Publications by authors named "Jim R Hughes"

53 Publications

Enhancers predominantly regulate gene expression during differentiation via transcription initiation.

Mol Cell 2021 Mar 3;81(5):983-997.e7. Epub 2021 Feb 3.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK; MRC WIMM Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. Electronic address:

Gene transcription occurs via a cycle of linked events, including initiation, promoter-proximal pausing, and elongation of RNA polymerase II (Pol II). A key question is how transcriptional enhancers influence these events to control gene expression. Here, we present an approach that evaluates the level and change in promoter-proximal transcription (initiation and pausing) in the context of differential gene expression, genome-wide. This combinatorial approach shows that in primary cells, control of gene expression during differentiation is achieved predominantly via changes in transcription initiation rather than via release of Pol II pausing. Using genetically engineered mouse models, deleted for functionally validated enhancers of the α- and β-globin loci, we confirm that these elements regulate Pol II recruitment and/or initiation to modulate gene expression. Together, our data show that gene expression during differentiation is regulated predominantly at the level of initiation and that enhancers are key effectors of this process.
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http://dx.doi.org/10.1016/j.molcel.2021.01.002DOI Listing
March 2021

Fra-1 regulates its target genes via binding to remote enhancers without exerting major control on chromatin architecture in triple negative breast cancers.

Nucleic Acids Res 2021 Feb 3. Epub 2021 Feb 3.

IGMM, Univ Montpellier, CNRS, Montpellier, France.

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.
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http://dx.doi.org/10.1093/nar/gkab053DOI Listing
February 2021

High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale.

Nat Commun 2021 01 22;12(1):531. Epub 2021 Jan 22.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods generally provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at limited numbers of loci. Due to technical limitations, generation of reproducible high-resolution interaction profiles has not been achieved at genome-wide scale. Here, to overcome this barrier, we systematically test each step of 3C and report two improvements over current methods. We show that up to 30% of reporter events generated using the popular in situ 3C method arise from ligations between two individual nuclei, but this noise can be almost entirely eliminated by isolating intact nuclei after ligation. Using Nuclear-Titrated Capture-C, we generate reproducible high-resolution genome-wide 3C interaction profiles by targeting 8055 gene promoters in erythroid cells. By pairing high-resolution 3C interaction calls with nascent gene expression we interrogate the role of promoter hubs and super-enhancers in gene regulation.
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http://dx.doi.org/10.1038/s41467-020-20809-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822813PMC
January 2021

DeepC: predicting 3D genome folding using megabase-scale transfer learning.

Nat Methods 2020 11 12;17(11):1118-1124. Epub 2020 Oct 12.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

Predicting the impact of noncoding genetic variation requires interpreting it in the context of three-dimensional genome architecture. We have developed deepC, a transfer-learning-based deep neural network that accurately predicts genome folding from megabase-scale DNA sequence. DeepC predicts domain boundaries at high resolution, learns the sequence determinants of genome folding and predicts the impact of both large-scale structural and single base-pair variations.
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http://dx.doi.org/10.1038/s41592-020-0960-3DOI Listing
November 2020

Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder.

Cell Stem Cell 2020 Nov 28;27(5):765-783.e14. Epub 2020 Sep 28.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stem.2020.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655526PMC
November 2020

Genetic and functional insights into CDA-I prevalence and pathogenesis.

J Med Genet 2021 Mar 9;58(3):185-195. Epub 2020 Jun 9.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

Background: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes and . Little is understood about either protein and it is unclear in which cellular pathways they participate.

Methods: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by . Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation.

Results: We identify six novel mutations and one novel mutation in and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells.

Conclusion: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.
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http://dx.doi.org/10.1136/jmedgenet-2020-106880DOI Listing
March 2021

Dynamics of the 4D genome during in vivo lineage specification and differentiation.

