Publications by authors named "Douglas R Higgs"

132 Publications

Publisher Correction: The relationship between genome structure and function.

Nat Rev Genet 2021 Oct 14. Epub 2021 Oct 14.

Laboratory of Gene Regulation, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

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http://dx.doi.org/10.1038/s41576-021-00425-wDOI Listing
October 2021

Testing the super-enhancer concept.

Nat Rev Genet 2021 Sep 3. Epub 2021 Sep 3.

Whitehead Institute for Biomedical Research, Cambridge, MA, USA.

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http://dx.doi.org/10.1038/s41576-021-00398-wDOI Listing
September 2021

Reactivation of a developmentally silenced embryonic globin gene.

Nat Commun 2021 07 21;12(1):4439. Epub 2021 Jul 21.

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

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.
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http://dx.doi.org/10.1038/s41467-021-24402-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295333PMC
July 2021

A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker.

Nat Commun 2021 06 21;12(1):3806. Epub 2021 Jun 21.

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

Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.
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http://dx.doi.org/10.1038/s41467-021-23980-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217497PMC
June 2021

Author Correction: Enhancer-promoter interactions and transcription.

Authors:
Douglas R Higgs

Nat Genet 2021 May;53(5):761

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

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http://dx.doi.org/10.1038/s41588-021-00815-0DOI Listing
May 2021

Enhancers predominantly regulate gene expression during differentiation via transcription initiation.

Mol Cell 2021 03 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

The relationship between genome structure and function.

Nat Rev Genet 2021 03 24;22(3):154-168. Epub 2020 Nov 24.

Laboratory of Gene Regulation, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Precise patterns of gene expression in metazoans are controlled by three classes of regulatory elements: promoters, enhancers and boundary elements. During differentiation and development, these elements form specific interactions in dynamic higher-order chromatin structures. However, the relationship between genome structure and its function in gene regulation is not completely understood. Here we review recent progress in this field and discuss whether genome structure plays an instructive role in regulating gene expression or is a reflection of the activity of the regulatory elements of the genome.
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http://dx.doi.org/10.1038/s41576-020-00303-xDOI Listing
March 2021

The mouse alpha-globin cluster: a paradigm for studying genome regulation and organization.

Curr Opin Genet Dev 2021 04 19;67:18-24. Epub 2020 Nov 19.

Laboratory of Gene Regulation, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. Electronic address:

The mammalian globin gene clusters provide a paradigm for studying the relationship between genome structure and function. As blood stem cells undergo lineage specification and differentiation to form red blood cells, the chromatin structure and expression of the α-globin cluster change. The gradual activation of the α-globin genes in well-defined cell populations has enabled investigation of the structural and functional roles of its enhancers, promoters and boundary elements. Recent studies of gene regulatory processes involving these elements at the mouse α-globin cluster have brought new insights into the general principles underlying the three-dimensional structure of the genome and its relationship to gene expression throughout time.
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http://dx.doi.org/10.1016/j.gde.2020.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100094PMC
April 2021

Recapitulation of erythropoiesis in congenital dyserythropoietic anaemia type I (CDA-I) identifies defects in differentiation and nucleolar abnormalities.

Haematologica 2020 10 29;Online ahead of print. Epub 2020 Oct 29.

Weatherall Institute of Molecular Medicine, Oxford University, Oxford.

The investigation of inherited disorders of erythropoiesis has elucidated many of the principles underlying the production of normal red blood cells and how this is perturbed in human disease. Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is a rare form of anaemia caused by mutations in two genes of unknown function: CDAN1 and CDIN1 (previously called C15orf41), whilst in some cases, the underlying genetic abnormality is completely unknown. Consequently, the pathways affected in CDA-I remain to be discovered. To enable detailed analysis of this rare disorder we have validated a culture system which recapitulates all of the cardinal haematological features of CDA-I, including the formation of the pathognomonic 'spongy' heterochromatin seen by electron microscopy. Using a variety of cell and molecular biological approaches we discovered that erythroid cells in this condition show a delay during terminal erythroid differentiation, associated with increased proliferation and widespread changes in chromatin accessibility. We also show that the proteins encoded by CDAN1 and CDIN1 are enriched in nucleoli which are structurally and functionally abnormal in CDA-I. Together these findings provide important pointers to the pathways affected in CDA-I which for the first time can now be pursued in the tractable culture system utilised here.
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http://dx.doi.org/10.3324/haematol.2020.260158DOI Listing
October 2020

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

Cell Stem Cell 2020 11 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 03 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

An evolutionarily ancient mechanism for regulation of hemoglobin expression in vertebrate red cells.

