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First published online December 1, 2003
doi: 10.1242/10.1242/dev.00886

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Recruitment of C. elegans dosage compensation proteins for gene-specific versus chromosome-wide repression

Howard Hughes Medical Institute, Department of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, CA 94720-3204, USA

View larger version (44K): [in a new window]   Fig. 1. Map of dpy-21 molecular lesions and levels of dpy-21 transcripts throughout development. (A) The intron-exon gene structure of dpy-21. The molecular changes for seven dpy-21 mutant alleles are indicated. Genetically, dpy-21(e428) is the most severe mutation, causing 17% larval lethality and dumpy, egg-laying defective adult survivors, some with a protruding vulva. (B) Northern blot of mRNA isolated from wild-type embryos, L1, L2, L3 and L4 larvae, and young adults without embryos. The blot was hybridized with a probe to the first 3347 nucleotides of the dpy-21 transcript and a myo-1 probe to measure pharyngeal myosin transcript levels as a loading control. The dpy-21 transcript is expressed throughout development, with the highest transcript levels occurring during embryogenesis.

View larger version (92K): [in a new window]   Fig. 2. The C-terminal domain of DPY-21 is conserved between species. In the alignment of the C terminus (amino acids 1224-1641), black indicates sequence identity, and gray represents sequence similarity. DPY-21 contains no identifiable motifs. However, the C-terminal region of DPY-21 appears to be conserved throughout evolution. No other significant similarity was found between DPY-21 and the putative homologs, as they are rendered in current data bases. The function of these DPY-21 homologs has not been determined. Locations of dpy-21 mutations are indicated by the + symbol.

View larger version (30K): [in a new window]   Fig. 3. DPY-21 associates physically with components of the dosage compensation complex. (A) Western blot of extracts from wild-type or dpy-21(e428) mutant gravid hermaphrodites serially diluted by 1.3-fold and probed with N-terminal antibodies to DPY-21 and antibodies to the loading control SMC-1, a protein involved in chromosome cohesion. Full-length DPY-21 (210 kDa) was present in extracts from wild-type but not dpy-21 mutant animals. A 60 kDa protein was variably detected in the dpy-21 mutant extract (asterisk). The apparent molecular weight of this protein is slightly larger than the 43 kDa protein predicted from the location of the e428 nonsense mutation at codon 394. The blot was also probed with antibodies to MIX-1, a protein involved in dosage compensation and chromosome condensation. Equivalent levels of MIX-1 in both mutant and wild-type extracts provide one example that dpy-21 mutations do not alter the stability of other dosage compensation proteins. (B) DPY-21 antibodies specifically precipitated SDC-3 from wild-type embryonic extracts (lane 3). SDC-3 was not immunoprecipitated by the preimmune sera (lane 1) or in a mock immunoprecipitation reaction that lacked antibodies (lane 2), showing specificity of the IP reactions. (C) DPY-27 antibodies strongly precipitated DPY-21 (lane 4). The precipitation of DPY-21 was specific, given that DPY-21 was not precipitated by the preimmune sera (lane 1) or antibodies to CBP-1, a DNA-associated CREB-binding protein (lane 2). DPY-21 antibodies failed to precipitate DPY-27 (lane 5), and DPY-26 antibodies only weakly precipitated DPY-21 (lane 3). These immunoprecipitation experiments indicate that DPY-21 interacts biochemically with other dosage compensation components but its association with the complex is probably not as robust as that of other members.

