X-chromosome target specificity diverged between dosage compensation mechanisms of two closely related Caenorhabditis species
- Howard Hughes Medical Institute, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
Figures
Figure 1 with 4 supplements
Conservation of X-chromosome dosage compensation machinery between C. briggsae and C. elegans.
(A) sdc-2 mutations cause XX-specific lethality in C. briggsae. Graph shows percent viability of wild-type and Cbr sdc-2 mutant XX and XO adults. Viability of homozygous XX and hemizygous XO Cbr …
Figure 1—figure supplement 1
Protein sequence alignment comparing SDC-2 proteins in C. elegans and C. briggsae.
In the sequence alignment, the red background indicates amino acid identity, and the red characters demark similarity. The predicted coiled-coil regions are delineated by blue brackets. The …
Figure 1—figure supplement 2
Conservation of SDC-2 and DPY-27 proteins in the Caenorhabditis genus.
Pairwise sequence comparisons between Caenorhabditis species show the percent of amino acid identity and similarity (in parenthesis) for (A) SDC-2 full-length protein, SDC-2 N-terminal domain, and …
Figure 1—figure supplement 3
DNA sequence changes mediated by genome editing.
(A) DNA sequences of mutant Cbr sdc-2 alleles that were created by genome editing using zinc-finger nucleases, as described in Wood et al., 2011. Mutations include short insertions (green) and …
Figure 1—figure supplement 4
Specificity of Cbr DPY-27 and MIX-1 antibodies.
(A) Western blot analysis comparing DPY-27 proteins in extracts made from twenty wild-type adult C. briggsae XX hermaphrodites (lane 1), adult XX hermaphrodites encoding a 3xFLAG-tagged version of …
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Figure 1—figure supplement 4—source data 1
Source data for DPY-27 and MIX-1 antibody specificity.
- https://cdn.elifesciences.org/articles/85413/elife-85413-fig1-figsupp4-data1-v2.zip
Figure 2
Conserved genetic hierarchy targets the C. briggsae dosage compensation complex (DCC) to the X chromosomes of hermaphrodites.
(A–E) Schematic depiction of the genetic hierarchy controlling sex-specific DCC recruitment to C. briggsae X chromosomes (left) paired with representative immunofluorescence experiments exemplifying …
Figure 3
sdc-2 controls dosage compensation and sex determination in C. briggsae.
(A) Diagram of the screening strategy to recover Cbr sdc-2 mutations as suppressors of the XO-specific lethality caused by a xol-1 mutation. Cbr xol-1 XX hermaphrodites were mated with males …
Figure 4 with 1 supplement
Identification of C. briggsae dosage compensation complex (DCC) recruitment elements on X.
(A) ChIP-seq profiles of Cbr SDC-2 and Cbr DPY-27 binding to X chromosomes. ChIP-seq experiments were performed using an anti-FLAG antibody to immunoprecipitate SDC-2 from a strain encoding …
Figure 4—figure supplement 1
ChIP-seq profiles of Cbr SDC-2 and Cbr DPY-27 binding to chromosomes X and V.
For ChIP-seq profiles of Cbr SDC-2 and Cbr DPY-27 binding to chromosomes X (A) and V (B), experiments were performed using an anti-FLAG antibody to immunoprecipitate SDC-2 from a strain encoding …
Figure 5
C. briggsae rex sites integrated into the C. elegans X chromosome by genome editing failed to recruit the C. elegans dosage compensation complex (DCC).
Binding of C. elegans DCC protein Cel SDC-3 and an IgG control were examined by ChIP-qPCR for Cel rex-32 at its endogenous location on X, and for six C. briggsae rex sites (Cbr rex-1, Cbr rex-2, Cbr …
Figure 6 with 1 supplement
Comparison of C. briggsae and C. elegans DNA motifs on X that occur within respective rex sites and recruit respective dosage compensation complex (DCC) complexes.
(A) Shown are the C. briggsae consensus motifs for the 13 bp MEX and 30 bp MEX II variants that recruit the DCC. Also shown are the C. elegans consensus motifs for the 12 bp MEX, 26 bp MEX II, and 9 …
Figure 6—figure supplement 1
C. briggsae SDC-2 ChIP-seq peak profiles for rex sites and non-rex sites on X.
(A, B) Each profile represents 2,000 bp centered on summit locations. (A) SDC-2 ChIP-seq profiles for all twelve Cbr rex sites. X coordinates for the peak summit locations are shown on the right, …
Figure 7 with 1 supplement
Enrichment of Cbr MEX and Cbr MEX II motifs on X chromosomes between C. briggsae and C. elegans.
