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. 2017 Sep 1;13(9):e1006966.
doi: 10.1371/journal.pgen.1006966. eCollection 2017 Sep.

5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells

Krzysztof Poterlowicz  1 , Joanne L Yarker  1 , Igor Malashchuk  1 , Brian R Lajoie  2 , Andrei N Mardaryev  1 , Michal R Gdula  3 , Andrey A Sharov  4 , Terumi Kohwi-Shigematsu  5 , Vladimir A Botchkarev  1   4 , Michael Y Fessing  1
Affiliations

5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells

Krzysztof Poterlowicz et al. PLoS Genet. .

Abstract

Mammalian genomes contain several dozens of large (>0.5 Mbp) lineage-specific gene loci harbouring functionally related genes. However, spatial chromatin folding, organization of the enhancer-promoter networks and their relevance to Topologically Associating Domains (TADs) in these loci remain poorly understood. TADs are principle units of the genome folding and represents the DNA regions within which DNA interacts more frequently and less frequently across the TAD boundary. Here, we used Chromatin Conformation Capture Carbon Copy (5C) technology to characterize spatial chromatin interaction network in the 3.1 Mb Epidermal Differentiation Complex (EDC) locus harbouring 61 functionally related genes that show lineage-specific activation during terminal keratinocyte differentiation in the epidermis. 5C data validated by 3D-FISH demonstrate that the EDC locus is organized into several TADs showing distinct lineage-specific chromatin interaction networks based on their transcription activity and the gene-rich or gene-poor status. Correlation of the 5C results with genome-wide studies for enhancer-specific histone modifications (H3K4me1 and H3K27ac) revealed that the majority of spatial chromatin interactions that involves the gene-rich TADs at the EDC locus in keratinocytes include both intra- and inter-TAD interaction networks, connecting gene promoters and enhancers. Compared to thymocytes in which the EDC locus is mostly transcriptionally inactive, these interactions were found to be keratinocyte-specific. In keratinocytes, the promoter-enhancer anchoring regions in the gene-rich transcriptionally active TADs are enriched for the binding of chromatin architectural proteins CTCF, Rad21 and chromatin remodeler Brg1. In contrast to gene-rich TADs, gene-poor TADs show preferential spatial contacts with each other, do not contain active enhancers and show decreased binding of CTCF, Rad21 and Brg1 in keratinocytes. Thus, spatial interactions between gene promoters and enhancers at the multi-TAD EDC locus in skin epithelial cells are cell type-specific and involve extensive contacts within TADs as well as between different gene-rich TADs, forming the framework for lineage-specific transcription.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. TAD organization of the large (>0.5 Mb) lineage-specific co-regulated gene loci in the mouse genome.
(a) Number of TADs occupied by the lineage-specific co-regulated gene loci in the mouse genome in the Embryonic Stem Cells (ESCs) depending of the locus size (based on the Hi-C data published in [9]). (b) Genome positions of the lineage-specific co-regulated gene loci in mouse genome and the position of the TAD borders occupied by these loci in the ESCs (mouse genome assembly mmu9), based on the Hi-C data published in [9]).
Fig 2
Fig 2. Gene-rich and gene-poor regions are organized into distinct TADs at the EDC locus in keratinocytes.
(a) Schematic structure of the 5.3 Mb genomic region containing the EDC locus on mouse chromosome 3 analysed using 5C technology in this manuscript (mm9/chr.3:89,900,000–95,200,000). (b) Alternating 5C probe design for the unique HindIII sites in the interrogated genomic regions. The position of the restriction sites interrogated by the forward primers are shown in blue, interrogated by the reverse primers are shown in red and the site for which the primers could not be designed are shown in green. (c) Heatmaps representing raw 5C data for both KC replicates. Reverse probes are plotted as columns and the forward probes as rows. Pearson’s correlation coefficient is also shown. (d) Heatmap representing the 5C data after the normalization and binning (bin size 150 kb, step size 15kb) in KCs. The position of TAD border midpoints (average for the midpoints calculated based on the insulation index analysis in two replicates independently) are identified by green lines under the heatmaps. Note the high frequency of the spatial contacts between the gene-poor TADs 2 and 5 (indicated by dashed rectangle on the heat map). The position of the regions covered by the BAC fish probes used in these studies, schematic map of the studied locus and insulation indexes profiles for two 5C library replicates are also shown. (e) Multi-colour 3D FISH analysis with BAC probes A (located at the 5’ border of TAD3, B (located at the 3’ border of TAD4) and C (located within TAD4) (left), or with BAC probe D (located