Publications

The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific services.

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Title / Authors / Details Open Access Download

Active turnover of DNA methylation during cell fate decisions.
Parry A, Rulands S, Reik W

DNA methylation is a key layer of epigenetic regulation. The deposition of methylation marks relies on the catalytic activity of DNA methyltransferases (DNMTs), and their active removal relies on the activity of ten-eleven translocation (TET) enzymes. Paradoxically, in important biological contexts these antagonistic factors are co-expressed and target overlapping genomic regions. The ensuing cyclic biochemistry of cytosine modifications gives rise to a continuous, out-of-thermal equilibrium transition through different methylation states. But what is the purpose of this intriguing turnover of DNA methylation? Recent evidence demonstrates that methylation turnover is enriched at gene distal regulatory elements, including enhancers, and can give rise to large-scale oscillatory dynamics. We discuss this phenomenon and propose that DNA methylation turnover might facilitate key lineage decisions.

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Nature reviews. Genetics, 1, 1,

PMID: 33024290

Generation and Characterization of Anti-Glucosepane Antibodies Enabling Direct Detection of Glucosepane in Retinal Tissue.
Streeter MD, Rowan S, Ray J, McDonald DM, Volkin J, Clark J, Taylor A, Spiegel DA

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

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ACS chemical biology, 1, 1,

PMID: 32975399

Landscape of Genome-Wide DNA Methylation of Colorectal Cancer Metastasis.
Ili C, Buchegger K, Demond H, Castillo-Fernandez J, Kelsey G, Zanella L, Abanto M, Riquelme I, López J, Viscarra T, García P, Bellolio E, Saavedra D, Brebi P

Colorectal cancer is a heterogeneous disease caused by both genetic and epigenetics factors. Analysing DNA methylation changes occurring during colorectal cancer progression and metastasis formation is crucial for the identification of novel epigenetic markers of patient prognosis. Genome-wide methylation sequencing of paired samples of colon (normal adjacent, primary tumour and lymph node metastasis) showed global hypomethylation and CpG island (CGI) hypermethylation of primary tumours compared to normal. In metastasis we observed high global and non-CGI regions methylation, but lower CGI methylation, compared to primary tumours. Gene ontology analysis showed shared biological processes between hypermethylated CGIs in metastasis and primary tumours. After complementary analysis with The Cancer Genome Atlas (TCGA) cohort, , , , and genes were found associated with poor survival. We mapped the methylation landscape of colon normal tissues, primary tumours and lymph node metastasis, being capable of identified methylation changes throughout the genome. Furthermore, we found five genes with potential for methylation biomarkers of poor prognosis in colorectal cancer patients.

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Cancers, 12, 9,

PMID: 32971738

Dominant mutations in ITPR3 cause Charcot-Marie-Tooth disease.
Rönkkö J, Molchanova S, Revah-Politi A, Pereira EM, Auranen M, Toppila J, Kvist J, Ludwig A, Neumann J, Bultynck G, Humblet-Baron S, Liston A, Paetau A, Rivera C, Harms MB, Tyynismaa H, Ylikallio E

ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot-Marie-Tooth disease gene.

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Annals of clinical and translational neurology, 1, 1,

PMID: 32949214

Open Access

Profiling DNA Methylation Genome-Wide in Single Cells.
Galvão A, Kelsey G

Single-cell bisulfite sequencing (scBS-seq) enables profiling of DNA methylation at single-nucleotide resolution and across all genomic features. It can explore methylation differences between cells in mixed cell populations and profile methylation in very rare cell types, such as mammalian oocytes and cells from early embryos. Here, we outline the scBS-seq protocol in a 96-well plate format applicable to studies of moderate throughput.

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Methods in molecular biology (Clifton, N.J.), 2214, 1,

PMID: 32944913

Inflammatory aortitis in a patient with type 2 hyper IgM syndrome.
Staels F, Betrains A, Willemsen M, Corvelyn A, Tousseyn T, Dierickx D, Humblet-Baron S, Liston A, Vanderschueren S, Schrijvers R

-

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Rheumatology (Oxford, England), 1, 1,

PMID: 32940674

Of Mosaicism and Mechanisms: How JAK1 Goes Awry.
Ross SH, Cantrell DA

Personalized medicines require understanding the molecular causes of disease. In this issue of Immunity, Gruber et al. reveal that a gain-of-function JAK1 genetic variant results in a mutant protein with mosaic expression that drives multi-organ immune dysregulation via kinase dependent and independent mechanisms. The work highlights how biochemistry can inform therapies to resolve complex immune disorders.