Nat Commun 2020 06 1;11(1):2722. Epub 2020 Jun 1.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Mammalian gene expression patterns are controlled by regulatory elements, which interact within topologically associating domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input chromosome conformation capture (3C) technique. We use this approach to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of specific contacts between enhancers and promoters. Our high-resolution data show that these enhancer-promoter interactions are not established prior to gene expression, but formed gradually during differentiation, concomitant with progressive upregulation of gene activity. Together, these results provide new insight into the close, interdependent relationship between chromatin architecture and gene regulation during development.
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http://dx.doi.org/10.1038/s41467-020-16598-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264236PMC
June 2020

Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.

Mol Cell 2020 06 23;78(5):960-974.e11. Epub 2020 Apr 23.

Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON K1H8L6, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H8L6, Canada. Electronic address:

Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.
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http://dx.doi.org/10.1016/j.molcel.2020.03.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344268PMC
June 2020

A Dynamic Folded Hairpin Conformation Is Associated with α-Globin Activation in Erythroid Cells.

Cell Rep 2020 02;30(7):2125-2135.e5

Dipartimento di Fisica, Università degli Studi di Napoli Federico II, and INFN Napoli, Complesso Universitario di Monte Sant'Angelo, 80126 Naples, Italy; Berlin Institute of Health (BIH), MDC-Berlin, 13125 Berlin, Germany. Electronic address:

We investigate the three-dimensional (3D) conformations of the α-globin locus at the single-allele level in murine embryonic stem cells (ESCs) and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent fluorescence in situ hybridization (FISH) data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ESCs to erythroid cells, associated with the activation of the globin genes. We find that in ESCs, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, whereas in erythroid cells the α-globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific gene-enhancer contacts, suggesting that beyond the interaction of CTCF sites, multiple molecular mechanisms cooperate to form an interacting domain.
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http://dx.doi.org/10.1016/j.celrep.2020.01.044DOI Listing
February 2020

A revised model for promoter competition based on multi-way chromatin interactions at the α-globin locus.

Nat Commun 2019 11 27;10(1):5412. Epub 2019 Nov 27.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Specific communication between gene promoters and enhancers is critical for accurate regulation of gene expression. However, it remains unclear how specific interactions between multiple regulatory elements contained within a single chromatin domain are coordinated. Recent technological advances which can detect multi-way chromatin interactions at single alleles can provide insights into how multiple regulatory elements cooperate or compete for transcriptional activation. Here, we use such an approach to investigate how interactions of the α-globin enhancers are distributed between multiple promoters in a mouse model in which the α-globin domain is extended to include several additional genes. Our data show that gene promoters do not form mutually exclusive interactions with enhancers, but all interact simultaneously in a single complex. These findings suggest that promoters do not structurally compete for interactions with enhancers, but form a regulatory hub structure, which is consistent with recent models of transcriptional activation occurring in non-membrane bound nuclear compartments.
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http://dx.doi.org/10.1038/s41467-019-13404-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881440PMC
November 2019

Nrf2 controls iron homeostasis in haemochromatosis and thalassaemia via Bmp6 and hepcidin.

Nat Metab 2019 05 13;1(5):519-531. Epub 2019 May 13.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK.

Iron is critical for life but toxic in excess because of iron-catalysed formation of pro-oxidants that cause tissue damage in a range of disorders. The Nrf2 transcription factor orchestrates cell-intrinsic protective antioxidant responses, and the peptide hormone hepcidin maintains systemic iron homeostasis, but is pathophysiologically decreased in haemochromatosis and beta-thalassaemia. Here, we show that Nrf2 is activated by iron-induced, mitochondria-derived pro-oxidants and drives Bmp6 expression in liver sinusoid endothelial cells, which in turn increases hepcidin synthesis by neighbouring hepatocytes. In Nrf2 knockout mice, the Bmp6-hepcidin response to oral and parenteral iron is impaired and iron accumulation and hepatic damage are increased. Pharmacological activation of Nrf2 stimulates the Bmp6-hepcidin axis, improving iron homeostasis in haemochromatosis and counteracting the inhibition of Bmp6 by erythroferrone in beta-thalassaemia. We propose that Nrf2 links cellular sensing of excess toxic iron to control of systemic iron homeostasis and antioxidant responses, and may be a therapeutic target for iron-associated disorders.
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http://dx.doi.org/10.1038/s42255-019-0063-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609153PMC
May 2019

DOT1L inhibition reveals a distinct subset of enhancers dependent on H3K79 methylation.