View larger version (70K): [in a new window]   Fig. 4. DPY-21 localizes to the X chromosomes of XX but not XO embryos, as expected for a component of the dosage compensation complex. (A-F) False-color confocal images of wild-type XX embryos (A,B), dpy-21(e428) mutant XX embryos (C), wild-type hermaphrodite gut nuclei (D), him-8(e1489) XO embryos (E) and him-8(e1489); xol-1(y9) mutant XO embryos (F) stained with DPY-21 antibodies (green), the DNA-intercalating dye 4',6 diamidino-2-phenylindole (DAPI) (blue) and an X-chromosome-specific marker (red) (either an X-chromosome-specific FISH probe or antibodies to SDC-3 or GFP, which identifies the DPY-27::GFP fusion protein used in D). (A-C,E,F) DPY-21 antibodies to amino acids 467-1102; (D) antibodies to the DPY-21 N terminus. The third column shows a merged image of the first two columns, and yellow indicates overlap in staining of DPY-21 and the X-chromosome marker. The fourth column shows the superimposition of DAPI images with images from the first two columns. Insets show the enlargement of a single nucleus indicated by the arrow. (A) In young embryos (<40 cells) that have not yet recruited the dosage compensation complex to X, DPY-21 is distributed throughout the nuclei. (B) In 40-cell stage embryos, DPY-21 exhibits a punctate pattern that is coincident with the X-localized SDC-3 protein. (C) Specificity of the DPY-21 antibody was shown in part by the absence of DPY-21 staining in dpy-21(e428) and dpy-21(y59) (data not shown), both of which contain an early amber stop mutation. SDC-3 localized to the X chromosomes of a dpy-21 mutant, indicating that DPY-21 is not essential for the recruitment of the dosage compensation complex to X. (D) The X-chromosome localization of DPY-21 is maintained throughout hermaphrodite development, as shown by the X-chromosome localization of DPY-21 in adult gut nuclei, which carry a DPY-27::GFP fusion protein. (E) DPY-21 is expressed, but fails to localize to the X chromosome of XO animals. (F) In xol-1(y9) mutant XO embryos, which have inappropriately activated dosage compensation, both DPY-21 and SDC-3 co-localize with the single X chromosome, indicating that the X-chromosome localization of DPY-21 is under the same sex-specific control as SDC-3. Scale bars: 5 µm.

View larger version (80K): [in a new window]   Fig. 5. DPY-21 is recruited to X chromosomes by components of the dosage compensation complex. (A-G) Confocal images of wild-type (A), sdc-2(y74) (B), sdc-3(y126) (C), dpy-26(y199) (D), dpy-27(y167) (E), dpy-28(s939) (F) and sdc-1(n485) (G) mutant embryos co-stained with DPY-21 antibodies to amino acids 467-1102 (green), DAPI (blue) and an X-chromosome-specific FISH probe (red) or SDC-3 antibodies (red). (A) In wild-type embryos, foci of DPY-21 staining co-localize with X chromosomes identified by FISH. (B-F) DPY-21 accumulates in dosage compensation mutant embryos, but foci of DPY-21 staining in sdc-2(y74) (B), sdc-3(y126) (C), dpy-26(y199) (D), dpy-27(y167) (E) and dpy-28(s939) (F) mutants are not coincident with the X chromosome. Thus DPY-21 requires sdc-2, sdc-3, dpy-26, dpy-27 and dpy-28 for its localization to X but not for its stability. (G) By contrast, neither DPY-21 nor SDC-3 requires sdc-1 for its localization to X. Insets show the enlargement of a single nucleus indicated by the arrow. Scale bars: 5 µm.

View larger version (25K): [in a new window]   Fig. 6. DPY-21 is not recruited to her-1 regulatory regions in XX animals, unlike other components of the dosage compensation complex. (A,B) False-color confocal immunofluorescence images of wild-type embryos (A) or gut cell nuclei (B) carrying her-1 extrachromosomal arrays that contain multiple copies of her-1 regulatory regions, lac operator repeats (lacO) and a transgene encoding a LacI-GFP fusion protein. LacI-GFP repressor binding to lacO permits array detection by GFP antibodies. For each embryo or gut cell nucleus, a single z-section is shown. Embryos and gut cell nuclei were stained with DAPI (gray) and antibodies to DPY-21 (green), SDC-3 (red) and GFP (blue). The fourth column shows the superimposition of the DPY-21 and SDC-3 images. (A) In embryos that have activated dosage compensation, both DPY-21 and SDC-3 co-localize with the X chromosome, which is denoted by an asterisk in the inset. By contrast, SDC-3, but not DPY-21, localizes to her-1 regulatory regions on the arrays. (B) In adult gut cell nuclei, DPY-21 co-localizes with SDC-3 on X chromosomes, but does not co-localize with SDC-3 on her-1 regulatory regions. Thus, DPY-21 participates directly in the chromosome-wide repression of X but not in the gene-specific repression of her-1. Scale bars: 5 µm.

View larger version (61K): [in a new window]   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.

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