Life Sciences Research for Lifelong Health

Publications bioinformatics

Title / Authors / Details Open Access Download

Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.
Schoenfelder S, Sugar R, Dimond A, Javierre BM, Armstrong H, Mifsud B, Dimitrova E, Matheson L, Tavares-Cadete F, Furlan-Magaril M, Segonds-Pichon A, Jurkowski W, Wingett SW, Tabbada K, Andrews S, Herman B, LeProust E, Osborne CS, Koseki H, Fraser P, Luscombe NM, Elderkin S

The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks. The strongest spatial network is composed of the four Hox gene clusters and early developmental transcription factor genes, the majority of which contact poised enhancers. Removal of Polycomb repression leads to disruption of promoter-promoter contacts in the Hox gene network. In contrast, promoter-enhancer contacts are maintained in the absence of Polycomb repression, with accompanying widespread acquisition of active chromatin signatures at network enhancers and pronounced transcriptional upregulation of network genes. Thus, PRC1 physically constrains developmental transcription factor genes and their enhancers in a silenced but poised spatial network. We propose that the selective release of genes from this spatial network underlies cell fate specification during early embryonic development.

+ View Abstract

Nature genetics, , 1546-1718, , 2015

PMID: 26323060

Open Access

Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression.
Strogantsev R, Krueger F, Yamazawa K, Shi H, Gould P, Goldman-Roberts M, McEwen K, Sun B, Pedersen R, Ferguson-Smith AC

Selective maintenance of genomic epigenetic imprints during pre-implantation development is required for parental origin-specific expression of imprinted genes. The Kruppel-like zinc finger protein ZFP57 acts as a factor necessary for maintaining the DNA methylation memory at multiple imprinting control regions in early mouse embryos and embryonic stem (ES) cells. Maternal-zygotic deletion of ZFP57 in mice presents a highly penetrant phenotype with no animals surviving to birth. Additionally, several cases of human transient neonatal diabetes are associated with somatic mutations in the ZFP57 coding sequence.

+ View Abstract

Genome biology, 16, 1474-760X, 112, 2015

PMID: 26025256

Open Access

Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C.
Mifsud B, Tavares-Cadete F, Young AN, Sugar R, Schoenfelder S, Ferreira L, Wingett SW, Andrews S, Grey W, Ewels PA, Herman B, Happe S, Higgs A, LeProust E, Follows GA, Fraser P, Luscombe NM, Osborne CS

Transcriptional control in large genomes often requires looping interactions between distal DNA elements, such as enhancers and target promoters. Current chromosome conformation capture techniques do not offer sufficiently high resolution to interrogate these regulatory interactions on a genomic scale. Here we use Capture Hi-C (CHi-C), an adapted genome conformation assay, to examine the long-range interactions of almost 22,000 promoters in 2 human blood cell types. We identify over 1.6 million shared and cell type-restricted interactions spanning hundreds of kilobases between promoters and distal loci. Transcriptionally active genes contact enhancer-like elements, whereas transcriptionally inactive genes interact with previously uncharacterized elements marked by repressive features that may act as long-range silencers. Finally, we show that interacting loci are enriched for disease-associated SNPs, suggesting how distal mutations may disrupt the regulation of relevant genes. This study provides new insights and accessible tools to dissect the regulatory interactions that underlie normal and aberrant gene regulation.

+ View Abstract

Nature genetics, , 1546-1718, , 2015

PMID: 25938943

Open Access

5-hydroxymethylcytosine marks promoters in colon that resist DNA hypermethylation in cancer.
Uribe-Lewis S, Stark R, Carroll T, Dunning MJ, Bachman M, Ito Y, Stojic L, Halim S, Vowler SL, Lynch AG, Delatte B, de Bony EJ, Colin L, Defrance M, Krueger F, Silva AL, Ten Hoopen R, Ibrahim AE, Fuks F, Murrell A

The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise.