Blood 2020 07;136(3):269-278

Department of Cell Biology, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands.

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.
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http://dx.doi.org/10.1182/blood.2020004826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531998PMC
July 2020

Enhancer-promoter interactions and transcription.

Authors:
Douglas R Higgs

Nat Genet 2020 05;52(5):470-471

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

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http://dx.doi.org/10.1038/s41588-020-0620-7DOI Listing
May 2020

An integrative view of the regulatory and transcriptional landscapes in mouse hematopoiesis.

Genome Res 2020 03 4;30(3):472-484. Epub 2020 Mar 4.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

Thousands of epigenomic data sets have been generated in the past decade, but it is difficult for researchers to effectively use all the data relevant to their projects. Systematic integrative analysis can help meet this need, and the VISION project was established for aldated ystematic ntegrati of epigenomic data in hematopoiesis. Here, we systematically integrated extensive data recording epigenetic features and transcriptomes from many sources, including individual laboratories and consortia, to produce a comprehensive view of the regulatory landscape of differentiating hematopoietic cell types in mouse. By using IDEAS as our ntegrative and iscriminative pigenome nnotation ystem, we identified and assigned epigenetic states simultaneously along chromosomes and across cell types, precisely and comprehensively. Combining nuclease accessibility and epigenetic states produced a set of more than 200,000 candidate -regulatory elements (cCREs) that efficiently capture enhancers and promoters. The transitions in epigenetic states of these cCREs across cell types provided insights into mechanisms of regulation, including decreases in numbers of active cCREs during differentiation of most lineages, transitions from poised to active or inactive states, and shifts in nuclease accessibility of CTCF-bound elements. Regression modeling of epigenetic states at cCREs and gene expression produced a versatile resource to improve selection of cCREs potentially regulating target genes. These resources are available from our VISION website to aid research in genomics and hematopoiesis.
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http://dx.doi.org/10.1101/gr.255760.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111515PMC
March 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

ATR-16 syndrome: mechanisms linking monosomy to phenotype.

J Med Genet 2020 06 31;57(6):414-421. Epub 2020 Jan 31.

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

Background: Deletions removing 100s-1000s kb of DNA, and variable numbers of poorly characterised genes, are often found in patients with a wide range of developmental abnormalities. In such cases, understanding the contribution of the deletion to an individual's clinical phenotype is challenging.

Methods: Here, as an example of this common phenomenon, we analysed 41 patients with simple deletions of ~177 to ~2000 kb affecting one allele of the well-characterised, gene dense, distal region of chromosome 16 (16p13.3), referred to as ATR-16 syndrome. We characterised deletion extents and screened for genetic background effects, telomere position effect and compensatory upregulation of hemizygous genes.

Results: We find the risk of developmental and neurological abnormalities arises from much smaller distal chromosome 16 deletions (~400 kb) than previously reported. Beyond this, the severity of ATR-16 syndrome increases with deletion size, but there is no evidence that critical regions determine the developmental abnormalities associated with this disorder. Surprisingly, we find no evidence of telomere position effect or compensatory upregulation of hemizygous genes; however, genetic background effects substantially modify phenotypic abnormalities.

Conclusions: Using ATR-16 as a general model of disorders caused by CNVs, we show the degree to which individuals with contiguous gene syndromes are affected is not simply related to the number of genes deleted but depends on their genetic background. We also show there is no critical region defining the degree of phenotypic abnormalities in ATR-16 syndrome and this has important implications for genetic counselling.
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http://dx.doi.org/10.1136/jmedgenet-2019-106528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279195PMC
June 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

Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of hematopoietic cells.

