Publications by authors named "Jelena Telenius"

26 Publications

  • Page 1 of 1

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

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

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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610627PMC
November 2020

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

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

Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo.

Dev Cell 2019 10;51(2):255-276.e7

University of Oxford, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford OX3 9DS, UK. Electronic address:

Precise control of developmental processes is encoded in the genome in the form of gene regulatory networks (GRNs). Such multi-factorial systems are difficult to decode in vertebrates owing to their complex gene hierarchies and dynamic molecular interactions. Here we present a genome-wide in vivo reconstruction of the GRN underlying development of the multipotent neural crest (NC) embryonic cell population. By coupling NC-specific epigenomic and transcriptional profiling at population and single-cell levels with genome/epigenome engineering in vivo, we identify multiple regulatory layers governing NC ontogeny, including NC-specific enhancers and super-enhancers, novel trans-factors, and cis-signatures allowing reverse engineering of the NC-GRN at unprecedented resolution. Furthermore, identification and dissection of divergent upstream combinatorial regulatory codes has afforded new insights into opposing gene circuits that define canonical and neural NC fates early during NC ontogeny. Our integrated approach, allowing dissection of cell-type-specific regulatory circuits in vivo, has broad implications for GRN discovery and investigation.
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http://dx.doi.org/10.1016/j.devcel.2019.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838682PMC
October 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

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

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

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

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

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

Genetic dissection of the α-globin super-enhancer in vivo.

Nat Genet 2016 08 4;48(8):895-903. Epub 2016 Jul 4.

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

Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5058437PMC
http://dx.doi.org/10.1038/ng.3605DOI Listing
August 2016

Multiplexed analysis of chromosome conformation at vastly improved sensitivity.

Nat Methods 2016 Jan 23;13(1):74-80. Epub 2015 Nov 23.

Medical Research Council, Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford, UK.

Methods for analyzing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging. In next-generation (NG) Capture-C, we have redesigned the Capture-C method to achieve unprecedented levels of sensitivity and reproducibility. NG Capture-C can be used to analyze many genetic loci and samples simultaneously. High-resolution data can be produced with as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. The method is straightforward to perform and should greatly facilitate the investigation of many questions related to gene regulation as well as the functional dissection of traits examined in genome-wide association studies.
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http://dx.doi.org/10.1038/nmeth.3664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724891PMC
January 2016

Molecular dynamics simulations of the interactions of medicinal plant extracts and drugs with lipid bilayer membranes.

FEBS J 2013 Jun 16;280(12):2785-805. Epub 2013 May 16.

MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark.

Several small drugs and medicinal plant extracts, such as the Indian spice extract curcumin, have a wide range of useful pharmacological properties that cannot be ascribed to binding to a single protein target alone. The lipid bilayer membrane is thought to mediate the effects of many such molecules directly via perturbation of the plasma membrane structure and dynamics, or indirectly by modulating transmembrane protein conformational equilibria. Furthermore, for bioavailability, drugs must interact with and eventually permeate the lipid bilayer barrier on the surface of cells. Biophysical studies of the interactions of drugs and plant extracts are therefore of interest. Molecular dynamics simulations, which can access time and length scales that are not simultaneously accessible by other experimental methods, are often used to obtain quantitative molecular and thermodynamic descriptions of these interactions, often with complementary biophysical measurements. This review considers recent molecular dynamics simulations of small drug-like molecules with membranes, and provides a biophysical description of possible routes of membrane-mediated pharmacological effects of drugs. The review is not exhaustive, and we focus on molecules containing aromatic ring-like structures to develop our hypotheses. We also show that some drugs and anesthetics may have an effect on the lipid bilayer analogous to that of cholesterol.
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http://dx.doi.org/10.1111/febs.12286DOI Listing
June 2013

Role of neutral lipids in tear fluid lipid layer: coarse-grained simulation study.

Langmuir 2012 Dec 26;28(49):17092-100. Epub 2012 Nov 26.

Department of Applied Physics, Aalto University School of Science, Finland.

Tear fluid lipid layer (TFLL) residing at the air-water interface of tears has been recognized to play an important role in the development of dry eye syndrome. Yet, the composition, structure, and mechanical properties of TFLL are only partly known. Here, we report results of coarse-grained simulations of a lipid layer comprising phospholipids, free fatty acids, cholesteryl esters, and triglycerides at the air-water interface to shed light on the properties of TFLL. We consider structural as well as dynamical properties of the lipid layer as a function of surface pressure. Simulations revealed that neutral lipids reside heterogeneously between phospholipids at relatively low pressures but form a separate hydrophobic phase with increasing surface pressure, transforming the initial lipid monolayer to a two-layered structure. When the model of TFLL was compared to a one-component phospholipid monolayer system, we found drastic differences in both structural and dynamical properties that explain the prominent role of neutral lipids as stabilizers of the TFLL. Based on our results, we suggest that neutral lipids are able to increase the stability of the TFLL by modulating its dynamical and structural behavior, which is important for the proper function of tear film.
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http://dx.doi.org/10.1021/la304366dDOI Listing
December 2012

Lessons from the biophysics of interfaces: lung surfactant and tear fluid.

