Fig. 7. SDC-3 localization to her-1 regulatory regions does not require DPY-27 or SDC-2, unlike its localization to X chromosomes. (A-E) False-color confocal immunofluorescence images of <30-cell wild-type embryos bearing extrachromosomal arrays carrying multiple copies of her-1 regulatory sites 2 and 3 (A,B), or dosage compensation mutant embryos carrying her-1 regulatory sites 1, 2 and 3 (C-E) plus lacO repeats and a transgene encoding LacI-GFP. Embryos were stained with DAPI (blue) and antibodies to SDC-3 (red) and GFP (green) (A,C-E). (A) In embryos with less than 30 cells, SDC-2 is not detectable (data not shown) and SDC-3 co-localizes with her-1 regulatory regions. (B) A single nucleus from an embryo stained with SDC-3 antibodies (red) and FISH probes specific to her-1 arrays (green) and X chromosomes (blue). Overlapping patterns of SDC-3 and her-1 array staining are indicated by yellow. In this single nucleus of a 10-cell embryo, SDC-3 localizes to her-1 regulatory regions at a time prior to its recruitment to X chromosomes. These results suggest that SDC-3 can localize to her-1 independently of SDC-2. (C) In xol-1 mutant embryos, her-1 transcription is repressed by the SDC proteins and SDC-3 was found localized to her-1 regulatory regions. (D) SDC-3 localizes to her-1 regulatory regions in xol-1 sdc-2 mutant embryos, indicating that SDC-3 does not require SDC-2 for its localization to her-1. By contrast, SDC-3 requires SDC-2 for its recruitment to X. The lack of SDC-2 protein was confirmed by staining with anti-SDC-2 antibodies (data not shown). (E) In dpy-27; xol-1 mutant embryos, SDC-3 is recruited to her-1 arrays, albeit in a mosaic pattern. (F) False-color confocal immunofluorescence images of an older sdc-3; xol-1 mutant embryo carrying extrachromosomal arrays of her-1 regulatory sequences and stained with SDC-2 antibodies. This image shows that SDC-2 requires SDC-3 for its localization to her-1 but its X localization is not perturbed by loss of SDC-3. Together, these results indicate that SDC-3 can bind to her-1 regulatory regions independently of DPY-27 and SDC-2. Moreover, the SDC protein required for recognition of her-1 regulatory sequences differs from that required for X-chromosome recognition. (G) Schematic of the her-1 gene and the binding sites for the dosage compensation complex. Transcription from the P1 promoter produces the functional male-specific her-1 transcript (1.2 kb) that includes four exons (blue). A second promoter resides within the second intron of her-1. This second promoter is co-regulated with the first promoter and makes a 0.8 kb transcript of unknown function that includes the last two exons. Insets show the enlargement of a single nucleus indicated by the arrow. Scale bars: 1 µm for B; 5 µm for A,C-F.