+ View Abstract

Genome biology, 16, 1474-760X, 69, 2015

PMID: 25853800

Open Access

The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.
Schoenfelder S, Furlan-Magaril M, Mifsud B, Tavares-Cadete F, Sugar R, Javierre BM, Nagano T, Katsman Y, Sakthidevi M, Wingett SW, Dimitrova E, Dimond A, Edelman LB, Elderkin S, Tabbada K, Darbo E, Andrews S, Herman B, Higgs A, LeProust E, Osborne CS, Mitchell JA, Luscombe NM, Fraser P

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

+ View Abstract

Genome research, 25, 1549-5469, 582-97, 2015

PMID: 25752748

Open Access

Social parasitism and the molecular basis of phenotypic evolution.
Cini A, Patalano S, Segonds-Pichon A, Busby GB, Cervo R, Sumner S

Contrasting phenotypes arise from similar genomes through a combination of losses, gains, co-option and modifications of inherited genomic material. Understanding the molecular basis of this phenotypic diversity is a fundamental challenge in modern evolutionary biology. Comparisons of the genes and their expression patterns underlying traits in closely related species offer an unrivaled opportunity to evaluate the extent to which genomic material is reorganized to produce novel traits. Advances in molecular methods now allow us to dissect the molecular machinery underlying phenotypic diversity in almost any organism, from single-celled entities to the most complex vertebrates. Here we discuss how comparisons of social parasites and their free-living hosts may provide unique insights into the molecular basis of phenotypic evolution. Social parasites evolve from a eusocial ancestor and are specialized to exploit the socially acquired resources of their closely-related eusocial host. Molecular comparisons of such species pairs can reveal how genomic material is re-organized in the loss of ancestral traits (i.e., of free-living traits in the parasites) and the gain of new ones (i.e., specialist traits required for a parasitic lifestyle). We define hypotheses on the molecular basis of phenotypes in the evolution of social parasitism and discuss their wider application in our understanding of the molecular basis of phenotypic diversity within the theoretical framework of phenotypic plasticity and shifting reaction norms. Currently there are no data available to test these hypotheses, and so we also provide some proof of concept data using the paper wasp social parasite/host system (Polistes sulcifer-Polistes dominula). This conceptual framework and first empirical data provide a spring-board for directing future genomic analyses on exploiting social parasites as a route to understanding the evolution of phenotypic specialization.

+ View Abstract

Frontiers in genetics, 6, , 32, 2015

PMID: 25741361

Open Access

The RNA-binding protein HuR is essential for the B cell antibody response.
Diaz-Muñoz MD, Bell SE, Fairfax K, Monzon-Casanova E, Cunningham AF, Gonzalez-Porta M, Andrews SR, Bunik VI, Zarnack K, Curk T, Heggermont WA, Heymans S, Gibson GE, Kontoyiannis DL, Ule J, Turner M

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

+ View Abstract

Nature immunology, 16, 1529-2916, 415-25, 2015

PMID: 25706746

Open Access

Global Reorganization of the Nuclear Landscape in Senescent Cells.
Chandra T, Ewels PA, Schoenfelder S, Furlan-Magaril M, Wingett SW, Kirschner K, Thuret JY, Andrews S, Fraser P, Reik W

Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF). However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs), somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation.

+ View Abstract

Cell reports, , 2211-1247, , 2015

PMID: 25640177

Open Access

Finding Our Way through Phenotypes
Deans AR, Lewis SE, Huala E, Anzaldo SS, Ashburner M, Balhoff JP, Blackburn DC, Blake JA, Burleigh JG, Chanet B, Cooper LD, Courtot M, Csösz S, Cui H, Dahdul W, Das S, Dececchi TA, Dettai A, Diogo R, Druzinsky RE, Dumontier M, Franz NM, Friedrich F, Gkoutos GV, Haendel M, Harmon LJ, Hayamizu TF, He Y, Hines HM, Ibrahim N, Jackson LM, Jaiswal P, James-Zorn C, Köhler S, Lecointre G, Lapp H, Lawrence CJ, Le Novère N, Lundberg JG, Macklin J, Mast AR, Midford PE, Mikó I, Mungall CJ, Oellrich A, Osumi-Sutherland D, Parkinson H, Ramírez MJ, Richter S, Robinson PN, Ruttenberg A, Schulz KS, Segerdell E, Seltmann KC, Sharkey MJ, Smith AD, Smith B, Specht CD, Squires RB, Thacker RW, Thessen A, Fernandez-Triana J, Vihinen M, Vize PD, Vogt L, Wall CE, Walls RL, Westerfeld M, Wharton RA, Wirkner CS, Woolley JB, Yoder MJ, Zorn AM, Mabee P