Nat Commun 2019 06 26;10(1):2803. Epub 2019 Jun 26.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Haematology Theme, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to demarcate potentially active enhancers. Here we reveal that putative enhancers marked with H3 lysine 79 (H3K79) di or trimethylation (me2/3) (which we name H3K79me2/3 enhancer elements or KEEs) can be found in multiple cell types. Mixed lineage leukemia gene (MLL) rearrangements (MLL-r) such as MLL-AF4 are a major cause of incurable acute lymphoblastic leukemias (ALL). Using the DOT1L inhibitor EPZ-5676 in MLL-AF4 leukemia cells, we show that H3K79me2/3 is required for maintaining chromatin accessibility, histone acetylation and transcription factor binding specifically at KEEs but not non-KEE enhancers. We go on to show that H3K79me2/3 is essential for maintaining enhancer-promoter interactions at a subset of KEEs. Together, these data implicate H3K79me2/3 as having a functional role at a subset of active enhancers in MLL-AF4 leukemia cells.
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http://dx.doi.org/10.1038/s41467-019-10844-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594956PMC
June 2019

High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates.

Methods Protoc 2018 Aug 1;1(3). Epub 2018 Aug 1.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DU, UK.

The emergence in recent years of DNA editing technologies-Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors-have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis.
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http://dx.doi.org/10.3390/mps1030029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481090PMC
August 2018

The bipartite TAD organization of the X-inactivation center ensures opposing developmental regulation of Tsix and Xist.

Nat Genet 2019 06 27;51(6):1024-1034. Epub 2019 May 27.

Institut Curie, CNRS UMR3215, INSERM U934, Paris, France.

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.
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http://dx.doi.org/10.1038/s41588-019-0412-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551226PMC
June 2019

Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains.

Nat Genet 2018 12 29;50(12):1744-1751. Epub 2018 Oct 29.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

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.
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http://dx.doi.org/10.1038/s41588-018-0253-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265079PMC
December 2018

A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions.

Nat Commun 2018 09 21;9(1):3849. Epub 2018 Sep 21.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Oxford University, Oxford, OX3 9DS, UK.

Self-interacting chromatin domains encompass genes and their cis-regulatory elements; however, the three-dimensional form a domain takes, whether this relies on enhancer-promoter interactions, and the processes necessary to mediate the formation and maintenance of such domains, remain unclear. To examine these questions, here we use a combination of high-resolution chromosome conformation capture, a non-denaturing form of fluorescence in situ hybridisation and super-resolution imaging to study a 70 kb domain encompassing the mouse α-globin regulatory locus. We show that this region forms an erythroid-specific, decompacted, self-interacting domain, delimited by frequently apposed CTCF/cohesin binding sites early in terminal erythroid differentiation, and does not require transcriptional elongation for maintenance of the domain structure. Formation of this domain does not rely on interactions between the α-globin genes and their major enhancers, suggesting a transcription-independent mechanism for establishment of the domain. However, absence of the major enhancers does alter internal domain interactions. Formation of a loop domain therefore appears to be a mechanistic process that occurs irrespective of the specific interactions within.
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http://dx.doi.org/10.1038/s41467-018-06248-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155075PMC
September 2018

HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis.

Nat Commun 2018 01 8;9(1):100. Epub 2018 Jan 8.

Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 8 College Road, Singapore, 169857, Singapore.

