Abstract
Chromosome conformation is an important feature of metazoan gene regulation1,2; however, enhancer–promoter contact remodeling during cellular differentiation remains poorly understood3. To address this, genome-wide promoter capture Hi-C (CHi-C)1,4 was performed during epidermal differentiation5. Two classes of enhancer–promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Mifsud, B. et al. Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C. Nat. Genet. 47, 598–606 (2015).
Dixon, J.R., Gorkin, D.U. & Ren, B. Chromatin domains: the unit of chromosome organization. Mol. Cell 62, 668–680 (2016).
Dixon, J.R. et al. Chromatin architecture reorganization during stem cell differentiation. Nature 518, 331–336 (2015).
Schoenfelder, S. et al. The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements. Genome Res. 25, 582–597 (2015).
Lopez-Pajares, V. et al. A lncRNA–MAF:MAFB transcription factor network regulates epidermal differentiation. Dev. Cell 32, 693–706 (2015).
Kagey, M.H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010).
Ji, X. et al. 3D chromosome regulatory landscape of human pluripotent cells. Cell Stem Cell 18, 262–275 (2016).
Ghavi-Helm, Y. et al. Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512, 96–100 (2014).
Jin, F. et al. A high-resolution map of the three-dimensional chromatin interactome in human cells. Nature 503, 290–294 (2013).
Phillips-Cremins, J.E. et al. Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153, 1281–1295 (2013).
Rao, S.S.P. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014).
Creyghton, M.P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).
Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).
Boglev, Y. et al. The unique and cooperative roles of the Grainy head-like transcription factors in epidermal development reflect unexpected target gene specificity. Dev. Biol. 349, 512–522 (2011).
Fuchs, E. & Green, H. The expression of keratin genes in epidermis and cultured epidermal cells. Cell 15, 887–897 (1978).
Montavon, T. et al. A regulatory archipelago controls Hox genes transcription in digits. Cell 147, 1132–1145 (2011).
Lonfat, N., Montavon, T., Darbellay, F., Gitto, S. & Duboule, D. Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci. Science 346, 1004–1006 (2014).
Adam, R.C. et al. Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice. Nature 521, 366–370 (2015).
Spitz, F. & Furlong, E.E.M. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012).
Li, L. et al. Widespread rearrangement of 3D chromatin organization underlies Polycomb-mediated stress-induced silencing. Mol. Cell 58, 216–231 (2015).
Dixon, J.R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).
Dowen, J.M. et al. Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159, 374–387 (2014).
Segre, J.A., Bauer, C. & Fuchs, E. Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat. Genet. 22, 356–360 (1999).
Boxer, L.D., Barajas, B., Tao, S., Zhang, J. & Khavari, P.A. ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes. Genes Dev. 28, 2013–2026 (2014).
Lopez, R.G. et al. C/EBPα and β couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation. Nat. Cell Biol. 11, 1181–1190 (2009).
Sen, G.L. et al. ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation. Dev. Cell 22, 669–677 (2012).
Schwartzman, O. et al. UMI-4C for quantitative and targeted chromosomal contact profiling. Nat. Methods 13, 685–691 (2016).
Samstein, R.M. et al. Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification. Cell 151, 153–166 (2012).
Kim, T.-H. et al. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity. Nature 506, 511–515 (2014).
Fossum, S.L. et al. Ets homologous factor regulates pathways controlling response to injury in airway epithelial cells. Nucleic Acids Res. 42, 13588–13598 (2014).
Kundaje, A. et al. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015).
Truong, A.B., Kretz, M., Ridky, T.W., Kimmel, R. & Khavari, P.A. p63 regulates proliferation and differentiation of developmentally mature keratinocytes. Genes Dev. 20, 3185–3197 (2006).
Kretz, M. et al. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493, 231–235 (2013).