Despite a large and multifaceted effort to understand the vast landscape of phenotypic data, their current form inhibits productive data analysis. The lack of a community-wide, consensus-based, human- and machine-interpretable language for describing phenotypes and their genomic and environmental contexts is perhaps the most pressing scientific bottleneck to integration across many key fields in biology, including genomics, systems biology, development, medicine, evolution, ecology, and systematics. Here we survey the current phenomics landscape, including data resources and handling, and the progress that has been made to accurately capture relevant data descriptions for phenotypes. We present an example of the kind of integration across domains that computable phenotypes would enable, and we call upon the broader biology community, publishers, and relevant funding agencies to support efforts to surmount today's data barriers and facilitate analytical reproducibility.

+ View Abstract

PLoS Biology, 13, 1, , 2015

PMID: 25562316

Open Access

Genome-wide bisulfite sequencing in zygotes identifies demethylation targets and maps the contribution of TET3 oxidation.
Peat JR, Dean W, Clark SJ, Krueger F, Smallwood SA, Ficz G, Kim JK, Marioni JC, Hore TA, Reik W

Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome. We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development. Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup.

+ View Abstract

Cell reports, 9, 2211-1247, 1990-2000, 2014

PMID: 25497087

Open Access

Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast.
F Cambuli, A Murray, W Dean, D Dudzinska, F Krueger, S Andrews, CE Senner, S Cook, M Hemberger

Embryonic (ES) and trophoblast (TS) stem cells reflect the first, irrevocable cell fate decision in development that is reinforced by distinct epigenetic lineage barriers. Nonetheless, ES cells can seemingly acquire TS-like characteristics upon manipulation of lineage-determining transcription factors or activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Here we have interrogated the progression of reprogramming in ES cell models with regulatable Oct4 and Cdx2 transgenes or conditional Erk1/2 activation. Although trans-differentiation into TS-like cells is initiated, lineage conversion remains incomplete in all models, underpinned by the failure to demethylate a small group of TS cell genes. Forced expression of these non-reprogrammed genes improves trans-differentiation efficiency, but still fails to confer a stable TS cell phenotype. Thus, even ES cells in ground-state pluripotency cannot fully overcome the boundaries that separate the first cell lineages but retain an epigenetic memory of their ES cell origin.

+ View Abstract

Nat Commun., 26, 5, 5538, 2014

PMID: 25423963

Open Access

The miR-155-PU.1 axis acts on Pax5 to enable efficient terminal B cell differentiation.
Lu D, Nakagawa R, Lazzaro S, Staudacher P, Abreu-Goodger C, Henley T, Boiani S, Leyland R, Galloway A, Andrews S, Butcher G, Nutt SL, Turner M, Vigorito E

A single microRNA (miRNA) can regulate the expression of many genes, though the level of repression imparted on any given target is generally low. How then is the selective pressure for a single miRNA/target interaction maintained across long evolutionary distances? We addressed this problem by disrupting in vivo the interaction between miR-155 and PU.1 in mice. Remarkably, this interaction proved to be key to promoting optimal T cell-dependent B cell responses, a previously unrecognized role for PU.1. Mechanistically, miR-155 inhibits PU.1 expression, leading to Pax5 down-regulation and the initiation of the plasma cell differentiation pathway. Additional PU.1 targets include a network of genes whose products are involved in adhesion, with direct links to B-T cell interactions. We conclude that the evolutionary adaptive selection of the miR-155-PU.1 interaction is exercised through the effectiveness of terminal B cell differentiation.