The repression of telomerase activity during cellular differentiation promotes replicative aging and functions as a physiological barrier for tumorigenesis in long-lived mammals, including humans. However, the underlying mechanisms remain largely unclear. Here we describe how miR-615-3p represses hTERT expression. mir-615-3p is located in an intron of the HOXC5 gene, a member of the highly conserved homeobox family of transcription factors controlling embryogenesis and development. Unexpectedly, we found that HoxC5 also represses hTERT expression by disrupting the long-range interaction between hTERT promoter and its distal enhancer. The 3'UTR of hTERT and its upstream enhancer region are well conserved in long-lived primates. Both mir-615-3p and HOXC5 are activated upon differentiation, which constitute a feed-forward loop that coordinates transcriptional and post-transcriptional repression of hTERT during cellular differentiation. Deregulation of HOXC5 and mir-615-3p expression may contribute to the activation of hTERT in human cancers.
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http://dx.doi.org/10.1038/s41467-017-02601-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758779PMC
January 2018

Low-input Capture-C: A Chromosome Conformation Capture Assay to Analyze Chromatin Architecture in Small Numbers of Cells.

Bio Protoc 2017 Dec;7(23)

Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.

Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This protocol describes two new low-input Capture-C approaches that can generate high-quality 3C interaction profiles from 10,000-20,000 cells, depending on the resolution used for analysis.
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http://dx.doi.org/10.21769/BioProtoc.2645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736099PMC
December 2017

Robust detection of chromosomal interactions from small numbers of cells using low-input Capture-C.

Nucleic Acids Res 2017 Dec;45(22):e184

Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin architecture in rare cell populations. We present a new low-input Capture-C approach that can generate high-quality 3C interaction profiles from 10 000-20 000 cells, depending on the resolution used for analysis. We also present a PCR-free, sequencing-free 3C technique based on NanoString technology called C-String. By comparing C-String and Capture-C interaction profiles we show that the latter are not skewed by PCR amplification. Furthermore, we demonstrate that chromatin interactions detected by Capture-C do not depend on the degree of cross-linking by performing experiments with varying formaldehyde concentrations.
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http://dx.doi.org/10.1093/nar/gkx1194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728395PMC
December 2017

Between form and function: the complexity of genome folding.

Hum Mol Genet 2017 10;26(R2):R208-R215

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS, UK.

It has been known for over a century that chromatin is not randomly distributed within the nucleus. However, the question of how DNA is folded and the influence of such folding on nuclear processes remain topics of intensive current research. A longstanding, unanswered question is whether nuclear organization is simply a reflection of nuclear processes such as transcription and replication, or whether chromatin is folded by independent mechanisms and this per se encodes function? Evidence is emerging that both may be true. Here, using the α-globin gene cluster as an illustrative model, we provide an overview of the most recent insights into the layers of genome organization across different scales and how this relates to gene activity.
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http://dx.doi.org/10.1093/hmg/ddx306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886466PMC
October 2017

Comparative analysis of three-dimensional chromosomal architecture identifies a novel fetal hemoglobin regulatory element.

Genes Dev 2017 08 15;31(16):1704-1713. Epub 2017 Sep 15.

Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.

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 and noncoding genes) and two distal chromosomal sites (HS5 and 3'HS1) that flank the locus. In contrast, in adult cells, the - region contacts the embryonic ε-globin gene, physically separating the fetal globin genes from the enhancer (locus control region [LCR]). Deletion of the 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 removal on chromatin architecture and gene expression closely mimic those of deleting the fetal globin repressor BCL11A, implicating BCL11A in the function of the 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 control elements.
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http://dx.doi.org/10.1101/gad.303461.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647940PMC
August 2017

Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints.

Genome Res 2017 10 13;27(10):1730-1742. Epub 2017 Sep 13.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, United Kingdom.

In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive -mer-based analysis of DNase footprints to determine any -mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome.
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http://dx.doi.org/10.1101/gr.220202.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630036PMC
October 2017

Deficiency Drives Enhancer Activation of Oncogenes in Clear Cell Renal Cell Carcinoma.

