor chromatin at the nuclear periphery. (A) Sketch shows the hsp-16.2/41 locus on chr V left arm (red triangle), 1.8 Mb from the telomere, and the MosSCI-inserted lacO repeat (tel V; black triangle), at ttTi9115, 170 kb from TG repeats (Towbin et al., 2012). Below is LMN-1-Dm-ID data showing terminal 4–5 Mb enriched for lamin association (Towbin et al., 2012). An expanded view of coding sequences shows the divergent hsp-16.2 and hsp-16.41 genes. (B) Quantitation of radial positioning of endogenous loci and GFP-lacI–tagged transgenes, as described in Materials and methods. Random localization = 33% in each zone. (C) The endogenous hsp-16.2/41 locus is enriched in zone 1 in early embryos and becomes more enriched after HS. FISH signal positions were quantified for the hsp-16.2 locus as in B. Only zone 1 values are shown (n = 229, 293, 281; P < 0.01 vs. random distribution for all). Red broken lines indicate a 33% or random distribution of the foci against which all values are compared. Asterisks indicate distributions significantly different from random, or different between indicated conditions. (D) Plasmids used to create small bombarded transgenes (tg). mCherry is driven by either the hsp-16.2 or the muscle-specific myo-3 promoter, co-bombarded with an array of 256 lacO sites and the unc-119+ marker. The copy number of the hsp-16.2 promoter is 1 (tg #1) or 74 (tg #2; Fig. S2). (E) Maximal Z projection of a partial 3D reconstruction of a 200-cell-stage embryo (GW421, expressing GFP-lacI) carrying a lacO-tagged transgene gwIs28[hsp-16.2::mCherry; 256xlacO; unc-119+]. The embryo is stained for GFP (anti-GFP, green), nuclear lamina (anti–LMN-1, red), and DNA (Hoechst, blue). Bar, 1 µm. (F) Quantitation of the GFP-lacI signal position for either the myo-3 transgene, or the two hsp-16.2 promoter–containing transgenes, as in B. Zone 1 values and asterisks are defined as in C. Hatched bars, after HS. Loci scored were (left to right) n = 275, 183, 503, 188, 226, and 200; p-values versus random = 0.47, 0.6 for myo-3; and P < 10−10 for hsp-16.2–containing transgenes. A Fisher’s exact test for significance before versus after HS for hsp-16.2 transgenes yielded P = 7.2 × 10−6, 7.5 × 10−3). (G) Sketch of DNA used for MosSCI insertion at ttTi5605 in mid–chr II: lacO sites and unc-119+ were integrated with or without the hsp-16.2 promoter driving mCherry. (H) Quantitation of the GFP-lacI signal for lacO only insertion, the ectopic hsp-16.2::mCherry construct at ttTi5605, and the MosSCI lacO insertion at tel V (see A). Method, bars, and asterisks are defined as in B and C. Numbers scored were (left to right) n = 117, 95, 309, 346, 213, and 204; p-values versus random = 0.17, 0.31, 0.02, 3 × 10−5, 3 × 10−12, and 2 × 10−10, respectively.](https://cdn.rupress.org/rup/content_public/journal/jcb/200/5/10.1083_jcb.201207024/5/jcb_201207024_fig2.jpeg?Expires=1771581264&Signature=fpxQD4ywhhBxBDnCtAAmO99hQzcsDMHkGxEwYuCc1MrEVnQY-hgG14kpT3Nvgbp0sin0z75HKj1CTTZB2li4QonakdoyKLAVSXjETTa5kDzNDA7ykCGP8rnI9I0Eh-wk~TOe-md4L1vWZkpQp3Pe1tdSUJ9R2BsRGe1Laea62yfhbc3HG6Pte-BjzzWvkrGpvv0kSjsSkmBPJ3w0QLRFsahkfe19-t30O7OPHCSABQvDh8KHFEdhDswFcAsubgK-WKsjWt2Kr50-IDfTwgS9A82f9v9IKWF1Z-WZ3rNRaz6VKGejmOgqAX7wdijfHiFNqLNukLOWBo1Dg~N5bYCLow__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The hsp-16.2 promoter is sufficient to anchor chromatin at the nuclear periphery. (A) Sketch shows the hsp-16.2/41 locus on chr V left arm (red triangle), 1.8 Mb from the telomere, and the MosSCI-inserted lacO repeat (tel V; black triangle), at ttTi9115, 170 kb from TG repeats (Towbin et al., 2012). Below is LMN-1-Dm-ID data showing terminal 4–5 Mb enriched for lamin association (Towbin et al., 2012). An expanded view of coding sequences shows the divergent hsp-16.2 and hsp-16.41 genes. (B) Quantitation of radial positioning of endogenous loci and GFP-lacI–tagged transgenes, as described in Materials and methods. Random localization = 33% in each zone. (C) The endogenous hsp-16.2/41 locus is enriched in zone 1 in early embryos and becomes more enriched after HS. FISH signal positions were quantified for the hsp-16.2 locus as in B. Only zone 1 values are shown (n = 229, 293, 281; P < 0.01 vs. random distribution for all). Red broken lines indicate a 33% or random distribution of the foci against which all values are compared. Asterisks indicate distributions significantly different from random, or different between indicated conditions. (D) Plasmids used to create small bombarded transgenes (tg). mCherry is driven by either the hsp-16.2 or the muscle-specific myo-3 promoter, co-bombarded with an array of 256 lacO sites and the unc-119+ marker. The copy number of the hsp-16.2 promoter is 1 (tg #1) or 74 (tg #2; Fig. S2). (E) Maximal Z projection of a partial 3D reconstruction of a 200-cell-stage embryo (GW421, expressing GFP-lacI) carrying a lacO-tagged transgene gwIs28[hsp-16.2::mCherry; 256xlacO; unc-119+]. The embryo is stained for GFP (anti-GFP, green), nuclear lamina (anti–LMN-1, red), and DNA (Hoechst, blue). Bar, 1 µm. (F) Quantitation of the GFP-lacI signal position for either the myo-3 transgene, or the two hsp-16.2 promoter–containing transgenes, as in B. Zone 1 values and asterisks are defined as in C. Hatched bars, after HS. Loci scored were (left to right) n = 275, 183, 503, 188, 226, and 200; p-values versus random = 0.47, 0.6 for myo-3; and P < 10−10 for hsp-16.2–containing transgenes. A Fisher’s exact test for significance before versus after HS for hsp-16.2 transgenes yielded P = 7.2 × 10−6, 7.5 × 10−3). (G) Sketch of DNA used for MosSCI insertion at ttTi5605 in mid–chr II: lacO sites and unc-119+ were integrated with or without the hsp-16.2 promoter driving mCherry. (H) Quantitation of the GFP-lacI signal for lacO only insertion, the ectopic hsp-16.2::mCherry construct at ttTi5605, and the MosSCI lacO insertion at tel V (see A). Method, bars, and asterisks are defined as in B and C. Numbers scored were (left to right) n = 117, 95, 309, 346, 213, and 204; p-values versus random = 0.17, 0.31, 0.02, 3 × 10−5, 3 × 10−12, and 2 × 10−10, respectively.
