Life Sciences Research for Lifelong Health


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Title / Authors / Details Open Access Download

Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system.
Bruno L, Ramlall V, Studer RA, Sauer S, Bradley D, Dharmalingam G, Carroll T, Ghoneim M, Chopin M, Nutt SL, Elderkin S, Rueda DS, Fisher AG, Siggers T, Beltrao P, Merkenschlager M

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

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Nature immunology, 20, 1529-2916, 1372-1380, 2019

PMID: 31451789

NAD cleavage activity by animal and plant TIR domains in cell death pathways.
Horsefield S, Burdett H, Zhang X, Manik MK, Shi Y, Chen J, Qi T, Gilley J, Lai JS, Rank MX, Casey LW, Gu W, Ericsson DJ, Foley G, Hughes RO, Bosanac T, von Itzstein M, Rathjen JP, Nanson JD, Boden M, Dry IB, Williams SJ, Staskawicz BJ, Coleman MP, Ve T, Dodds PN, Kobe B

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

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Science (New York, N.Y.), 365, 1095-9203, 793-799, 2019

PMID: 31439792

Epigenetic Regulation of Vascular Smooth Muscle Cells by Histone H3 Lysine 9 Dimethylation Attenuates Target Gene-Induction by Inflammatory Signaling.
Harman JL, Dobnikar L, Chappell J, Stokell BG, Dalby A, Foote K, Finigan A, Freire-Pritchett P, Taylor AL, Worssam MD, Madsen RR, Loche E, Uryga A, Bennett MR, Jørgensen HF

Vascular inflammation underlies cardiovascular disease. Vascular smooth muscle cells (VSMCs) upregulate selective genes, including MMPs (matrix metalloproteinases) and proinflammatory cytokines upon local inflammation, which directly contribute to vascular disease and adverse clinical outcome. Identification of factors controlling VSMC responses to inflammation is therefore of considerable therapeutic importance. Here, we determine the role of Histone H3 lysine 9 di-methylation (H3K9me2), a repressive epigenetic mark that is reduced in atherosclerotic lesions, in regulating the VSMC inflammatory response. Approach and Results: We used VSMC-lineage tracing to reveal reduced H3K9me2 levels in VSMCs of arteries after injury and in atherosclerotic lesions compared with control vessels. Intriguingly, chromatin immunoprecipitation showed H3K9me2 enrichment at a subset of inflammation-responsive gene promoters, including MMP3, MMP9, MMP12, and IL6, in mouse and human VSMCs. Inhibition of G9A/GLP, the primary enzymes responsible for H3K9me2, significantly potentiated inflammation-induced gene induction in vitro and in vivo without altering NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell) and MAPK signaling. Rather, reduced G9A/GLP activity enhanced inflammation-induced binding of transcription factors NFκB-p65 and cJUN to H3K9me2 target gene promoters MMP3 and IL6. Taken together, these results suggest that promoter-associated H3K9me2 directly attenuates the induction of target genes in response to inflammation in human VSMCs.

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Arteriosclerosis, thrombosis, and vascular biology, , 1524-4636, ATVBAHA119312765, 2019

PMID: 31434493

A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites.
McCormick B, Craig HE, Chu JY, Carlin LM, Canel M, Wollweber F, Toivakka M, Michael M, Astier AL, Norton L, Lilja J, Felton JM, Sasaki T, Ivaska J, Hers I, Dransfield I, Rossi AG, Vermeren S

Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.

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Journal of immunology (Baltimore, Md. : 1950), 203, 1550-6606, 1579-1588, 2019

PMID: 31427445

Open Access

An intergenic non-coding RNA promoter required for histone modifications in the human β-globin chromatin domain.
Debrand E, Chakalova L, Miles J, Dai YF, Goyenechea B, Dye S, Osborne CS, Horton A, Harju-Baker S, Pink RC, Caley D, Carter DRF, Peterson KR, Fraser P

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human β-globin genes. Mutational analyses in β-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult δ- and β-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring ε- and γ-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of β-like globin gene transcription during development.

