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Summary Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation., Graphical Abstract, Highlights • Extreme long-range enhancer clusters overlap PRS patient mutations at the SOX9 locus • PRS enhancers drive stage-specific SOX9 expression in the cranial neural crest • Mandible development has heightened sensitivity to perturbation of SOX9 gene dosage • Deletion of mouse EC1.45 leads to quantitative changes in mandible morphology, Non-coding mutations over a megabase from SOX9 cause the craniofacial disorder Pierre Robin sequence (PRS). Long et al. leverage a human neural crest model to demonstrate that PRS is caused by loss of extreme long-range enhancers active during a restricted developmental window and explore mechanisms underlying the specificity of disease manifestations.

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues., Globin loci harbor genes that are expressed embryonically and silenced postnatally. Here the authors show that zeta-globin silencing depends upon selective hypoacetylation of its TAD subdomain, which blocks its interaction with the alpha-globin super-enhancer, and zeta-globin can be reactivated by acetylation.

The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other’s TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes. Swapping the Xist/Tsix transcriptional units and placing their promoters in each other’s topologically associating domain shows that the topological partitioning of the X-inactivation center is critical to ensure proper X inactivation during development.

Chromatin structure is tightly intertwined with transcription regulation. Here we compared the chromosomal architectures of fetal and adult human erythroblasts and found that, globally, chromatin structures and compartments A/B are highly similar at both developmental stages. At a finer scale, we detected distinct folding patterns at the developmentally controlled β-globin locus. Specifically, new fetal stage-specific contacts were uncovered between a region separating the fetal (γ) and adult (δ and β) globin genes (encompassing the HBBP1 and BGLT3 noncoding genes) and two distal chromosomal sites (HS5 and 3′HS1) that flank the locus. In contrast, in adult cells, the HBBP1–BGLT3 region contacts the embryonic ε-globin gene, physically separating the fetal globin genes from the enhancer (locus control region [LCR]). Deletion of the HBBP1 region in adult cells alters contact landscapes in ways more closely resembling those of fetal cells, including increased LCR–γ-globin contacts. These changes are accompanied by strong increases in γ-globin transcription. Notably, the effects of HBBP1 removal on chromatin architecture and gene expression closely mimic those of deleting the fetal globin repressor BCL11A, implicating BCL11A in the function of the HBBP1 region. Our results uncover a new critical regulatory region as a potential target for therapeutic genome editing for hemoglobinopathies and highlight the power of chromosome conformation analysis in discovering new cis control elements.

The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression.

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel–Lindau (VHL) tumor suppressor. Roles for noncoding cis-regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profiles, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specific aspects of malignancy. Superenhancer profiling identified ZNF395 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivo. VHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α–HIF1β heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter–enhancer interactions. Subtype-specific driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression. Significance: Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered cis-regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL, a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specific promoter–enhancer complexes. Cancer Discov; 7(11); 1284–305. ©2017 AACR. See related commentary by Ricketts and Linehan, p. 1221. This article is highlighted in the In This Issue feature, p. 1201

Chromosome conformation capture (3C) methods are central to understanding the link between nuclear structure and function, and the physical interactions between distal regulatory elements and promoters. However, no one method is appropriate to address all biological questions, as each variant differs markedly in resolution, reproducibility, throughput and biases. A thorough appreciation of the strengths and weaknesses of each technique is critical when choosing the correct method for a specific application or for gauging how best to interpret different sources of data. In addition, the analysis method must be carefully considered, as this choice can profoundly affect the output. In this Review, we describe and compare the different available 3C-based approaches, with a focus on the analysis of mammalian genomes.

© 2016 Nature America, Inc. All rights reserved. Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

Regulatory enhancer elements in solid tumours remain poorly characterized. Here we apply micro-scale chromatin profiling to survey the distal enhancer landscape of primary gastric adenocarcinoma (GC), a leading cause of global cancer mortality. Integrating 110 epigenomic profiles from primary GCs, normal gastric tissues and cell lines, we highlight 36,973 predicted enhancers and 3,759 predicted super-enhancers respectively. Cell-line-defined super-enhancers can be subclassified by their somatic alteration status into somatic gain, loss and unaltered categories, each displaying distinct epigenetic, transcriptional and pathway enrichments. Somatic gain super-enhancers are associated with complex chromatin interaction profiles, expression patterns correlated with patient outcome and dense co-occupancy of the transcription factors CDX2 and HNF4α. Somatic super-enhancers are also enriched in genetic risk SNPs associated with cancer predisposition. Our results reveal a genome-wide reprogramming of the GC enhancer and super-enhancer landscape during tumorigenesis, contributing to dysregulated local and regional cancer gene expression., Gene expression is regulated by enhancers and super-enhancers, which can be identified by chromatin profiling. Here, the authors surveyed gastric cancer samples and cell lines to identify enhancer elements exhibiting somatic alterations.