Rinaldi, L. et al. Dnmt3a and Dnmt3b associate with enhancers to regulate human epidermal stem cell homeostasis. Cell Stem Cell 19, 491–501 (2016).
Servant, N. et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. Genome Biol. 16, 259 (2015).
Huang, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Bao, X. et al. A novel ATAC–seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63. Genome Biol. 16, 284 (2015).
Wingett, S. et al. HiCUP: pipeline for mapping and processing Hi-C data. F1000Res 4, 1310 (2015).
Cairns, J. et al. CHiCAGO: robust detection of DNA looping interactions in Capture Hi-C data. Genome Biol. 17, 127 (2016).
Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Klein, F.A. et al. FourCSeq: analysis of 4C sequencing data. Bioinformatics 31, 3085–3091 (2015).
Freire-Pritchett, P. et al. Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells. eLife 6, e21926 (2017).
Landt, S.G. et al. ChIP–seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 22, 1813–1831 (2012).
Acknowledgements
We thank J. Wysocka, A. Oro, R. Flynn, O. Wapinski, and A. Brunet for presubmission review. This work was supported by the US Veterans Affairs Office of Research and Development and NIH/NIAMS grant AR45192 to P.A.K. and by NIH grant U01 HG007919 to M.S., H.Y.C., W.J.G., A.K., and P.A.K. This work was supported in part by funding from the Biotechnology and Biological Sciences Research Council (BB/J004480/1) and the European Community's Seventh Framework Programme (MODHEP consortium 259743) to P.F.
Author information
Authors and Affiliations
Contributions
B.C.B., A.J.R., and M.F.-M. designed and executed experiments, analyzed data, and wrote the manuscript. I.H., M.R.M., D.S.K., L.D.B., Z.Z., M. Spivakov, and V.L.-P. executed experiments, analyzed data, and contributed to design of experimentation. J.C., M. Spivakov, and S.W.W. developed and executed the CHiCAGO analysis pipeline. W.J.G., H.Y.C., M. Snyder, A.K., P.F., and H.Y.C. contributed to experimental design and analysis. P.A.K. designed experiments, analyzed data, and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Characterization of Hi-C/CHi-C dynamics and enhancer identification.
(a) Scatterplot of Hi-C read counts supporting contacts between domain boundaries. Contacts enclosing domains containing differentially expressed genes are highlighted. (b) Prevalence of differentially expressed genes associated with dynamic domain contacts and dynamic enhancer–promoter contacts. Increasing and decreasing contact sets refer to contacts significantly gained and lost, respectively, on day 3 or day 6 versus day 0 (dynamic contact calls by edgeR, FDR < 0.1, minimum of 15 reads supporting contact in at least one condition). (c) Metaplots of ChIP–seq signal from the ENCODE Project, derived from normal human epidermal keratinocyte (NHEK) cells. H3K27ac peaks identified in this study. TSS distal indicates peaks greater than 5 kb away from a TSS. TSS-proximal peaks exhibit the expected promoter-associated H3K4me1-low, H3K4me3-high signature, while TSS-distal peaks exhibit the expected enhancer-associated H3K4me1-high, H3K4me3-low signature. (d) Distribution of enhancer reporter activity from H3K27ac-marked regions. Measurements of 50 putative H3K27ac-marked enhancer elements were made in progenitor keratinocytes. The proportion of elements showing different fold inductions during differentiation over empty control vector is shown. (e) Proportion of enhancer–promoter and promoter–promoter contacts contained within domains. The high-confidence set corresponds to contacts supported by greater than 15 reads in any replicate of CHi-C (this set was used for edgeR differential contact analysis).
Supplementary Figure 2 Characterization of CHi-C dynamics and relationship with dynamic gene expression.