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Immunity, 53, 3,

PMID: 32937149

LifeTime and improving European healthcare through cell-based interceptive medicine.
Rajewsky N, Almouzni G, Gorski SA, Aerts S, Amit I, Bertero MG, Bock C, Bredenoord AL, Cavalli G, Chiocca S, Clevers H, De Strooper B, Eggert A, Ellenberg J, Fernández XM, Figlerowicz M, Gasser SM, Hubner N, Kjems J, Knoblich JA, Krabbe G, Lichter P, Linnarsson S, Marine JC, Marioni J, Marti-Renom MA, Netea MG, Nickel D, Nollmann M, Novak HR, Parkinson H, Piccolo S, Pinheiro I, Pombo A, Popp C, Reik W, Roman-Roman S, Rosenstiel P, Schultze JL, Stegle O, Tanay A, Testa G, Thanos D, Theis FJ, Torres-Padilla ME, Valencia A, Vallot C, van Oudenaarden A, Vidal M, Voet T,

LifeTime aims to track, understand and target human cells during the onset and progression of complex diseases and their response to therapy at single-cell resolution. This mission will be implemented through the development and integration of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during progression from health to disease. Analysis of such large molecular and clinical datasets will discover molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. Timely detection and interception of disease embedded in an ethical and patient-centered vision will be achieved through interactions across academia, hospitals, patient-associations, health data management systems and industry. Applying this strategy to key medical challenges in cancer, neurological, infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

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Nature, 1, 1,

PMID: 32894860

Frizzled-Dependent Planar Cell Polarity without Secreted Wnt Ligands.
Yu JJS, Maugarny-Calès A, Pelletier S, Alexandre C, Bellaiche Y, Vincent JP, McGough IJ

Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here, we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.

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Developmental cell, 54, 5,

PMID: 32888416

Open Access

Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.
Guarnieri G, Sarchielli E, Comeglio P, Herrera-Puerta E, Piaceri I, Nacmias B, Benelli M, Kelsey G, Maggi M, Gallina P, Vannelli GB, Morelli A

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and β-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

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International journal of molecular sciences, 21, 17,

PMID: 32854421

SBML Level 3: an extensible format for the exchange and reuse of biological models.
Keating SM, Waltemath D, König M, Zhang F, Dräger A, Chaouiya C, Bergmann FT, Finney A, Gillespie CS, Helikar T, Hoops S, Malik-Sheriff RS, Moodie SL, Moraru II, Myers CJ, Naldi A, Olivier BG, Sahle S, Schaff JC, Smith LP, Swat MJ, Thieffry D, Watanabe L, Wilkinson DJ, Blinov ML, Begley K, Faeder JR, Gómez HF, Hamm TM, Inagaki Y, Liebermeister W, Lister AL, Lucio D, Mjolsness E, Proctor CJ, Raman K, Rodriguez N, Shaffer CA, Shapiro BE, Stelling J, Swainston N, Tanimura N, Wagner J, Meier-Schellersheim M, Sauro HM, Palsson B, Bolouri H, Kitano H, Funahashi A, Hermjakob H, Doyle JC, Hucka M,

Systems biology has experienced dramatic growth in the number, size, and complexity of computational models. To reproduce simulation results and reuse models, researchers must exchange unambiguous model descriptions. We review the latest edition of the Systems Biology Markup Language (SBML), a format designed for this purpose. A community of modelers and software authors developed SBML Level 3 over the past decade. Its modular form consists of a core suited to representing reaction-based models and packages that extend the core with features suited to other model types including constraint-based models, reaction-diffusion models, logical network models, and rule-based models. The format leverages two decades of SBML and a rich software ecosystem that transformed how systems biologists build and interact with models. More recently, the rise of multiscale models of whole cells and organs, and new data sources such as single-cell measurements and live imaging, has precipitated new ways of integrating data with models. We provide our perspectives on the challenges presented by these developments and how SBML Level 3 provides the foundation needed to support this evolution.