Long non-coding (lnc) RNAs can regulate gene expression and protein functions. However, the proportion of lncRNAs with biological activities among the thousands expressed in mammalian cells is controversial. We studied Lockd (LncRNA downstream of Cdkn1b), a 434 nt polyadenylated lncRNA originating 4 kilobases (kb) 3′ to the Cdkn1b gene. Deletion of the 25 kb Lockd locus reduced Cdkn1b transcription by approximately 70% in an erythroid cell line. In contrast, homozygous insertion of a polyadenylation cassette 80 bp downstream of the Lockd transcription start site reduced the entire lncRNA transcript level by > 90%, with no effect on Cdkn1b transcription. The Lockd promoter contains a DNase hypersensitive site, binds numerous transcription factors, and physically associates with the Cdkn1b promoter in chromosomal conformation capture studies. Thus, the Lockd gene positively regulates Cdkn1b transcription through an enhancer-like cis element, while the lncRNA itself is dispensable, which may be the case for other lncRNAs.

The alpha-globin genes are located in the subtelomeric region of the short arm of chromosome 16. Expression of each allele is controlled by 4 distal cis-acting enhancers (MCS-R1-4) located 48, 40, 33 and 10kb upstream of the duplicated structural alpha genes . Experimental and clinical studies have shown that R1 and R2 are the most important enhancers of alpha-globin production. Here we present an individual with alpha thalassemia who inherited one copy of chromosome 16 from which both alpha genes have been deleted (--SEA). This individual's other copy of chromosome 16 harbours a small deletion of R2. Thus all alpha-globin expression in this individual originates from one copy of the alpha cluster in which R1 drives expression of the two alpha-globin genes in cis. Remarkably, this patient maintains a relatively normal degree of oxygenation despite a severe degree of alpha thalassemia. This female patient was born in Surinam and her mother and father were of Indian and Indonesian origins respectively. She most recently presented, aged 26 yrs, with tiredness and shortness of breath on exertion. She had no previously diagnosed medical conditions, but had received two blood transfusions as a child, both prompted by concurrent infections. Her growth and development were normal. She had suffered one miscarriage at 13 weeks gestation aged 25 yrs. There was nothing of note in the family history and the patient is currently employed as a clerical worker. Cardiovascular examination was normal with no hepatosplenomegaly. Her CBC was consistent with HbH disease, with a Hb of 73g/L, Hct 25%, MCV 45fl, MCH 13.2pg with a blood film showing marked microcytosis anisopoikilocytosis, target cells and 30.7% HbH inclusions on Brilliant Cressyl Blue staining. HPLC showed evidence of a fast-moving band, HbA 88.4% and HbA2 1.3%, HbE 5.1%. MLPA analysis showed that the father and proband both carried heterozygous deletions of R2, and the mother and proband both carried the --SEA deletion on one allele. Due to the severity of the anaemia and the striking red cell indices it was highly likely that she had intact regulatory elements in cis with the --SEA deletion on one allele, and on the other, a regulatory network missing R2 lying upstream of the two intact alpha-globin genes To investigate this further, CD34+ cells were extracted from the patient's peripheral blood and differentiated using a liquid culture system (Trakarnsanga et al. Nature Comms. 2017). The alpha/non-alpha-globin synthetic ratio of mRNA as measured by qPCR at peak globin-production was markedly reduced compared to WT cells and those of a patient carrying the --SEA deletion on one allele alone: WT 1.26, SEA 0.65, Patient 0.11. The upstream enhancers are associated with regions of open chromatin that can be detected by chromatin accessibility assays. To see whether the deletion of R2 had caused new regulatory elements to appear or whether accessibility of the existing elements was altered, ATAC-seq was performed on Day 13 of differentiation. This showed a reduction in the peak at R2 in keeping with its deletion on one allele, but showed no change in the peak height of R1 compared to that in the individual with the --SEA deletion alone or WT. To assess chromatin interactions across the domain Capture-C, a sensitive assay to detect interactions between selected regions of chromatin, was performed (Davies et al. Nature Methods 2016). Compared to WT, when capturing from the alpha-globin promoters interactions with R1, R3 and R4 were observed with no interactions with R2 detected, in keeping with the hypothesised distribution of deletions. No interactions in trans between the intact R2 and alpha-globin genes were detected. Previous cases of deletions of the upstream alpha-globin regulatory element have either involved deletions of the regulatory elements on one allele only, or involved both alleles but with a full complement of alpha genes. Our findings show that whilst a severe HbH phenotype was observed, it is possible to maintain transfusion independence with only R1, R3, R4 and two alpha-globin genes. Considering the ATAC-seq and Capture-C findings, as well as comparison with the homologous locus in mouse it is likely that the bulk of regulation in this patient occurs via R1. The observed phenotype is consistent with redundancy within the regulatory elements of the alpha gene locus, which may enable continued expression of key cell-specific genes in the event of adverse genotypic events. Disclosures van Beers: RR Mechatronics: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Pfizer: Research Funding.

