A complex regulatory landscape involved in the development of mammalian external genitals
- School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland
- Department of Genetics and Evolution, University of Geneva, Switzerland
- Collège de France, France
Figures
Figure 1 with 1 supplement
Transcription of Hoxd genes in developing GT.
(A) Quantification of Hoxd genes transcript levels by RNA-seq (FPKM values) in GT at E12.5 (Amândio et al., 2016), E16.5 and E18.5. (B) RT-qPCR of Hoxd13 mRNAs in different stages of GT development. …
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Figure 1—source data 1
Hoxgenes expression values (FPKM and RT-qPCR) during GT development.
- https://cdn.elifesciences.org/articles/52962/elife-52962-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Hox genes expression profile during GT development.
Bar plots show the quantification of Hoxa, Hoxb and Hoxc genes transcripts by RNA-seq (FPKM values) in GT cells at E12.5, E16.5 and E18.5. The gene cluster is indicated on top of each bar plot.
Figure 2 with 1 supplement
Chromatin topology of C-DOM during GT development.
(A) Schematic representation of the two regulatory landscapes, with the centromeric (C-DOM) and telomeric (T-DOM) TADs flanking the HoxD cluster (black box), which acts as a TAD boundary. Gray boxes …
Figure 2—figure supplement 1
Chromatin interactions in C-DOM during GT development.
4C-seq profiles at the HoxD cluster and C-DOM, using GT cells at E12.5, E13.5, E15.5, E17.5 and forebrain cells. Coordinates (mm10): chr2:73815520–74792376. GT2 (upper panel, blue line) and island V …
Figure 3
Various segments of C-DOM contribute to Hoxd13 transcription in the GT.
(A) The gray tracks show ATAC-seq profiles of E10.5 CR (average of two biological replicates, track 1), E13.5 GT (average of three biological replicates, track 2) and E17.5 GT (average of two …
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Figure 3—source data 1
RT-qPCR values ofhoxdgenes in wildtype and deletion alleles.
- https://cdn.elifesciences.org/articles/52962/elife-52962-fig3-data1-v2.xlsx
Figure 4
Deletion of the Prox enhancer.
(A) Schematic representation of the HoxD cluster and the C-DOM with the deletion of the Prox sequence (Del(Prox)). (B) X-gal staining showing the activity of the Prox enhancer at E13.5. (C) Hoxd13 …
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Figure 4—source data 1
RT-qPCR values ofHoxd13in wildtype andDel(Prox)mutant GTs.
- https://cdn.elifesciences.org/articles/52962/elife-52962-fig4-data1-v2.xlsx
Figure 5
Activity of C-DOM regulatory elements in vivo.
(A) Schematic representation of C-DOM and the HoxD cluster. Previously characterized enhancers are shown as black boxes and red arrowheads point to the SB and rel5 breakpoints. (B) ATAC-seq profile …
Figure 6 with 2 supplements
Serial deletions of single cis-regulatory elements.
(A) Schematic representation of the alleles generated by CRISPR-Cas9 editing in vivo. The extents of sequences to be deleted were selected based on ATAC-seq, H3K27ac ChIP-seq, 4C-seq datasets and …
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Figure 6—source data 1
RT-qPCR values ofHoxd13in wildtype and deletion alleles.
- https://cdn.elifesciences.org/articles/52962/elife-52962-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Deletion of the GT2 enhancer.
(A) ATAC-seq profiles of HoxD and C-DOM in wildtype and mutant Del(GT2) E13.5 GTs. Coordinates (mm10): chr2:73815520–74792376. The wildtype track is the average of three biological replicates and …
Figure 6—figure supplement 2
Alleles generated by CRISPR-Cas9.
Sanger sequencing results of F0 animals for all alleles generated. Scissors indicate CRISPR-Cas9 mediated breakpoints flanking each regulatory region. SgRNA sequences are marked in red or in green. …
Figure 7 with 1 supplement
Deletion and inversion of the island V CTCF site in vivo.
(A) CTCF ChIP-seq profiles of wildtype and Del(V) mutant E13.5 GTs. CUT and RUN of mutant Inv(V) E13.5 GT. The upper track shows the orientations of the CTCF motives (red and blue arrowheads). The …
Figure 7—figure supplement 1
Chromatin accessibility in the deletion and inversion of the island V.
(A) ATAC-seq profiles of wildtype and mutant Del(V) and Inv(V) E13.5 GTs. Coordinates (mm10): chr2:73815520–74792376. The wildtype track is the average of three biological replicates and the Del(V) …
Figure 8
HOX13 protein binding in C-DOM.
(A) HOX13 CUT and RUN profiles using E10.5 CR cells (red) and GT cells at E13.5 (Blue). The blue box represents the HoxD cluster and gray boxes are non-Hox genes. The profile encompasses 4 Mb and …
Additional files
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Supplementary file 1
List of genotyping primers.
- https://cdn.elifesciences.org/articles/52962/elife-52962-supp1-v2.xlsx
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Supplementary file 2
List of sgRNAs.
- https://cdn.elifesciences.org/articles/52962/elife-52962-supp2-v2.xlsx
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Supplementary file 3
List of fosmids.
- https://cdn.elifesciences.org/articles/52962/elife-52962-supp3-v2.xlsx
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Supplementary file 4
List of primers used for recombineering.
- https://cdn.elifesciences.org/articles/52962/elife-52962-supp4-v2.xlsx
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Supplementary file 5
List of 4C-seq primers.
- https://cdn.elifesciences.org/articles/52962/elife-52962-supp5-v2.xlsx
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Supplementary file 6
List of datasets and replicate status.
- https://cdn.elifesciences.org/articles/52962/elife-52962-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52962/elife-52962-transrepform-v2.docx