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PloS one, 14, 1932-6203, e0217532, 2019

PMID: 31412036

Open Access

Aldosterone, STX and amyloid-β peptides modulate GPER (GPR30) signalling in an embryonic mouse hippocampal cell line (mHippoE-18).
Evans PD

The GPCR, GPER, mediates many of the rapid, non-genomic actions of 17β-estradiol in multiple tissues, including the nervous system. Controversially, it has also been suggested to be activated by aldosterone, and by the non-steroidal diphenylacrylamide compound, STX, in some preparations. Here, the ability of the GPER agonist, G-1, and aldosterone in the presence of the mineralocorticoid receptor antagonist, eplerenone, to potentiate forskolin-stimulated cyclic AMP levels in the hippocampal clonal cell line, mHippoE-18, are compared. Both stimulatory effects are blocked by the GPER antagonist G36, by PTX, (suggesting the involvement of Gi/o G proteins), by BAPTA-AM, (suggesting they are calcium sensitive), by wortmannin (suggesting an involvement of PI3Kinase) and by soluble amyloid-β peptides. STX also stimulates cyclic AMP levels in mHippoE-18 cells and these effects are blocked by G36 and PTX, as well as by amyloid-β peptides. This suggests that both aldosterone and STX may modulate GPER signalling in mHippoE-18 cells.

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Molecular and cellular endocrinology, 496, 1872-8057, 110537, 2019

PMID: 31404576

Open Access

ZFP57 regulation of transposable elements and gene expression within and beyond imprinted domains.
Shi H, Strogantsev R, Takahashi N, Kazachenka A, Lorincz MC, Hemberger M, Ferguson-Smith AC

KRAB zinc finger proteins (KZFPs) represent one of the largest families of DNA-binding proteins in vertebrate genomes and appear to have evolved to silence transposable elements (TEs) including endogenous retroviruses through sequence-specific targeting of repressive chromatin states. ZFP57 is required to maintain the post-fertilization DNA methylation memory of parental origin at genomic imprints. Here we conduct RNA-seq and ChIP-seq analyses in normal and ZFP57 mutant mouse ES cells to understand the relative importance of ZFP57 at imprints, unique and repetitive regions of the genome.

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Epigenetics & chromatin, 12, 1756-8935, 49, 2019

PMID: 31399135

Open Access

Transcriptome analysis identifies a robust gene expression program in the mouse intestinal epithelium on aging.
Kazakevych J, Stoyanova E, Liebert A, Varga-Weisz P

The intestinal epithelium undergoes constant regeneration driven by intestinal stem cells. How old age affects the transcriptome in this highly dynamic tissue is an important, but poorly explored question. Using transcriptomics on sorted intestinal stem cells and adult enterocytes, we identified candidate genes, which change expression on aging. Further validation of these on intestinal epithelium of multiple middle-aged versus old-aged mice highlighted the consistent up-regulation of the expression of the gene encoding chemokine receptor Ccr2, a mediator of inflammation and several disease processes. We observed also increased expression of Strc, coding for stereocilin, and dramatically decreased expression of Rps4l, coding for a ribosome subunit. Ccr2 and Rps4l are located close to the telomeric regions of chromosome 9 and 6, respectively. As only few genes were differentially expressed and we did not observe significant protein level changes of identified ageing markers, our analysis highlights the overall robustness of murine intestinal epithelium gene expression to old age.

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Scientific reports, 9, 2045-2322, 10410, 2019

PMID: 31320724

Open Access

RNA proximity sequencing reveals the spatial organization of the transcriptome in the nucleus.
Morf J, Wingett SW, Farabella I, Cairns J, Furlan-Magaril M, Jiménez-García LF, Liu X, Craig FF, Walker S, Segonds-Pichon A, Andrews S, Marti-Renom MA, Fraser P

The global, three-dimensional organization of RNA molecules in the nucleus is difficult to determine using existing methods. Here we introduce Proximity RNA-seq, which identifies colocalization preferences for pairs or groups of nascent and fully transcribed RNAs in the nucleus. Proximity RNA-seq is based on massive-throughput RNA barcoding of subnuclear particles in water-in-oil emulsion droplets, followed by cDNA sequencing. Our results show RNAs of varying tissue-specificity of expression, speed of RNA polymerase elongation and extent of alternative splicing positioned at varying distances from nucleoli. The simultaneous detection of multiple RNAs in proximity to each other distinguishes RNA-dense from sparse compartments. Application of Proximity RNA-seq will facilitate study of the spatial organization of transcripts in the nucleus, including non-coding RNAs, and its functional relevance.