IUBMB Life 2020 01 25;72(1):27-38. Epub 2019 Nov 25.

Department of Hematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

Members of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model-driven, integrative fashion. We have formed the collaborative multi-lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for 20 cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions-along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis-regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for the regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.
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http://dx.doi.org/10.1002/iub.2195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972633PMC
January 2020

Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors.

Methods Protoc 2018 Jul 30;1(3). Epub 2018 Jul 30.

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

The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4-7 fold higher than starting cell numbers within 9-12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations.
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http://dx.doi.org/10.3390/mps1030028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481050PMC
July 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-Cell Proteomics Reveal that Quantitative Changes in Co-expressed Lineage-Specific Transcription Factors Determine Cell Fate.

Cell Stem Cell 2019 05 14;24(5):812-820.e5. Epub 2019 Mar 14.

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

Hematopoiesis provides an accessible system for studying the principles underlying cell-fate decisions in stem cells. Proposed models of hematopoiesis suggest that quantitative changes in lineage-specific transcription factors (LS-TFs) underlie cell-fate decisions. However, evidence for such models is lacking as TF levels are typically measured via RNA expression rather than by analyzing temporal changes in protein abundance. Here, we used single-cell mass cytometry and absolute quantification by mass spectrometry to capture the temporal dynamics of TF protein expression in individual cells during human erythropoiesis. We found that LS-TFs from alternate lineages are co-expressed, as proteins, in individual early progenitor cells and quantitative changes of LS-TFs occur gradually rather than abruptly to direct cell-fate decisions. Importantly, upregulation of a megakaryocytic TF in early progenitors is sufficient to deviate cells from an erythroid to a megakaryocyte trajectory, showing that quantitative changes in protein abundance of LS-TFs in progenitors can determine alternate cell fates.
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http://dx.doi.org/10.1016/j.stem.2019.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886472PMC
May 2019

Potential new approaches to the management of the Hb Bart's hydrops fetalis syndrome: the most severe form of α-thalassemia.

Hematology Am Soc Hematol Educ Program 2018 11;2018(1):353-360

Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom.

The α-thalassemia trait, associated with deletions removing both α-globin genes from 1 chromosome (genotype ζ αα/ζ--), is common throughout Southeast Asia. Consequently, many pregnancies in couples of Southeast Asian origin carry a 1 in 4 risk of producing a fetus inheriting no functional α-globin genes (ζ--/ζ--), leading to hemoglobin (Hb) Bart's hydrops fetalis syndrome (BHFS). Expression of the embryonic α-globin genes (ζ-globin) is normally limited to the early stages of primitive erythropoiesis, and so when the ζ-globin genes are silenced, at ∼6 weeks of gestation, there should be no α-like globin chains to pair with the fetal γ-globin chains of Hb, which consequently form nonfunctional tetramers (γ) known as Hb Bart's. When deletions leave the ζ-globin gene intact, a low level of ζ-globin gene expression continues in definitive erythroid cells, producing small amounts of Hb Portland (ζγ), a functional form of Hb that allows the fetus to survive up to the second or third trimester. Untreated, all affected individuals die at these stages of development. Prevention is therefore of paramount importance. With improvements in early diagnosis, intrauterine transfusion, and advanced perinatal care, there are now a small number of individuals with BHFS who have survived, with variable outcomes. A deeper understanding of the mechanism underlying the switch from ζ- to α-globin expression could enable persistence or reactivation of embryonic globin synthesis in definitive cells, thereby providing new therapeutic options for such patients.
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http://dx.doi.org/10.1182/asheducation-2018.1.353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6246003PMC
November 2018

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

How to Tackle Challenging ChIP-Seq, with Long-Range Cross-Linking, Using ATRX as an Example.