+ View Abstract

The Journal of experimental medicine, 211, 1540-9538, 2183-98, 2014

PMID: 25288398

Open Access

Complex spike patterns in olfactory bulb neuronal networks.
Nicol AU, Segonds-Pichon A, Magnusson MS

T-pattern analysis is a procedure developed for detecting non-randomly recurring hierarchical and multiordinal real-time sequential patterns (T-patterns).

+ View Abstract

Journal of neuroscience methods, 239, 1872-678X, 11-7, 2015

PMID: 25256643

Resetting transcription factor control circuitry toward ground-state pluripotency in human.
Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, Reik W, Bertone P, Smith A

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

+ View Abstract

Cell, 158, 1097-4172, 1254-69, 2014

PMID: 25215486

Open Access

Unbiased analysis of potential targets of breast cancer susceptibility loci by Capture Hi-C.
Dryden NH, Broome LR, Dudbridge F, Johnson N, Orr N, Schoenfelder S, Nagano T, Andrews S, Wingett S, Kozarewa I, Assiotis I, Fenwick K, Maguire SL, Campbell J, Natrajan R, Lambros M, Perrakis E, Ashworth A, Fraser P, Fletcher O

Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21, and 9q31.2. We identified interaction peaks between putative regulatory elements ("bait fragments") within the captured regions and "targets" that included both protein-coding genes and long noncoding (lnc) RNAs over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2, and MYC; and target lncRNAs included DIRC3, PVT1, and CCDC26. For one gene desert, we were able to define two SNPs (rs12613955 and rs4442975) that were highly correlated with the published risk variant and that mapped within the bait end of an interaction peak. In vivo ChIP-qPCR data show that one of these, rs4442975, affects the binding of FOXA1 and implicate this SNP as a putative functional variant.

+ View Abstract

Genome research, 24, 1549-5469, 1854-68, 2014

PMID: 25122612

Open Access

Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity.
Smallwood SA,Lee HJ,Angermueller C,Krueger F,Saadeh H,Peat J,Andrews SR,Stegle O,Reik W,Kelsey G

We report a single-cell bisulfite sequencing (scBS-seq) method that can be used to accurately measure DNA methylation at up to 48.4% of CpG sites. Embryonic stem cells grown in serum or in 2i medium displayed epigenetic heterogeneity, with '2i-like' cells present in serum culture. Integration of 12 individual mouse oocyte datasets largely recapitulated the whole DNA methylome, which makes scBS-seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

+ View Abstract

Nature methods, 11, 1548-7105, 817-20, 2014

PMID: 25042786

Open Access

Olfactory bulb encoding during learning under anesthesia.
Nicol AU, Sanchez-Andrade G, Collado P, Segonds-Pichon A, Kendrick KM

Neural plasticity changes within the olfactory bulb are important for olfactory learning, although how neural encoding changes support new associations with specific odors and whether they can be investigated under anesthesia, remain unclear. Using the social transmission of food preference olfactory learning paradigm in mice in conjunction with in vivo microdialysis sampling we have shown firstly that a learned preference for a scented food odor smelled on the breath of a demonstrator animal occurs under isofluorane anesthesia. Furthermore, subsequent exposure to this cued odor under anesthesia promotes the same pattern of increased release of glutamate and gamma-aminobutyric acid (GABA) in the olfactory bulb as previously found in conscious animals following olfactory learning, and evoked GABA release was positively correlated with the amount of scented food eaten. In a second experiment, multiarray (24 electrodes) electrophysiological recordings were made from olfactory bulb mitral cells under isofluorane anesthesia before, during and after a novel scented food odor was paired with carbon disulfide. Results showed significant increases in overall firing frequency to the cued-odor during and after learning and decreases in response to an uncued odor. Analysis of patterns of changes in individual neurons revealed that a substantial proportion (>50%) of them significantly changed their response profiles during and after learning with most of those previously inhibited becoming excited. A large number of cells exhibiting no response to the odors prior to learning were either excited or inhibited afterwards. With the uncued odor many previously responsive cells became unresponsive or inhibited. Learning associated changes only occurred in the posterior part of the olfactory bulb. Thus olfactory learning under anesthesia promotes extensive, but spatially distinct, changes in mitral cell networks to both cued and uncued odors as well as in evoked glutamate and GABA release.