(a) Normalized CHi-C read count profile for contacts from the GRHL1 promoter (normalized to total reads associated with the viewpoint fragment in each condition, days 0–6 of differentiation). The gray dotted line marks the position of the viewpoint fragment. (b) As in a, for contacts from the KRT1 promoter. (c) Heat map of CHi-C q scores for 1,481 distal genomic region contacts displaying decreasing contact with target promoters during epidermal cell differentiation. Contacts are sorted in order of enhancer–promoter distance (edgeR, FDR < 0.1, fold change > 2). (d) Enrichment of overlap between dynamic contacts and gene subsets by dynamic expression. Enrichment >1.0 represents the scenario in which the contact and gene subsets co-occurred more frequently than expected by chance (empirical FDR, N.S., P > 0.05, *P < 0.05). (e) Enriched GO terms for induced or repressed gene sets displaying either gained (left) or lost (right) enhancer–promoter (EP) contacts.
Supplementary Figure 3 Identification and characterization of gene sets based on contact dynamics.
(a) Pie chart representation of three classes of genes induced at day 6 versus day 0 of differentiation. Genes are classified by association with stable or gained contacts in differentiation. (b) Example promoter viewpoint CHi-C read count profiles for genes of each of three classes of induced genes. (c) Enrichment values for GO terms enriched at FDR < 0.01 in at least one of three gene classes. An asterisk indicates that the term was significantly enriched in that specific gene set. (d) Box-and-whisker plots representing expression fold change on day 6 versus day 0 of differentiation for all genes or classes of induced genes (two-sided KS test, GS versus G P = 0.9503, GS versus S P = 0.4915). (e) Box-and-whisker plots representing the z score of expression on day 6 versus other differentiation days and collection of ENCODE cell types (Online Methods) for all genes or each class of induced genes (two-sided KS test, GS versus G P = 0.7501, GS versus S P = 0.2.536 × 10−3). KS test, n.s., P > 0.05, *P < 0.05, **P < 0.01.
Supplementary Figure 4 Chromatin state and dynamics of induced gene-linked enhancers and promoters.
(a) Top, heat map of chromHMM chromatin state segmentation emission probabilities for NHEKs from the Roadmap Epigenomics Project. State codes are accompanied by descriptions. Bottom, enrichment fold changes of putative repressive distal regions in select enhancer (genic enhancer and enhancer) and repressive (bivalent enhancer and repressed Polycomb) chromHMM states. (b) Normalized CHi-C read count profile for contacts from the PRDM1 promoter (normalized to total reads associated with the viewpoint fragment in each condition). The gray dotted line marks the position of the viewpoint fragment. (c) H3K27ac at genomic regions as in Figure 2c showing H3K27ac at promoter elements in contact with the promoters of induced differentiation genes.
Supplementary Figure 5 Developmental and lineage specificity of induced gene-linked enhancers.
(a) Heat map of H3K27ac ChIP–seq signal at enhancers contacting induced genes and exhibiting gained H3K27ac in differentiation. z score is calculated for ChIP–seq signal across rows (cell types). Column labels correspond to the following: ES, H1 hESC human embryonic stem cells; GM, GM12878 immortalized B lymphocytes; HS, human skeletal muscle cells and myoblasts; HU, HUVEC human umbilical vein endothelial cells; LF, NHLF normal human lung fibroblasts; OS, OSTEO human primary osteoblasts; HM, HMEC human mammary epithelial cells; NH, NHEK normal human epidermal keratinocytes; D0, day 0; D3, day 3; D6, day 6. (b) Box-and-whisker plots representing the heat map in d (two-sided KS test, D0 versus hES P = 7.292 × 10−9, D3 versus hES P < 2.2 × 10−16, D6 versus hES P < 2.2 × 10−16). (c) Similar to d, including enhancers contacting induced genes and exhibiting no significant dynamics of H3K27ac in differentiation. (d) Box-and-whisker plots representing the heat map in f (two-sided KS test, D0 versus hES P < 2.2 × 10−16, D3 versus hES P < 2.2 × 10−16, D6 versus hES P < 2.2 × 10−16). (e) Normalized CHi-C read count profile for contacts from the DSC2 promoter (normalized to total reads associated with the viewpoint fragment in each condition). The gray dotted line marks the position of the viewpoint fragment. NHEK and hESC H3K27ac signal tracks and peak calls are taken directly from the ENCODE portal of the UCSC Genome Browser. (f) Box-and-whisker plots representing CHi-C read count z scores for induced gene-associated contacts gained in epidermal differentiation (two-sided KS test, D6 versus hES P < 2.2 × 10−16, D3 versus hES P < 2.2 × 10−16). (g) Similar to f for induced gene-associated contacts that are stable in epidermal differentiation (two-sided KS test, D0 versus hES P < 2.2 × 10−16, D3 versus hES P < 2.2 × 10−16, D6 versus hES P < 2.2 × 10−16). (h) Similar to e, for profiles of the SOX21 promoter, which is expressed specifically in hESCs. KS test, **P < 1 × 10−5, ***P < 1 × 10−15.