Current methods for analysing chromosome conformation in mammalian cells are either insensitive and low resolution or low throughput. Since available methods are both expensive and relatively difficult to perform and analyse they are not widely used outside of specialised laboratories. Here we have redesigned the Capture-C method producing a new approach, called next generation (NG) Capture-C. This produces unprecedented levels of sensitivity and reproducibility, which can be used to analyse any number of genetic loci and/or many samples in a single experiment. NG Capture-C is straightforward to perform, requiring only standard reagents and access to conventional next generation sequencing platforms. Importantly, high-resolution data can be produced on as few as 100,000 cells and SNPs can be used to generate allele specific tracks. The method should therefore greatly facilitate the task of linking SNPs identified by genome wide association studies with the genes they influence. The complete and detailed protocol presented here, with new publicly available tools for library design and data analysis, will allow most laboratories to analyse chromatin conformation at levels of sensitivity and throughput that were previously impossible.

Krûppel-like factor-1 (KLF1) is a C2H2 zinc finger transcription factor which is essential for broad erythroid gene expression and erythropoiesis in vivo. A number of studies have shown ∼700 genes are poorly expressed when KLF1 is absent [1-8]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice [9,10], and partly responsible for congenital anemia in humans and mice with dominant mutations in KLF1 [11,12]. To determine whether KLF1-dependent genes are direct or indirect targets of KLF1, we have previously performed global ChIP-seq experiments identifying 945-1350 regions of KLF1 occupancy in the mouse genome [7]. About 15% of these regions fall within the promoters of KLF1 target genes but surprisingly, most are thousands of kilobases distant from any known gene. Many of these distant sites exhibit co-occupancy with other transcriptional regulators involved in erythropoiesis, including GATA1. Approximately half of the KLF1 occupied sites are found within regions of mono-methylation of lysine 4 on histone 3 (H3K4me1). These regions are devoid of histones tri-methylated at the same residue (H3K4me3). This methylation signature is commonly associated with regions of the genome that act as transcriptional enhancers [13,14] and many are also bound by the co-activator, p300. The nature and function of these distant sites, particularly those without enhancer marks, is interesting as they may shed light on novel mechanisms of action of KLF1 and associated transcription factors. The transcriptional machinery of the cell, including many transcription factors is found in large sub-nuclear compartments called transcriptional factories [15]. KLF1 has been found localized to a subset of these in erythroid cells. KLF1 is also required for long-range looping of the β-globin gene into these transcription factories [16]. Other erythroid genes involved in the production of a functional haemoglobin molecule such as α-globin and haem synthesis enzymes are often found in the same transcription factory. This strongly suggests KLF1 can employ this sub-nuclear machine to co-ordinate the transcriptional output from many genes and thereby direct erythroid cell differentiation. To explore the function of KLF1-bound loci, we have performed multiplexed chromosome conformation capture (3C) coupled with sequencing (Capture-seq) using a tamoxifen responsive, KLF1 inducible cell line to investigate the role of KLF1 in chromosomal looping. In addition, we have analysed primary transcriptional output of KLF1 target genes by nascent RNA-seq. As expected β-globin and a-globin transcription is rapidly induced, becoming detectable within 5 minutes. However, the transcriptional response of dematin and a set direct KLF1 target genes is much slower. Thus, the mechanism of KLF1 transcriptional activation differs between target gene loci. We find a dynamic role of KLF1-dependent chromosomal looping and transcriptional co-factor recruitment required to effect gene transcription during erythropoiesis. We will discuss models of differentiation transcription regulation by KLF1. References: 1. Drissen R, et al. (2005). Molecular and Cellular Biology 25: 5205–5214. 2. Funnell APW, et al. (2007). Molecular and Cellular Biology 27: 2777–2790. 3. Hodge D, et al. (2006). Blood 107: 3359–3370. 4. Pilon AM, et al. (2008). Molecular and Cellular Biology 28: 7394–7401. 5. Siatecka M, et al. (2010). PNAS 107: 15151–15156. 6. Siatecka M, Bieker JJ (2011). Blood 118: 2044–2054. 7. Tallack MR, et al. (2010). Genome Res 20: 1052–1063. 8. Tallack MR, Perkins AC (2010). IUBMB Life 62: 886–890. 9. Perkins AC, Sharpe AH, Orkin SH (1995). Nature 375: 318–322. 10. Nuez B, et al. (1995). Nature 375: 316–318. 11. Arnaud L, S et al. (2010). Am J Hum Genet 87: 721–727. 12. Borg J, et al. (2011). Haematologica 96: 635–638. 13. Zentner GE, et al. (2011). Genome Res 21: 1273–1283. 14. Pekowska A, et al. (2011). EMBO J 30: 4198–4210. 15. Osborne CS, et al. (2004). Nat Genet 36: 1065–1071. 16. Schoenfelder S, et al. (2010). Nat Genet 42: 53–61. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

The three-dimensional (3D) organization of chromosomes can be probed using methods like Capture-C. However, it is unclear how such population-level data relate to the organization within a single cell, and the mechanisms leading to the observed interactions are still largely obscure. We present a polymer modeling scheme based on the assumption that chromosome architecture is maintained by protein bridges, which form chromatin loops. To test the model, we perform FISH experiments and compare with Capture-C data. Starting merely from the locations of protein binding sites, our model accurately predicts the experimentally observed chromatin interactions, revealing a population of 3D conformations. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0909-0) contains supplementary material, which is available to authorized users.