(A, B) Graphs show the enrichment (y-axis) of Cbr MEX (A) or Cbr MEX II (B) variants (x-axis) on X chromosomes compared to autosomes in the C. briggsae (green circles) and C. elegans (orange …
Figure 7—figure supplement 1
The C. briggsae X chromosome is not enriched for the C. elegans MEX (A) or MEX II (B) motifs that are highly enriched on Cel X chromosomes and pivotal for DCC binding to Cel X chromosomes in vivo.
The descriptions of these graphs are the same as those presented in the legend to Figure 7. (C) Graph shows the Cbr SDC-2 RPKM signal from ChIP-seq experiments as a function of the distance from Cel …
Figure 8 with 1 supplement
Combinatorial clustering of MEX and MEX II motifs in Cbr rex-1 facilitates dosage compensation complex (DCC) binding to the endogenous rex-1 site on X.
(A) Shown is an enlargement of the SDC-2 ChIP-seq peak profile for Cbr rex-1 with its associated MEX (purple) and MEX II (green) motifs and their ln(P) scores. (B) DPY-27 ChIP-seq analysis was …
Figure 8—figure supplement 1
Clustering of MEX and MEX II motifs in Cbr rex-1 confers dosage compensation complex (DCC) binding in vivo.
(A) Shown is an enlargement of the SDC-2 ChIP-seq peak profile for Cbr rex-1 with its associated MEX and MEX II motifs and their ln(P) scores. Numbers between motifs indicate the base pairs …
Figure 9 with 1 supplement
MEX and MEX II motifs are critical for dosage compensation complex (DCC) binding to Cbr rex-4 in vivo.
(A) Shown is an enlargement of the SDC-2 ChIP-seq profile for rex-4, a schematic of the MEX (purple) and MEX II (green) motifs in rex-4, and the location of primers (E and F, dashed lines) to …
Figure 9—figure supplement 1
MEX and MEX II motifs are critical for SDC-2 binding to Cbr rex-4 in vivo.
This figure extends the analysis of SDC-2 binding at rex-4 in wild-type and rex-4 mutant strains presented in Figure 9 by including SDC-2 ChIP-qPCR analysis at intervals extending all along the …
Figure 10 with 1 supplement
Both MEX II motifs are critical for dosage compensation complex (DCC) binding to Cbr rex-3 in vivo.
(A) Shown is an enlargement of SDC-2 ChIP-seq profile for Cbr rex-3 with its associated MEX II motifs (green) and their ln(P) scores. Motifs are separated by 178 bp. Locations of primers (F and G, …
Figure 10—figure supplement 1
Both MEX II motifs are critical for dosage compensation complex (DCC) binding to Cbr rex-3 in vivo.
This figure extends the analysis of SDC-2 binding at rex-3 in wild-type and rex-3 mutant strains in Figure 10 by including SDC-2 ChIP-qPCR analysis at intervals extending all along the SDC-2 entire …
Figure 11 with 1 supplement
Multiple MEX motifs in Cbr rex-7 contribute to dosage compensation complex (DCC) binding in vivo.
(A) Shown is an enlargement of SDC-2 ChIP-seq profile for Cbr rex-7 with its associated MEX motifs (purple) and their ln(P) scores. Motifs are separated by 85 bp and 22 bp. Locations of primers (D …
Figure 11—figure supplement 1
Multiple MEX motifs in Cbr rex-7 contribute to dosage compensation complex (DCC) binding in vivo.
This figure extends the analysis of SDC-2 binding at rex-7 in wild-type and rex-7 mutant strains in Figure 11 by including SDC-2 ChIP-qPCR analysis at intervals extending all along the SDC-2 entire …
Figure 12
Functional divergence of X motifs demonstrated by C. elegans dosage compensation complex (DCC) binding studies in vivo and in vitro to Cel rex sites engineered to replace Cel motifs with Cbr MEX and MEX II motifs.
(A) Comparison of DNA sequences for the two MEX II motifs in wild-type Cel rex-39 (Cel ln[P] of –21.23 and –20.74) with the Cbr MEX II motifs (Cbr ln[P] of –20.04 and Cel ln[P] > –9 for both) that …
Figure 13
A nucleotide position in the consensus Cbr MEX motif can act as a critical determinant for whether Cel dosage compensation complex (DCC) binds in vivo and in vitro.