Supplementary Figure 6 Characterization of contact dynamics and cohesin association of induced gene-linked enhancers.
(a) Contact strength between promoter elements in contact with the promoters of induced differentiation genes as in Figure 2d. Box-and-whisker plots represent the median and interquartile range of the change in contact q score between day 6 and day 0. Contact sets are defined by H3K27ac dynamics at the promoter locus (bait HindIII fragment) or distal promoter. Dotted lines denote day 0 for either H3K27ac signal or differentiation gene mRNA expression; solid lines denote day 6. (b) Enrichment of genomic features in 10-kb regions surrounding domain boundaries relative to 10-kb regions in domain centers. (c) SMC1A ChIP–seq metaplot signal at H3K27 premarked and H3K27ac gained putative enhancers that contact induced genes as well as CTCF peaks. Error bands represent 98% boostrapped confidence intervals. (d) Normalized CHi-C read count profile for contacts from the HOPX promoter (normalized to total reads associated with the viewpoint fragment in each condition). The gray dotted line marks the position of the viewpoint fragment. (e) MA plot (Online Methods) of SMC1A ChIP–seq signal at enhancer loci on day 3 versus day 0. Loci identified as significantly dynamic are highlighted (edgeR, FDR < 0.1, fold change > 0). (f) MA plot of SMC1A ChIP–seq signal at enhancer loci on day 6 versus day 0. Loci identified as significantly dynamic are highlighted (edgeR, FDR < 0.1, fold change > 0).
Supplementary Figure 7 Developmental context of transcription factor depletion and further characterization of effect on chromatin conformation.
(a) Enrichment of enhancer subsets versus all enhancers in super-enhancers identified on day 0, day 3, or day 6. (b) Relative expression by RT–qPCR for KLF4, ZNF750, and several known target genes in siRNA-treated cells. Error bars represent s.d. across two technical replicates (independent samples from a pool of siRNA-treated cells). (c) Principal-component analysis of H3K27ac ChIP–seq signal in a panel of ENCODE cell types, normal epidermal differentiation time points, and differentiating epidermal cells treated with control or transcription factor–targeting siRNA. CTRi, control siRNA; KLFi, KLF4 siRNA; ZNFi, ZNF750 siRNA. Epidermal cells, including transcription factor–depleted cells, cluster closely. (d) Relative expression by published data (Dev. Cell 22, 669–677, 2012) for genes analyzed by UMI-4C in siRNA-treated cells. (e) UMI-4C profile of interactions anchored by the PRDM1 promoter in control and KLF4- or ZNF750-knockdown conditions. Error bands represent s.e.m. between replicates (independent samples of siRNA-treated cells).