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Nature biotechnology, 37, 1546-1696, 793-802, 2019

PMID: 31267103

Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes.
Miguel-Escalada I, Bonàs-Guarch S, Cebola I, Ponsa-Cobas J, Mendieta-Esteban J, Atla G, Javierre BM, Rolando DMY, Farabella I, Morgan CC, García-Hurtado J, Beucher A, Morán I, Pasquali L, Ramos-Rodríguez M, Appel EVR, Linneberg A, Gjesing AP, Witte DR, Pedersen O, Grarup N, Ravassard P, Torrents D, Mercader JM, Piemonti L, Berney T, de Koning EJP, Kerr-Conte J, Pattou F, Fedko IO, Groop L, Prokopenko I, Hansen T, Marti-Renom MA, Fraser P, Ferrer J

Genetic studies promise to provide insight into the molecular mechanisms underlying type 2 diabetes (T2D). Variants associated with T2D are often located in tissue-specific enhancer clusters or super-enhancers. So far, such domains have been defined through clustering of enhancers in linear genome maps rather than in three-dimensional (3D) space. Furthermore, their target genes are often unknown. We have created promoter capture Hi-C maps in human pancreatic islets. This linked diabetes-associated enhancers to their target genes, often located hundreds of kilobases away. It also revealed >1,300 groups of islet enhancers, super-enhancers and active promoters that form 3D hubs, some of which show coordinated glucose-dependent activity. We demonstrate that genetic variation in hubs impacts insulin secretion heritability, and show that hub annotations can be used for polygenic scores that predict T2D risk driven by islet regulatory variants. Human islet 3D chromatin architecture, therefore, provides a framework for interpretation of T2D genome-wide association study (GWAS) signals.

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Nature genetics, 51, 1546-1718, 1137-1148, 2019

PMID: 31253982

Open Access

Common and distinct transcriptional signatures of mammalian embryonic lethality.
Collins JE, White RJ, Staudt N, Sealy IM, Packham I, Wali N, Tudor C, Mazzeo C, Green A, Siragher E, Ryder E, White JK, Papatheodoru I, Tang A, Füllgrabe A, Billis K, Geyer SH, Weninger WJ, Galli A, Hemberger M, Stemple DL, Robertson E, Smith JC, Mohun T, Adams DJ, Busch-Nentwich EM

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.

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Nature communications, 10, 2041-1723, 2792, 2019

PMID: 31243271

Open Access

Fetal and trophoblast PI3K p110α have distinct roles in regulating resource supply to the growing fetus in mice.
López-Tello J, Pérez-García V, Khaira J, Kusinski LC, Cooper WN, Andreani A, Grant I, Fernández de Liger E, Lam BY, Hemberger M, Sandovici I, Constancia M, Sferruzzi-Perri AN

Studies suggest that placental nutrient supply adapts according to fetal demands. However, signaling events underlying placental adaptations remain unknown. Here we demonstrate that phosphoinositide 3-kinase p110α in the fetus and the trophoblast interplay to regulate placental nutrient supply and fetal growth. Complete loss of fetal p110α caused embryonic death, whilst heterozygous loss resulted in fetal growth restriction and impaired placental formation and nutrient transport. Loss of trophoblast p110α resulted in viable fetuses, abnormal placental development and a failure of the placenta to transport sufficient nutrients to match fetal demands for growth. Using RNA-seq we identified genes downstream of p110α in the trophoblast that are important in adapting placental phenotype. Using CRISPR/Cas9 we showed loss of p110α differentially affects gene expression in trophoblast and embryonic stem cells. Our findings reveal important, but distinct roles for p110α in the different compartments of the conceptus, which control fetal resource acquisition and growth.

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eLife, 8, 2050-084X, , 2019

PMID: 31241463

Open Access

Inhibition of Phosphoinositide-3-Kinase Signaling Promotes the Stem Cell State of Trophoblast.
Lee CQE, Bailey A, Lopez-Tello J, Sferruzzi-Perri AN, Okkenhaug K, Moffett A, Rossant J, Hemberger M