(A) Shown are DNA sequences of three wild-type or mutant Cel or Cbr MEX motifs within Cel rex-33 assayed for Cel SDC-3 binding in vivo (B) and Cel SDC-2 binding in vitro (C). The ln(P) scores for …
Tables
Table 1
MALDI-TOF identification of Cbr MIX-1 peptides.
| m/z Submitted | MH+ Matched | Delta ppm | Peptide | MissedCleavage | Database Sequence |
|---|---|---|---|---|---|
| 916.47 | 916.46 | 9.5 | 674–680 | 0 | (K)YHENVVR(L) |
| 1163.59 | 1163.58 | 3.3 | 375–384 | 1 | (K)LRGELEGMSR(G) |
| 1214.65 | 1214.66 | –3.6 | 631–641 | 0 | (R)VLIESQCLPGR(R) |
| 1224.63 | 1224.62 | 8.8 | 713–723 | 1 | (R)EVAYTDGVKSR(T) |
| 1263.74 | 1263.74 | –0.87 | 524–534 | 0 | (R)DVEGLVLHLIR(L) |
| 1285.69 | 1285.69 | –2.8 | 631–641 | 0 | (R)VLIESQCLPGR(R) |
| 1350.69 | 1350.70 | –8.9 | 656–666 | 0 | (R)YTIINDQSLQR(A) |
| 1881.97 | 1881.98 | –2.3 | 134–150 | 0 | (R)GVGLNVNNPHFLIMQGR(I) |
| 1886.89 | 1886.91 | –6.8 | 86–101 | 0 | (K)QSPFGMDHLDELVVQR(H) |
| 2064.01 | 2064.00 | 3.4 | 460–477 | 0 | (K)ITQQVQSLGYNADEDVQR(R) |
| 2377.18 | 2377.16 | 5.6 | 385–415 | 1 | (R)GTVTNDKGEHVSLETYIQETR(A) |
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This table lists the mass-to-charge ratio (m/z) of measured peptides, the predicted masses (MH+ Matched), and the deviation from predicted masses (Delta ppm). The ID of each measured peptide is described by the residue range within full-length MIX-1 (Peptide) and its corresponding amino acid sequence (Database Sequence). The number of uncut tryptic peptide bonds is listed for each peptide (Missed Cleavage).
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In addition to MIX-1, MALDI-TOF analysis of excised protein bands in the molecular weight range of condensin subunits excised from an SDS-PAGE gel revealed peptides corresponding to four common high-molecular weight contaminants: the three vitellogenin yolk proteins VIT-2, VIT-4, VIT-5, and CBG14234, an ortholog of VIT-4. No protein bands corresponding to the molecular weights of SDC-2 or SDC-3 were visible on the SDS-PAGE gel.
Table 2
Results of DCC recruitment assays in vivo.
(A) Cbr rex DNA fragments assayed in C. briggsae and (B) Identical Cel rex DNA fragments assayed in C. elegans and in C. briggsae.
| (A) C. briggsae DCC binds C. briggsae DCC recruitment sites. | |||||
|---|---|---|---|---|---|
| Cbr rex Site | Cbr Chr X Peak Position | Cbr SDC-2 RPKM | Cbr Array Assay in vivo % Recruitment (No. of Nuclei) | ||
| rex-1 | 10,780,533 | 2890 | 92% | (59) | |
| rex-2 | 12,642,866 | 999 | 90% | (101) | |
| rex-3 | 19,468,721 | 3219 | 88% | (74) | |
| rex-4 | 6,358,591 | 3915 | 85% | (68) | |
| rex-5 | 3,153,011 | 3562 | 98% | (45) | |
| rex-6 | 18,811,390 | 2203 | 74% | (68) | |
| rex-7 | 8,026,460 | 2964 | 97% | (65) | |
| rex-8 | 16,578,214 | 3217 | 37% | (52) | |
| rex-9 | 3,135,562 | 1029 | 85% | (62) | |
| rex-10 | 895,450 | 3605 | 80% | (55) | |
| rex-11 | 4,563,250 | 830 | 89% | (54) | |
| rex-12 | 19,564,937 | 1786 | 79% | (77) | |
| flat 2 | 11,762,995 | 2890 | 6% | (48) | |
| flat 3 | 20,918,257 | 999 | 0% | (144) | |
| (B) C. briggsae DCC does not bind C. elegans DCC recruitment sites. | |||||
| Cel rex Site | Cel Chr X Peak Position | Cel Array Assay in vivo % Recruitment (No. of Nuclei) | Cbr Array Assay in vivo % Recruitment (No. of Nuclei) | ||
| rex-4 | 11,522,205 | 100% | (16) | 1% | (116) |
| rex-33 | 6,296,501 | 100% | (63) | 0% | (53) |
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(A) Extrachromosomal arrays composed of DNA fragments (2 kb) that were PCR-amplified from C. briggsae X chromosome regions corresponding to Cbr SDC-2 ChIP-seq peaks were tested for their ability to recruit the Cbr DCC. Gut nuclei from C. briggsae transgenic lines were scored for the presence of the array using a FISH probe against the myo-2::gfp vector and the presence or absence of DCC binding to the array by immunofluorescence signal using Cbr DPY-27 antibodies. The % recruitment is the percentage of total scored array-bearing nuclei that showed DPY-27 bound to the array.