Cancer Discov 2017 11 11;7(11):1284-1305. Epub 2017 Sep 11.

Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, Singapore.

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau () tumor suppressor. Roles for noncoding -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 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination and 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 may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression. Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered -regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of , 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. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0375DOI Listing
November 2017

Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia.

Nat Commun 2017 09 4;8(1):424. Epub 2017 Sep 4.

Medical Research Council (MRC) Molecular Hematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.

β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia.β-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.
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http://dx.doi.org/10.1038/s41467-017-00479-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583283PMC
September 2017

Hepcidin is regulated by promoter-associated histone acetylation and HDAC3.

Nat Commun 2017 09 1;8(1):403. Epub 2017 Sep 1.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3.Hepcidin controls systemic iron levels by inhibiting intestinal iron absorption and iron recycling. Here, Pasricha et al. demonstrate that the hepcidin-chromatin locus displays HDAC3-mediated reversible epigenetic modifications during both erythropoiesis and iron deficiency.
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http://dx.doi.org/10.1038/s41467-017-00500-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581335PMC
September 2017

DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease.

Proc Natl Acad Sci U S A 2017 09 21;114(36):E7526-E7535. Epub 2017 Aug 21.

Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, United Kingdom;

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.
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http://dx.doi.org/10.1073/pnas.1703087114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594649PMC
September 2017

Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo.

Nat Cell Biol 2017 Aug 24;19(8):952-961. Epub 2017 Jul 24.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK.

The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.
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http://dx.doi.org/10.1038/ncb3573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540176PMC
August 2017

Functional characterisation of -regulatory elements governing dynamic expression in the early mouse embryo.

Development 2017 04 7;144(7):1249-1260. Epub 2017 Feb 7.

The Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK

The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The -acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue-specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable, and only VPE is required for optimal expression Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500 kb) pre-formed compartment in ESCs and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a and four additional putative enhancers display increased chromatin accessibility in DE that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of the target genes and is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.
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http://dx.doi.org/10.1242/dev.147322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399628PMC
April 2017

How best to identify chromosomal interactions: a comparison of approaches.

Nat Methods 2017 01;14(2):125-134

Medical Research Council (MRC) Molecular Hematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford, United Kingdom.

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.
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http://dx.doi.org/10.1038/nmeth.4146DOI Listing
January 2017

MLL-AF4 binds directly to a BCL-2 specific enhancer and modulates H3K27 acetylation.

Exp Hematol 2017 03 14;47:64-75. Epub 2016 Nov 14.

Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, Headington, Oxford, UK. Electronic address:

Survival rates for children and adults carrying mutations in the Mixed Lineage Leukemia (MLL) gene continue to have a very poor prognosis. The most common MLL mutation in acute lymphoblastic leukemia is the t(4;11)(q21;q23) chromosome translocation that fuses MLL in-frame with the AF4 gene producing MLL-AF4 and AF4-MLL fusion proteins. Previously, we found that MLL-AF4 binds to the BCL-2 gene and directly activates it through DOT1L recruitment and increased H3K79me2/3 levels. In the study described here, we performed a detailed analysis of MLL-AF4 regulation of the entire BCL-2 family. By measuring nascent RNA production in MLL-AF4 knockdowns, we found that of all the BCL-2 family genes, MLL-AF4 directly controls the active transcription of both BCL-2 and MCL-1 and also represses BIM via binding of the polycomb group repressor 1 (PRC1) complex component CBX8. We further analyzed MLL-AF4 activation of the BCL-2 gene using Capture-C and identified a BCL-2-specific enhancer, consisting of two clusters of H3K27Ac at the 3' end of the gene. Loss of MLL-AF4 activity results in a reduction of H3K79me3 levels in the gene body and H3K27Ac levels at the 3' BCL-2 enhancer, revealing a novel regulatory link between these two histone marks and MLL-AF4-mediated activation of BCL-2.
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http://dx.doi.org/10.1016/j.exphem.2016.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333536PMC
March 2017