Trophoblast stem cells (TSCs) are a heterogeneous cell population despite the presence of fibroblast growth factor (FGF) and transforming growth factor β (TGFB) as key growth factors in standard culture conditions. To understand what other signaling cascades control the stem cell state of mouse TSCs, we performed a kinase inhibitor screen and identified several novel pathways that cause TSC differentiation. Surprisingly, inhibition of phosphoinositide-3-kinase (PI3K) signaling increased the mRNA and protein expression of stem cell markers instead, and resulted in a tighter epithelial colony morphology and fewer differentiated cells. PI3K inhibition could not substitute for FGF or TGFB and did not affect phosphorylation of extracellular signal-regulated kinase, and thus acts independently of these pathways. Upon removal of PI3K inhibition, TSC transcription factor levels reverted to normal TSC levels, indicating that murine TSCs can reversibly switch between these two states. In summary, PI3K inhibition reduces the heterogeneity and seemingly heightens the stem cell state of TSCs as indicated by the simultaneous upregulation of multiple key marker genes and cell morphology. Stem Cells 2019;37:1307-1318.

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Stem cells (Dayton, Ohio), 37, 1549-4918, 1307-1318, 2019

PMID: 31233251

Chicdiff: a computational pipeline for detecting differential chromosomal interactions in Capture Hi-C data.
Cairns J, Orchard WR, Malysheva V, Spivakov M

Capture Hi-C is a powerful approach for detecting chromosomal interactions involving, at least on one end, DNA regions of interest, such as gene promoters. We present Chicdiff, an R package for robust detection of differential interactions in Capture Hi-C data. Chicdiff enhances a state-of-the-art differential testing approach for count data with bespoke normalisation and multiple testing procedures that account for specific statistical properties of Capture Hi-C. We validate Chicdiff on published Promoter Capture Hi-C data in human Monocytes and CD4+ T cells, identifying multitudes of cell type-specific interactions, and confirming the overall positive association between promoter interactions and gene expression.

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Bioinformatics (Oxford, England), , 1367-4811, , 2019

PMID: 31197313

Calcium Signaling and Tissue Calcification.
Proudfoot D

Calcification is a regulated physiological process occurring in bones and teeth. However, calcification is commonly found in soft tissues in association with aging and in a variety of diseases. Over the last two decades, it has emerged that calcification occurring in diseased arteries is not simply an inevitable build-up of insoluble precipitates of calcium phosphate. In some cases, it is an active process in which transcription factors drive conversion of vascular cells to an osteoblast or chondrocyte-like phenotype, with the subsequent production of mineralizing "matrix vesicles." Early studies of bone and cartilage calcification suggested roles for cellular calcium signaling in several of the processes involved in the regulation of bone calcification. Similarly, calcium signaling has recently been highlighted as an important component in the mechanisms regulating pathological calcification. The emerging hypothesis is that ectopic/pathological calcification occurs in tissues in which there is an imbalance in the regulatory mechanisms that actively prevent calcification. This review highlights the various ways that calcium signaling regulates tissue calcification, with a particular focus on pathological vascular calcification.

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Cold Spring Harbor perspectives in biology, 11, 1943-0264, , 2019

PMID: 31138543

Lipopolysaccharide-induced neuroinflammation induces presynaptic disruption through a direct action on brain tissue involving microglia-derived interleukin 1 beta.
Sheppard O, Coleman MP, Durrant CS

Systemic inflammation has been linked to synapse loss and cognitive decline in human patients and animal models. A role for microglial release of pro-inflammatory cytokines has been proposed based on in vivo and primary culture studies. However, mechanisms are hard to study in vivo as specific microglial ablation is challenging and the extracellular fluid cannot be sampled without invasive methods. Primary cultures have different limitations as the intricate multicellular architecture in the brain is not fully reproduced. It is essential to confirm proposed brain-specific mechanisms of inflammatory synapse loss directly in brain tissue. Organotypic hippocampal slice cultures (OHSCs) retain much of the in vivo neuronal architecture, synaptic connections and diversity of cell types whilst providing convenient access to manipulate and sample the culture medium and observe cellular reactions.