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(B) Identical DNA fragments encoding individual C. elegans DCC recruitment sites (rex) were injected into C. elegans and C. briggsae to create extrachromosomal arrays containing multiple copies of the rex site. Gut nuclei from C. elegans or C. briggsae transgenic lines were scored for the presence of the array using a FISH probe against the myo-2::gfp vector and for the presence or absence of DCC binding to the array by immunofluorescence signal from the species-matched DPY-27 antibody. The % recruitment is the percentage of total scored array-bearing nuclei that showed DCC binding to the array.
Table 3
Motifs within rex sites.
The ln(P) values for MEX II motifs are underlined, and the values for MEX motifs are not underlined.
| Cbr rex Site | Chr X Peak Position | SDC-2 RPKM | Cbr MEX motif ln(P) < –12 Cbr MEX II ln(P) < –12 |
|---|---|---|---|
| rex-1 | 10,780,533 | 2890 | –15.57 (13 bp) –15.57 (106 bp) –14.63 (14 bp) –14.47 (93 bp) –27.58 |
| rex-2 | 12,642,866 | 999 | –14.25 (73 bp) –22.69 |
| rex-3 | 19,468,721 | 3219 | –12.36 (178 bp) –20.04 |
| rex-4 | 6,358,591 | 3915 | –19.09 (33 bp) –13.80 |
| rex-5 | 3,153,011 | 3562 | –18.98 |
| rex-6 | 18,811,390 | 2203 | –15.43 (289 bp) –13.35 |
| rex-7 | 8,026,460 | 2964 | –18.72 (85 bp) –12.26 (22 bp) –12.58 |
| rex-8 | 16,578,214 | 3217 | –13.00 (60 bp) –14.31 (69 bp) –13.22 (23 bp) –13.52 |
| rex-9 | 3,135,562 | 1029 | –12.8 |
| rex-10 | 895,450 | 3605 | –12.60 (63 bp) –14.68 |
| rex-11 | 4,563,250 | 830 | |
| rex-12 | 19,564,937 | 1786 |
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Listed are the rex sites analyzed in this study and their motifs. Motif cutoffs used include MEX with ln(P) < –12 and MEX II with ln(P) < –12. The distances between adjacent motifs (in bp) is listed in parenthesis between motifs. Also listed are the coordinates (in bp) with the maximum SDC-2 ChIP-seq signal in each rex site and the maximum SDC-2 ChIP signal in reads per kilobase per million reads mapped (RPKM) within a 50 bp window. MEX and MEX II are not likely to be the only DNA sequence features within rex sites that contribute to DCC binding, since rex-11 and rex-12 lack these motifs with ln(P) values < –12.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/85413/elife-85413-mdarchecklist1-v2.pdf
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Supplementary file 1
List of alleles and strains used in this study.
- https://cdn.elifesciences.org/articles/85413/elife-85413-supp1-v2.docx
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Supplementary file 2
List of primers.
- https://cdn.elifesciences.org/articles/85413/elife-85413-supp2-v2.docx
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Supplementary file 3
Chromosome-specific BACs used to generate FISH probes.
- https://cdn.elifesciences.org/articles/85413/elife-85413-supp3-v2.docx
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Supplementary file 4
List of target-specific sequences for guide RNAs used in CRISPR / Cas9 genome editing experiments.
- https://cdn.elifesciences.org/articles/85413/elife-85413-supp4-v2.docx
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Supplementary file 5
DNA sequences of repair templates used in CRISPR / Cas9 genome editing experiments.
- https://cdn.elifesciences.org/articles/85413/elife-85413-supp5-v2.docx
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Supplementary file 6
DNA templates used for in vitro DCC binding assays.
- https://cdn.elifesciences.org/articles/85413/elife-85413-supp6-v2.docx
