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Comparative Study
. 2015 Jun 16;112(24):7542-7.
doi: 10.1073/pnas.1505463112. Epub 2015 Jun 1.

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Carlos Gómez-Marín  1 , Juan J Tena  1 , Rafael D Acemel  1 , Macarena López-Mayorga  1 , Silvia Naranjo  1 , Elisa de la Calle-Mustienes  1 , Ignacio Maeso  1 , Leonardo Beccari  2 , Ivy Aneas  3 , Erika Vielmas  4 , Paola Bovolenta  2 , Marcelo A Nobrega  3 , Jaime Carvajal  1 , José Luis Gómez-Skarmeta  5
Affiliations
Comparative Study

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Carlos Gómez-Marín et al. Proc Natl Acad Sci U S A. .

Abstract

Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.

Keywords: CTCF; Six cluster; TAD; evolution; regulatory landscapes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The six3a/six2a cluster in divided in two developmentally stable 3D compartments. (A) The first, third, and fourth tracks show 4C-seq data on whole zebrafish embryos at 24 hpf from six3a (red), six2a (dark blue), and Enhancer III (Enh III, light blue) viewpoints, respectively. The second track shows the difference between the number of reads in the 4C-seqs from the six3a and six2a viewpoints. Black and gray signals indicate positive or negative differences, respectively. The fifth track shows accumulative reads along the region for six3a (red) and six2a (blue). Differences of these accumulative reads are shown in the track below. The asterisk marks the border region. (B) 4C-seq on whole zebrafish embryos at dome, 80% epiboly, 24 and 48 hpf from the six3a and six2a viewpoints (black triangles). Contact percentages for each gene on the two 3D compartments are indicated.
Fig. S1.
Fig. S1.
The six3a/six2a locus is partitioned in two regulatory landscapes harboring several tissue specific enhancers. (A) High-resolution circular chromosome conformation capture (4C-seq) in whole zebrafish embryos at 24 hpf from different viewpoints (black triangles) along the six3a/six2a genomic region. The genomic position region in which there is a maximum difference between the accumulative contacts from the six3a and six2a viewpoints is shown with an asterisk, and the two 3D compartments are shaded in red and blue, respectively. The bottom tracks show the distribution of the H3K27ac (pink) and H3K4me3 (green) histone marks and the genes along this genomic region. Above the H3K27ac track, the black lines show the different regions tested for enhancer activity. The Enhancer III region used as a 4C-seq viewpoint in the fourth track is highlighted with a light blue rectangle. (B) Close up of the region around the six3a and six2a genes marked by a dotted rectangle in A. The different identified enhancers are numbered from I to VI. The last blue track shows evolutionary conserved regions. (C) Transgenic embryos from anterior (Left) or lateral (Right) views showing GFP expression driven by the different H3K27ac positive regions tested. The vector used in the transgenic assays to test the activity of regions II and VI contains a midbrain enhancer used as a positive control of transgenesis. This expression domain (red arrow) is therefore independent of the region under evaluation. Regions I to IV drive the reporter in six3a-expressing territories, whereas regions V and VI activate GFP in six2a domains.
Fig. 2.
Fig. 2.
All four zebrafish six clusters are divided in two different 3D compartments. (A–D) 4C-seq on whole zebrafish embryos at 24 hpf from the different six genes at each cluster (black triangles). Border regions are indicated by an asterisk, and the two 3D compartments are shaded in red and blue. Contact percentages for each gene on the two 3D compartments are shown. Expression patterns of each gene at 24 hpf are shown for each gene below the 4C-seq tracks. (A) six3a/six2a cluster. (B) six3b/six2b cluster. (C) six6a/six1a/six4a cluster. (D) sixba/six1b/six4b cluster.
Fig. S2.
Fig. S2.
Developmental dynamics of chromatin contacts, H3K27ac and H3K4me3 histone marks, and RNA-seq at the different zebrafish six clusters. (A–C) six3b/six2b (A), six6a/six1a/six4a (B), and six6b/six1b/six4b (C) clusters. In all clusters, the frequency of contacts at each side of the boundary (dashed lines and asterisks) is indicated for each promoter viewpoint.
Fig. S3.
Fig. S3.
4C-seq replicas at the same stage are similar to 4C-seq at different developmental stages. (A and B) Comparison between 4C-seq replicas at a single stage and 4C-seq at different developmental stages in the six3a/six2a (A) and six6b/six1b/six4b (B) clusters. (Right) The correlation coefficients between 4C-seq datasets. (C) Correlation coefficient between 4C-seq dataset from replicas of the same gene at the same stage, same gene at different stages, genes within the same TAD, or genes at different TADs.