+ View Abstract

Frontiers in behavioral neuroscience, 8, , 193, 2014

PMID: 24926241

Open Access

The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues.
AK Johnsson, Y Dai, M Nobis, MJ Baker, EJ McGhee, S Walker, JP Schwarz, S Kadir, JP Morton, KB Myant, DJ Huels, A Segonds-Pichon, OJ Sansom, KI Anderson, P Timpson, HC Welch

The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.

+ View Abstract

Cell reports, 6, 6, 1153-64, 2014

PMID: 24630994
DOI: 10.1016/j.celrep.2014.02.024

Open Access

A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation.
Iurlaro M, Ficz G, Oxley D, Raiber EA, Bachman M, Booth MJ, Andrews S, Balasubramanian S, Reik W

DNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts.

+ View Abstract

Genome biology, 14, 1465-6914, R119, 2013

PMID: 24156278

Open Access

FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency.
G Ficz, TA Hore, F Santos, HJ Lee, W Dean, J Arand, F Krueger, D Oxley, YL Paul, J Walter, SJ Cook, S Andrews, MR Branco, W Reik

Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

+ View Abstract

Cell stem cell, 13, 3, 351-9, 2013

PMID: 23850245
DOI: 10.1016/j.stem.2013.06.004

Open Access

Detailed molecular characterisation of acute myeloid leukaemia with a normal karyotype using targeted DNA capture.
Conte N, Varela I, Grove C, Manes N, Yusa K, Moreno T, Segonds-Pichon A, Bench A, Gudgin E, Herman B, Bolli N, Ellis P, Haddad D, Costeas P, Rad R, Scott M, Huntly B, Bradley A, Vassiliou GS

Advances in sequencing technologies are giving unprecedented insights into the spectrum of somatic mutations underlying acute myeloid leukaemia with a normal karyotype (AML-NK). It is clear that the prognosis of individual patients is strongly influenced by the combination of mutations in their leukaemia and that many leukaemias are composed of multiple subclones, with differential susceptibilities to treatment. Here, we describe a method, employing targeted capture coupled with next-generation sequencing and tailored bioinformatic analysis, for the simultaneous study of 24 genes recurrently mutated in AML-NK. Mutational analysis was performed using open source software and an in-house script (Mutation Identification and Analysis Software), which identified dominant clone mutations with 100% specificity. In each of seven cases of AML-NK studied, we identified and verified mutations in 2-4 genes in the main leukaemic clone. Additionally, high sequencing depth enabled us to identify putative subclonal mutations and detect leukaemia-specific mutations in DNA from remission marrow. Finally, we used normalised read depths to detect copy number changes and identified and subsequently verified a tandem duplication of exons 2-9 of MLL and at least one deletion involving PTEN. This methodology reliably detects sequence and copy number mutations, and can thus greatly facilitate the classification, clinical research, diagnosis and management of AML-NK.

+ View Abstract

Leukemia, 27, 1476-5551, 1820-5, 2013

PMID: 23702683

Open Access

Deficiency in spliceosome-associated factor CTNNBL1 does not affect ongoing cell cycling but delays exit from quiescence and results in embryonic lethality in mice.
Chandra A, van Maldegem F, Andrews S, Neuberger MS, Rada C