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Journal of neuroinflammation, 16, 1742-2094, 106, 2019

PMID: 31103036

Open Access

Immunodeficiency, autoimmune thrombocytopenia and enterocolitis caused by autosomal recessive deficiency of PIK3CD-encoded phosphoinositide 3-kinase δ.
Swan DJ, Aschenbrenner D, Lamb CA, Chakraborty K, Clark J, Pandey S, Engelhardt KR, Chen R, Cavounidis A, Ding Y, Krasnogor N, Carey CD, Acres M, Needham S, Cant AJ, Arkwright PD, Chandra A, Okkenhaug K, Uhlig HH, Hambleton S

Haematologica, , 1592-8721, , 2019

PMID: 31073077

Open Access

Butyrate Protects Mice from Clostridium difficile-Induced Colitis through an HIF-1-Dependent Mechanism.
Fachi JL, Felipe JS, Pral LP, da Silva BK, Corrêa RO, de Andrade MCP, da Fonseca DM, Basso PJ, Câmara NOS, de Sales E Souza ÉL, Dos Santos Martins F, Guima SES, Thomas AM, Setubal JC, Magalhães YT, Forti FL, Candreva T, Rodrigues HG, de Jesus MB, Consonni SR, Farias ADS, Varga-Weisz P, Vinolo MAR

Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection.

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Cell reports, 27, 2211-1247, 750-761.e7, 2019

PMID: 30995474

Open Access

A SUV39H1-low chromatin state characterises and promotes migratory properties of cervical cancer cells.
Rodrigues C, Pattabiraman C, Vijaykumar A, Arora R, Narayana SM, Kumar RV, Notani D, Varga-Weisz P, Krishna S

Metastatic progression is a major cause of mortality in cervical cancers, but factors regulating migratory and pre-metastatic cell populations remain poorly understood. Here, we sought to assess whether a SUV39H1-low chromatin state promotes migratory cell populations in cervical cancers, using meta-analysis of data from The Cancer Genome Atlas (TCGA), immunohistochemistry, genomics and functional assays. Cervical cancer cells sorted based on migratory ability in vitro have low levels of SUV39H1 protein, and SUV39H1 knockdown in vitro enhanced cervical cancer cell migration. Further, TCGA SUV39H1-low tumours correlated with poor clinical outcomes and showed gene expression signatures of cell migration. SUV39H1 expression was examined within biopsies, and SUV39H1 cells within tumours also demonstrated migratory features. Next, to understand genome scale transcriptional and chromatin changes in migratory populations, cell populations sorted based on migration in vitro were examined using RNA-Seq, along with ChIP-Seq for H3K9me3, the histone mark associated with SUV39H1. Migrated populations showed SUV39H1-linked migratory gene expression signatures, along with broad depletion of H3K9me3 across gene promoters. We show for the first time that a SUV39H1-low chromatin state associates with, and promotes, migratory populations in cervical cancers. Our results posit SUV39H1-low cells as key populations for prognosis estimation and as targets for novel therapies.

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Experimental cell research, , 1090-2422, , 2019

PMID: 30772380

Open Access

Targeting PI3Kδ Function For Amelioration of Murine Chronic Graft-Versus-Host Disease.
Paz K, Flynn R, Du J, Tannheimer S, Johnson AJ, Dong S, Stark AK, Okkenhaug K, Panoskaltsis-Mortari A, Sage PT, Sharpe AH, Luznik L, Ritz J, Soiffer RJ, Cutler CS, Koreth J, Antin JH, Miklos DB, MacDonald KP, Hill GR, Maillard I, Serody JS, Murphy WJ, Munn DH, Feser C, Zaiken M, Vanhaesebroeck B, Turka LA, Byrd JC, Blazar BR

Chronic graft-versus-host disease is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T-cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center -promoting Tfollicular helper cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ, a lipid kinase, is critical for activated T-cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T-cells that become Tfhs is required for cGVHD in a non-sclerodermatous multi-organ system disease model that includes bronchiolitis obliterans, dependent upon GC B-cells, Tfhs, and counterbalanced by Tfollicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B-cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow derived GC B-cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443 that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of FDA-approved PI3Kδ inhibitors for cGVHD therapy in patients. This article is protected by copyright. All rights reserved.

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American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, , 1600-6143, , 2019

PMID: 30748099

The PI3K p110δ Isoform Inhibitor Idelalisib Preferentially Inhibits Human Regulatory T Cell Function.
Chellappa S, Kushekhar K, Munthe LA, Tjønnfjord GE, Aandahl EM, Okkenhaug K, Taskén K