within gene-poor TAD2) and E (located within gene-poor TAD5) (right) in basal epidermal keratinocytes. Representative single optical sections are shown. Scale bars are 2μm. (f) Box plots showing median, 25% quartile, 75% quartile with whiskers indicating maximum and minimum for spatial distances between the centres of the regions covered by probes A and B, probes B and C, as well as probes D and E before (in μm) and after normalization to the average nuclear radius (in % of average nuclear radius) in basal epidermal keratinocytes in situ. The distances between the centres of the regions covered by the probes A and B (located within TAD3) are significantly shorter than the distances between loci covered by the probes B and C (located within TAD4). The indicated p-values for pair-wise comparison are calculated using Mann-Whitney U-test, n = 60 alleles for each interrogated locus. The distances between the centres of the regions covered by the probes D and E (located in the gene poor TADs 2 and 5 respectively) are similar to the much closer regions covered by the probes B and C (located in the adjacent gene-rich TAD3 and TAD4).
Fig 3
Fig 3. 5C looping interactions at the EDC locus involve gene promoters and enhancers in keratinocytes.
(a) Vent diagram indicating the overlap of the significant 5C interactions (q<0.05) between the 5C library replicates and pie chart showing the number of all “true” intra-TAD (red) and inter-TAD (green) 5C interactions in KCs. (b) Pie-chart indicating number of 5C interactions connecting two regions anchoring transcription start sites (TSSs) within 5kb (promoter-promoter interactions); one contacting region anchoring a TSS within 5kb and the other contacting region not anchoring a TSS within 5 kb (promoter-non promoter interactions); and both contacting regions not anchoring TSSs within 5kb (non-promoter–non promoter interactions). (c) Genome browser images of the normalized ChIP-seq signals for H3K4me1 and H3K27ac enrichment as well as the position of the putative gene enhancers at the EDC containing locus in KCs aligned to the schematic locus map. Genome browser images of the normalized ChIP-seq signals for several enhancer regions at small scale are provided as examples. See Materials and Methods section for details of ChIP-seq peak calling and pursing the putative enhancers. The TAD border midpoints are indicated by the green lines.
Fig 4
Fig 4. Spatial interaction networks between gene enhancers and promoters at the EDC locus in keratinocytes.
(a) 5C looping interactions between gene enhancers (top line) and promoters (bottom line) (in red) and gene enhancers with regions not containing gene promoters (bottom line) (in bleu), TAD border midpoints are indicated by the vertical green lines. Schematic organization of the EDC locus is also shown. (b) 5C looping interactions involving Ivl and S100a11 gene promoters (bottom line) with their enhancers (top line), TAD border midpoints are indicated by the vertical green lines. (c) 5C looping interactions between the enhancer cluster E2/E3 (top line) and its putative target gene promoters (bottom line), TAD border midpoints are indicated by the vertical green lines. (d) 5C looping interactions between the enhancer E9 (top line) and its putative target gene promoters. (e) Scaling plot showing the normalized average counts versus genomic distances for the 5C looping interaction between gene promoters and enhancers within the TADs (red), and between the TADs (blue).
Fig 5
Fig 5. Chromatin architectural proteins CTCF, Rad21 and ATP-dependent chromatin remodeller Brg1 are enriched in the regions involved in the promoter-enhancer spatial interactions at the EDC locus.
(a) Schematic map of the EDC containing locus and genome browser view of the normalized ChIP-seq signals for the indicated proteins. The TAD border midpoints are indicated by the green vertical lines. (b) Number of the called ChIP-Seq peaks for the indicated proteins in the individual TADs (c) Percentage of all “true” 5C looping interactions anchoring the regions bound by the indicated proteins. (d) Results of the enrichment analysis of 5C interactions anchoring the regions bound by the indicated proteins in comparison to all the background interactions at the EDC containing locus.–log10 of the p-values are shown (exact Fisher test). (e) Percentage of the significant 5C interactions anchored to the regions bound by the proteins indicated in the left column that are also anchored to the regions bound by the proteins indicated in the top row. (f) Percentage of 5C interaction involving gene enhancers that are anchor the regions bound by the indicated proteins.(g) Results of the enrichment analysis for the 5C interactions involving gene enhancers that are anchored to the regions bound by the indicated proteins in comparison to all background interactions in the EDC locus.–log10 of the p-values are shown (exact Fisher test).
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