CTNNBL1 is an armadillo-repeat protein that associates with the CDC5L/Prp19 complex of the spliceosome. Unlike the majority of spliceosomal proteins (and despite having no obvious homologs), CTNNBL1 is inessential for cell viability as revealed by studies in both vertebrate B cell lines and in fission yeast. Here, however, we show that ablation of CTNNBL1 in the mouse germline results in mid-gestation embryonic lethality but that lineage-specific CTNNBL1 ablation in early B cell precursors does not affect the production and abundance of mature B lymphocytes. However, CTNNBL1-deficient resting B lymphocytes show sluggish exit from quiescence on cell activation, although once entry into cycle has initiated, proliferation and differentiation in response to mitogenic stimuli continue largely unaffected. A similar sluggish exit from quiescence is also observed on reprovision of nutrients to nitrogen-starved CTNNBL1-deficient yeast. The results indicate that, whereas other RNA splicing-associated factors have been connected to cell cycle progression, CTNNBL1 plays no essential role in cycling cells but does fulfill an evolutionarily conserved function in helping cells to undergo efficient exit from quiescence following activation.

+ View Abstract

Cell cycle (Georgetown, Tex.), 12, 1551-4005, 732-42, 2013

PMID: 23343763

Open Access

Base-pair resolution DNA methylome of the EBV-positive Endemic Burkitt lymphoma cell line DAUDI determined by SOLiD bisulfite-sequencing.
Kreck B, Richter J, Ammerpohl O, Barann M, Esser D, Petersen BS, Vater I, Murga Penas EM, Bormann Chung CA, Seisenberger S, Lee Boyd V, Smallwood S, Drexler HG, Macleod RA, Hummel M, Krueger F, Häsler R, Schreiber S, Rosenstiel P, Franke A, Siebert R

Leukemia, 27, 1476-5551, 1751-3, 2013

PMID: 23307032

Open Access

DNA methylation profiles define stem cell identity and reveal a tight embryonic-extraembryonic lineage boundary.
CE Senner, F Krueger, D Oxley, S Andrews, M Hemberger

Embryonic (ES) and epiblast (EpiSC) stem cells are pluripotent but committed to an embryonic lineage fate. Conversely, trophoblast (TS) and extraembryonic endoderm (XEN) stem cells contribute predominantly to tissues of the placenta and yolk sac, respectively. Here we show that each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct developmental origin, TS and XEN cells share key epigenomic hallmarks, chiefly characterized by robust DNA methylation of embryo-specific developmental regulators, as well as a subordinate role of 5-hydroxymethylation. We also observe a substantial methylation reinforcement of pre-existing epigenetic repressive marks that specifically occurs in extraembryonic stem cells compared to in vivo tissue, presumably due to continued high Dnmt3b expression levels. These differences establish a major epigenetic barrier between the embryonic and extraembryonic stem cell types. In addition, epigenetic lineage boundaries also separate the two extraembryonic stem cell types by mutual repression of key lineage-specific transcription factors. Thus, global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells. Our detailed methylation profiles identify a cohort of developmentally regulated sequence elements, such as orphan CpG islands, that will be most valuable to uncover novel transcriptional regulators and pivotal "gatekeeper" genes in pluripotency and lineage differentiation.

+ View Abstract

Stem cells (Dayton, Ohio), 30, 12, 2732-45, 2012

PMID: 23034951
DOI: 10.1002/stem.1249

Open Access

Large scale loss of data in low-diversity illumina sequencing libraries can be recovered by deferred cluster calling.
F Krueger, SR Andrews, CS Osborne

Massively parallel DNA sequencing is capable of sequencing tens of millions of DNA fragments at the same time. However, sequence bias in the initial cycles, which are used to determine the coordinates of individual clusters, causes a loss of fidelity in cluster identification on Illumina Genome Analysers. This can result in a significant reduction in the numbers of clusters that can be analysed. Such low sample diversity is an intrinsic problem of sequencing libraries that are generated by restriction enzyme digestion, such as e4C-seq or reduced-representation libraries. Similarly, this problem can also arise through the combined sequencing of barcoded, multiplexed libraries. We describe a procedure to defer the mapping of cluster coordinates until low-diversity sequences have been passed. This simple procedure can recover substantial amounts of next generation sequencing data that would otherwise be lost.

+ View Abstract

PloS one, 6, 1, e16607, 2011

PMID: 21305042
DOI: 10.1371/journal.pone.0016607