In chronic lymphocytic leukemia (CLL), signaling through several prosurvival B cell surface receptors activates the PI3K signaling pathway. Idelalisib is a highly selective PI3K (PI3Kδ) isoform-specific inhibitor effective in relapsed/refractory CLL and follicular lymphoma. However, severe autoimmune adverse effects in association with the use of idelalisib in the treatment of CLL, particularly as a first-line therapy, gave indications that idelalisib may preferentially target the suppressive function of regulatory T cells (Tregs). On this background, we examined the effect of idelalisib on the function of human Tregs ex vivo with respect to proliferation, TCR signaling, phenotype, and suppressive function. Our results show that human Tregs are highly susceptible to PI3Kδ inactivation using idelalisib compared with CD4 and CD8 effector T cells (Teffs) as evident from effects on anti-CD3/CD28/CD2-induced proliferation (order of susceptibility [IC]: Treg [.5 μM] > CD4 Teff [2.0 μM] > CD8 Teff [6.5 μM]) and acting at the level of AKT and NF-κB phosphorylation. Moreover, idelalisib treatment of Tregs altered their phenotype and reduced their suppressive function against CD4 and CD8 Teffs. Phenotyping Tregs from CLL patients treated with idelalisib supported our in vitro findings. Collectively, our data show that human Tregs are more dependent on PI3Kδ-mediated signaling compared with CD4 and CD8 Teffs. This Treg-preferential effect could explain why idelalisib produces adverse autoimmune effects by breaking Treg-mediated tolerance. However, balancing effects on Treg sensitivity versus CD8 Teff insensitivity to idelalisib could still potentially be exploited to enhance inherent antitumor immune responses in patients.

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Journal of immunology (Baltimore, Md. : 1950), , 1550-6606, , 2019

PMID: 30692213

Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication.
Sima J, Chakraborty A, Dileep V, Michalski M, Klein KN, Holcomb NP, Turner JL, Paulsen MT, Rivera-Mulia JC, Trevilla-Garcia C, Bartlett DA, Zhao PA, Washburn BK, Nora EP, Kraft K, Mundlos S, Bruneau BG, Ljungman M, Fraser P, Ay F, Gilbert DM

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.

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Cell, , 1097-4172, , 2018

PMID: 30595451

Publisher Correction: Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels.
Dobnikar L, Taylor AL, Chappell J, Oldach P, Harman JL, Oerton E, Dzierzak E, Bennett MR, Spivakov M, Jørgensen HF

The original version of this Article contained errors in the author affiliations.Martin R. Bennett was incorrectly associated with Nuclear Dynamics Programme, Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK. This has now been corrected in both the PDF and HTML versions of the Article. Furthermore, Phoebe Oldach was incorrectly associated with Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.This has now been corrected in the HTML version of the Article. The PDF version of the Article was correct at the time of publication.

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Nature communications, 9, 2041-1723, 5401, 2018

PMID: 30559342

Open Access

Author Correction: Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks.
Choy MK, Javierre BM, Williams SG, Baross SL, Liu Y, Wingett SW, Akbarov A, Wallace C, Freire-Pritchett P, Rugg-Gunn PJ, Spivakov M, Fraser P, Keavney BD

In the original version of the Article, the gene symbol for tissue factor pathway inhibitor was inadvertently given as 'TFP1' instead of 'TFPI'. This has now been corrected in both the PDF and HTML versions of the Article.

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Nature communications, 9, 2041-1723, 4792, 2018

PMID: 30420621

Open Access

Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels.
Dobnikar L, Taylor AL, Chappell J, Oldach P, Harman JL, Oerton E, Dzierzak E, Bennett MR, Spivakov M, Jørgensen HF

Vascular smooth muscle cells (VSMCs) show pronounced heterogeneity across and within vascular beds, with direct implications for their function in injury response and atherosclerosis. Here we combine single-cell transcriptomics with lineage tracing to examine VSMC heterogeneity in healthy mouse vessels. The transcriptional profiles of single VSMCs consistently reflect their region-specific developmental history and show heterogeneous expression of vascular disease-associated genes involved in inflammation, adhesion and migration. We detect a rare population of VSMC-lineage cells that express the multipotent progenitor marker Sca1, progressively downregulate contractile VSMC genes and upregulate genes associated with VSMC response to inflammation and growth factors. We find that Sca1 upregulation is a hallmark of VSMCs undergoing phenotypic switching in vitro and in vivo, and reveal an equivalent population of Sca1-positive VSMC-lineage cells in atherosclerotic plaques. Together, our analyses identify disease-relevant transcriptional signatures in VSMC-lineage cells in healthy blood vessels, with implications for disease susceptibility, diagnosis and prevention.

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Nature communications, 9, 2041-1723, 4567, 2018

PMID: 30385745

Open Access