Methods Mol Biol 2018 ;1832:105-130

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

Chromatin immunoprecipitation coupled with high-throughput, next-generation DNA sequencing (ChIP-seq) has enabled researchers to establish the genome-wide patterns of chromatin modifications and binding of chromatin-associated proteins. Well-established protocols produce robust ChIP-seq data for many proteins by sequencing the DNA obtained following immunoprecipitation of fragmented chromatin using a wide range of specific antibodies. In general, the quality of these data mainly depends on the specificity and avidity of the antibody used. However, even using optimal antibodies, ChIP-seq can become more challenging when the protein associates with chromatin via protein-protein interactions rather than directly binding DNA. An example of such a protein is the alpha-thalassaemia mental retardation X-linked (ATRX) protein; a chromatin remodeler that associates with the histone chaperone DAXX, in the deposition of the replication-independent histone variant H3.3 and plays an important role in maintaining chromatin integrity. Inherited mutations of ATRX cause syndromal mental retardation (ATR-X Syndrome) whereas acquired mutations are associated with myelodysplasia, acute myeloid leukemia (ATMDS syndrome), and a range of solid tumors. Therefore, high quality ChIP-seq data have been needed to analyze the genome-wide distribution of ATRX, to advance our understanding of its normal role and to comprehend how mutations contribute to human disease. Here, we describe an optimized ChIP-seq protocol for ATRX which can also be used to produce high quality data sets for other challenging proteins which are indirectly associated with DNA and complement the ChIP-seq toolkit for genome-wide analyses of histone chaperon complexes and associated chromatin remodelers. Although not a focus of this chapter, we will also provide some insight for the analysis of the large dataset generated by ChIP-seq. Even though this protocol has been fully optimized for ATRX, it should also provide guidance for efficient ChIP-seq analysis, using the appropriate antibodies, for other proteins interacting indirectly with DNA.
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http://dx.doi.org/10.1007/978-1-4939-8663-7_6DOI Listing
April 2019

Molecular Basis and Genetic Modifiers of Thalassemia.

Hematol Oncol Clin North Am 2018 04;32(2):177-191

Molecular Hematology Unit, Medical Research Council (MRC), Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK; National Institute for Health Research, Oxford Biomedical Research Centre, Blood Theme, Oxford University Hospitals, Headington, Oxford OX3 9DU, UK. Electronic address:

Thalassemia is a disorder of hemoglobin characterized by reduced or absent production of one of the globin chains in human red blood cells with relative excess of the other. Impaired synthesis of β-globin results in β-thalassemia, whereas defective synthesis of α-globin leads to α-thalassemia. Despite being a monogenic disorder, thalassemia exhibits remarkable clinical heterogeneity that is directly related to the intracellular imbalance between α- and β-like globin chains. Novel insights into the genetic modifiers have contributed to the understanding of the correlation between genotype and phenotype and are being explored as therapeutic pathways to cure this life-limiting disease.
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http://dx.doi.org/10.1016/j.hoc.2017.11.003DOI Listing
April 2018

Phenotypic and molecular characterization of a serum-free miniature erythroid differentiation system suitable for high-throughput screening and single-cell assays.

Exp Hematol 2018 04 9;60:10-20. Epub 2018 Jan 9.

Medical Research Council (MRC) Molecular Hematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; Oxford National Institute for Health Research Biomedical Research Centre, Blood Theme, Oxford University Hospital, John Radcliffe Hospital, Headington, Oxford, United Kingdom. Electronic address:

In vitro erythroid differentiation systems are used to study the mechanisms underlying normal and abnormal erythropoiesis and to test the effects of various extracellular factors on erythropoiesis. The use of serum or conditioned medium in liquid cultures and the seeding of cultures with heterogeneous peripheral blood mononuclear cells confound the reproducibility of these systems. Newer erythroid differentiation culture systems have overcome some of these limitations by using a fully defined, serum-free medium and initiating cultures using purified CD34 cells. Although widely used in bulk cultures, these protocols have not been rigorously tested in high-throughput or single-cell assays. Here, we describe a serum-free erythroid differentiation system suitable for small-scale and single-cell experiments. This system generates large numbers of terminally differentiated erythroid cells of very high purity. Here we have adapted this culture system to a 96-well format and have developed a protocol to grow erythroid colonies from single erythroid progenitors in minute culture volumes.
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http://dx.doi.org/10.1016/j.exphem.2018.01.001DOI Listing
April 2018

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
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