Supplementary Figure 8 Discovery and characterization of EHF as a novel regulator of epidermal differentiation
(a) Comparative approach to identify transcription factor motifs enriched in H3K27ac premarked enhancers relative to H3K27ac gained enhancers in contact with induced differentiation genes. (b) Heat map of Pearson correlations for mRNA expression values derived from RNA-seq (Roadmap Epigenomics). Hierarchical clustering was used to identify sets of cell types labeled by names (columns) and group IDs (rows). (c) Gene expression fold change of EHF and other transcription factors during differentiation by RNA-seq. Day 6 was compared with day 0. (d) Western blot of EHF at days 0, 3, and 6 of differentiation for primary human keratinocytes derived from two independent donors. (e) Quantification of the western blot in d, with normalization to actin loading control.
Supplementary Figure 9 Characterization of EHF and functional effects of EHF depletion on chromatin.
(a) RT–qPCR of EHF and differentiation-specific genes in organotypic epidermis depleted of EHF using two independent siRNAs targeting EHF versus scrambled controls. Error bars represent s.d. across two biological replicates (independent organotypic tissues). (b) Quantification of differentiation protein immunofluorescence signal area within the epidermis across technical replicates for control and two biological replicate organotypic epidermal tissues depleted of EHF (Student’s t test, one-sided, Rep 1 versus Control P = 3.54 × 10−2, Rep 2 versus Control P = 9.3 × 10−3; *P < 0.05, **P < 0.01). (c) Heat map of RNA-seq z scores for all differentially expressed genes in triplicate biological replicate EHF-depleted (EHFi) versus control (CTRi) organotypic epidermal tissue. GO enrichments correspond to genes exhibiting increased expression with EHF depletion. (d) ChIP–qPCR of EHF, day 0 and day 3, demonstrating enrichment at a panel of enhancers in contact with promoters of genes induced in differentiation. Genes are noted in the heat map in b. Error bars represent s.d. across biological replicates (independent samples of cells from a single culture). (e) Top motifs identified by HOMER in EHF ChIP–seq peaks relative to a random background. (f) Box-and-whisker plots representing the median and interquartile range of the change in contact q score between day 3 and day 0. Contact sets are defined by EHF binding and H3K27ac dynamics at the promoter locus (bait HindIII fragment) or enhancer (empirical FDR, *P < 0.01). (g) RT–qPCR of DSG3 in control and EHF-depleted cells. Error bars represent s.d. across technical replicates. (h) UMI-4C profile of interactions anchored by the DSG3 promoter in control and EHF-knockdown conditions. Error bands represent s.e.m. between biological replicates.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9. (PDF 1596 kb)
Supplementary Table 1
HiC of domain-forming contacts. (XLSX 886 kb)
Supplementary Table 2
RNA-seq dynamic gene sets. (XLSX 196 kb)
Supplementary Table 3
CHi-C dynamic contact sets. (XLSX 734 kb)
Supplementary Table 4
Gained and stable enhancer–promoter contact-based gene sets. (XLSX 51 kb)
Supplementary Table 5
ChIP–seq H3K27ac dynamic enhancer sets. (XLSX 317 kb)
Supplementary Table 6
ChIP–qPCR and UMI-4C oligonucleotide sequences. (XLSX 43 kb)
Supplementary Table 7
RNA-seq EHF-regulated gene sets. (XLSX 44 kb)
Source data
Rights and permissions
About this article
Cite this article
Rubin, A., Barajas, B., Furlan-Magaril, M. et al. Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation. Nat Genet 49, 1522–1528 (2017). https://doi.org/10.1038/ng.3935
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ng.3935
This article is cited by
-
The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer
Genome Biology (2024)
-
Enhancer–promoter interactions become more instructive in the transition from cell-fate specification to tissue differentiation
Nature Genetics (2024)
-
The urothelial gene regulatory network: understanding biology to improve bladder cancer management
Oncogene (2024)
-
Increased enhancer–promoter interactions during developmental enhancer activation in mammals
Nature Genetics (2024)
-
Cellular reprogramming is driven by widespread rewiring of promoter-enhancer interactions
BMC Biology (2023)