Fig. 3.
Fig. 3.
Conserved subdivision of Six clusters in two 3D compartments along deuterostome evolution. (A–C) 4C-seq in whole mouse (A and B) or sea urchin (C) embryos from the different Six genes as viewpoints. Border regions are indicated by an asterisk, and the two 3D compartments are shaded in red and blue. Contact percentages for each gene on the two 3D compartments is shown. In murine clusters, HiC data from mouse ES cells is shown below. HiC data show that clusters are divided into the two TADs also defined by 4C-seq. Border regions identified by 4C-seq coincide in both clusters with TADs borders. (Right) Expression patterns of the different genes. (D) Transient transgenic fish embryos injected with the full six2a-GFP/six3a-mCherry BAC (Left) or a TAD-deleted version (Δ18kb) of the same BAC (Right) at 72 h hpf. Arrowheads point to the brain expression domain characteristic of the six3a gene.
Fig. S4.
Fig. S4.
3D chromatin configuration of the mouse Six3/Six2 cluster at two different developmental stages. 4C-seq from Six3 and Six2 viewpoints (black triangles) in whole embryos at stages E14.5 and E9.5. The genomic region in which there is maximal difference between the accumulative contacts from the Six3 and Six2 viewpoints is shown with a dashed line, and the two 3D compartments are shaded in red and blue, respectively. The percentage of contacts for each gene on the two 3D compartments is indicated. Below are shown the two TADs detected by HiC data from mouse ES cells (red and blue triangles).
Fig. 4.
Fig. 4.
Diverging CTCF sites are signature of TAD borders. (A–C) Mouse (A), zebrafish (B), and sea urchin (C) Six6/Six1/Six6 cluster. From bottom to top, all panels show the genes at their corresponding genomic regions, the difference between Six6 and Six1 4C-seq signals, and the orientation of CTCF sites represented by arrowheads (purple and yellow correspond to sites in minus or plus strands, respectively). Note that the difference between Six6 and Six1 4C-seq signals clearly reveals the TAD border determined by HiC from mouse ES cells (upper track in A). A also shows the genomic distribution of CTCF in three different mouse cell types (second track). B shows ATAC-seq data from 24-hpf zebrafish embryos (first track). (D and E) Upper graph in each panel shows the number (y axis) and orientation of CTCF motifs along 50 kb (x axis) at each side of the human (D) and mouse (E) TAD borders. Below each graph, a boxplot show the enrichment of CTCF in diverging orientations at each side of the borders.
Fig. S5.
Fig. S5.
Diverging CTCF sites are signature of TAD borders and are not associated with promoters. (A and B) These panels show, from top to bottom, HiC data from human ES cells, the genomic distribution of CTCF in three different cell types, the orientation of CTCF sites represented by arrowheads (purple and yellow correspond to sites in minus or plus strands, respectively) at the boundary regions, and the genes at the Six2/Six3 (A) and Six6/Six1/Six4 (B) clusters. (C) Mouse Six3/Six2 cluster showing HiC data from mouse ES cells, the genomic distribution of CTCF in three different cell types, the difference between Six3 and Six2 4C-seq signals, the orientation of CTCF sites, and the genes around this genomic region. (D–F) From top to bottom, ATAC-seq peaks from 24-hpf zebrafish embryos, difference between Six genes 4C-seq signals, orientation of CTCF sites represented by arrowheads, and genes at the six6b/six1b/six4b (D), six3a/six2a (E), and six3b/six2b (F) clusters. (G and H) (Upper) Number (y axis) and orientation (purple and yellow bars correspond to CTCF sites at the plus or minus strands, respectively) of CTCF sites along 50 kb (x axis) at each side of human (D) and mouse (E) randomly selected promoters (1,000). (Lower) Boxplot shows the enrichment of CTCF in diverging orientations at each side of the boundaries. The differences observed between the mean relative position of the motifs in both strands were statistically significant in the boundary-centered windows (P = 3.27E−113 in human, P = 1.75E−118 in mouse; Fig. 4), but not in the promoter-centered windows (P = 0.107 in human, P = 0.066 in mouse).
Fig. S6.
Fig. S6.
ATAC-seq signal at CTCF sites in zebrafish. The different panels show the ATAC-seq peaks at each CTCF sites in the different zebrafish Six cluster, as indicated.
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