Prog Retin Eye Res 2011 May 23;30(3):204-15. Epub 2011 Feb 23.

Helsinki Eye Lab, Department of Ophthalmology, University of Helsinki, Helsinki, Finland.

The purpose of this review is to provide insight into the biophysical properties and functions of tear fluid and lung surfactant--two similar fluids covering the epithelium of two distinctive organs. Both fluids form a layer-like structure that essentially comprise of an aqueous layer next to the epithelium and an anterior lipid layer at the air-water interface. The aqueous layers contain soluble proteins and metabolites, and they are responsible for the host defence system and nutrition of the organ. However, many proteins also interact with the lipid layer and are important for the surface-active function of the fluid film. The lipid layer of lung surfactant comprises mainly of phospholipids, especially phosphatidylcholines, and only small amounts of non-polar lipids, mainly cholesterol. In contrast, tear fluid lipid layer comprises of a mixture of polar and non-polar lipids. However, the relative proportion and the spectrum of different polar and non-polar lipids seem to be more extensive in tear fluid than in lung surfactant. The differing lipid compositions generate distinctive lipid layer structures. Despite the structural differences, these lipid layers decrease the surface tension of the air-water interface. The structure of the tear film lipid layer also minimises the evaporation of the tear fluid. In lung surfactant surface activity is crucial for the function of the organ, as the lipid layer prevents the collapse of the lung alveoli during the compression-expansion cycle of breathing. Similarly the tear film experiences a compression-expansion cycle during blinking. The dynamics of this cycle have been studied to a lesser extent and are not as clear as those of lung surfactant. The common structure and properties suggest a similar behaviour under rapid compression-expansion for both fluids.
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http://dx.doi.org/10.1016/j.preteyeres.2011.02.002DOI Listing
May 2011

Molecular organization of the tear fluid lipid layer.

Biophys J 2010 Oct;99(8):2559-67

Department of Ophthalmology, University of Helsinki, Finland.

The tear fluid protects the corneal epithelium from drying out as well as from invasion by pathogens. It also provides cell nutrients. Similarly to lung surfactant, it is composed of an aqueous phase covered by a lipid layer. Here we describe the molecular organization of the anterior lipid layer of the tear film. Artificial tear fluid lipid layers (ATFLLs) composed of egg yolk phosphatidylcholine (60 mol %), free fatty acids (20 mol %), cholesteryl oleate (10 mol %), and triglycerides (10 mol %) were deposited on the air-water interface and their physico-chemical behavior was compared to egg-yolk phosphatidylcholine monolayers by using Langmuir-film balance techniques, x-ray diffraction, and imaging techniques as well as in silico molecular level simulations. At low surface pressures, ATFLLs were organized at the air-water interface as heterogeneous monomolecular films. Upon compression the ATFLLs collapsed toward the air phase and formed hemispherelike lipid aggregates. This transition was reversible upon relaxation. These results were confirmed by molecular-level simulations of ATFLL, which further provided molecular-scale insight into the molecular distributions inside and dynamics of the tear film. Similar type of behavior is observed in lung surfactant but the folding takes place toward the aqueous phase. The results provide novel information of the function of lipids in the tear fluid.
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http://dx.doi.org/10.1016/j.bpj.2010.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956218PMC
October 2010

Visualization of complex processes in lipid systems using computer simulations and molecular graphics.

Methods Mol Biol 2009 ;580:317-38

Department of Applied Physics, Helsinki University of Technology, Espoo, Finland.

Computer simulation has become an increasingly popular tool in the study of lipid membranes, complementing experimental techniques by providing information on structure and dynamics at high spatial and temporal resolution. Molecular visualization is the most powerful way to represent the results of molecular simulations, and can be used to illustrate complex transformations of lipid aggregates more easily and more effectively than written text. In this chapter, we review some basic aspects of simulation methodologies commonly employed in the study of lipid membranes and we describe a few examples of complex phenomena that have been recently investigated using molecular simulations. We then explain how molecular visualization provides added value to computational work in the field of biological membranes, and we conclude by listing a few molecular graphics packages widely used in scientific publications.
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http://dx.doi.org/10.1007/978-1-60761-325-1_18DOI Listing
January 2010

Structural basis of mechanochemical coupling in a hexameric molecular motor.

J Biol Chem 2008 Feb 5;283(6):3607-3617. Epub 2007 Dec 5.

Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Viikki Biocenter P. O. Box 65, Helsinki FIN-00014, Finland. Electronic address:

The P4 protein of bacteriophage phi12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small approximately 1 A changes in the ATP-binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at the atomic level. Here we study in atomic detail the mechanochemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, consisting of superfamily 4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNA-binding site that moves along the mechanical reaction coordinate. The lever is flanked by gamma-phosphate sensors (Asn-234 and Ser-252) that report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg-279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This ensures cooperative sequential hydrolysis that is tightly coupled to mechanical motion. Given the structural conservation, the mutated residues may play similar roles in other hexameric helicases and related molecular motors.
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http://dx.doi.org/10.1074/jbc.M706366200DOI Listing
February 2008
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