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Molecular systems biology, 16, 8,

PMID: 32845085

Imprints in the history of epigenetics.
Kelsey G

No abstract available

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Nature reviews. Molecular cell biology, 1, 1,

PMID: 32839539

Hypermethylation and reduced expression of Gtl2, Rian and Mirg at the Dlk1-Dio3 imprinted locus as a marker for poor developmental potential of mouse embryonic stem cells.
Schacker M, Cheng YH, Eckersley-Maslin M, Snaith RM, Colledge WH

Mouse embryonic stem cells (ESCs) have played a crucial role in biomedical research where they can be used to elucidate gene function through the generation of genetically modified mice. A critical requirement for the success of this technology is the ability of ESCs to contribute to viable chimaeras with germ-line transmission of the genetically modified allele. We have identified several ESC clones that cause embryonic death of chimaeras at mid to late gestation stages. These clones had a normal karyotype, were pathogen free and their in vitro differentiation potential was not compromised. Chimaeric embryos developed normally up to E13.5 but showed a significant decrease in embryo survival by E17.5 with frequent haemorrhaging. We investigated the relationship between the ESCs transcriptional and epigenomic state and their ability to contribute to viable chimaeras. RNA sequencing identified four genes (Gtl2, Rian, Mirg and Rtl1as) located in the Dlk1-Dio3 imprinted locus that were expressed at lower levels in the compromised ESC clones and this was confirmed by qRT-PCR. Bisulphite sequencing analysis showed significant hypermethylation at the Dlk1-Dio3 imprinted locus with no consistent differences in methylation patterns at other imprinted loci. Treatment of the compromised ESCs with 5-azacytidine reactivated stable expression of Gtl2 and rescued the lethal phenotype but only gave low level chimaeras.

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Stem cell research, 48, 1,

PMID: 32822966

Cbls boost B cells.
Linterman MA

T cell regulation of antibody-mediated immunity is critical for health. In this issue of JEM, Li et al. (https://doi.org/10.1084/jem.20191537) identify the Cbl family of E3 ubiquitin ligases as B cell-intrinsic gatekeepers of T cell-dependent humoral immunity.

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The Journal of experimental medicine, 217, 9,

PMID: 32813871

Open Access

Naive Pluripotent Stem Cells Exhibit Phenotypic Variability that Is Driven by Genetic Variation.
Ortmann D, Brown S, Czechanski A, Aydin S, Muraro D, Huang Y, Tomaz RA, Osnato A, Canu G, Wesley BT, Skelly DA, Stegle O, Choi T, Churchill GA, Baker CL, Rugg-Gunn PJ, Munger SC, Reinholdt LG, Vallier L

Variability among pluripotent stem cell (PSC) lines is a prevailing issue that hampers not only experimental reproducibility but also large-scale applications and personalized cell-based therapy. This variability could result from epigenetic and genetic factors that influence stem cell behavior. Naive culture conditions minimize epigenetic fluctuation, potentially overcoming differences in PSC line differentiation potential. Here we derived PSCs from distinct mouse strains under naive conditions and show that lines from distinct genetic backgrounds have divergent differentiation capacity, confirming a major role for genetics in PSC phenotypic variability. This is explained in part through inconsistent activity of extra-cellular signaling, including the Wnt pathway, which is modulated by specific genetic variants. Overall, this study shows that genetic background plays a dominant role in driving phenotypic variability of PSCs.

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Cell stem cell, 1, 1,

PMID: 32795399

Open Access

Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity.
Mabbitt PD, Loreto A, Déry MA, Fletcher AJ, Stanley M, Pao KC, Wood NT, Coleman MP, Virdee S

MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2-E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2-E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix-coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration.

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Nature chemical biology, 1, 1,

PMID: 32747811

Mitochondrial Oxidative Damage Underlies Regulatory T Cell Defects in Autoimmunity.
Alissafi T, Kalafati L, Lazari M, Filia A, Kloukina I, Manifava M, Lim JH, Alexaki VI, Ktistakis NT, Doskas T, Garinis GA, Chavakis T, Boumpas DT, Verginis P

Regulatory T cells (Tregs) are vital for the maintenance of immune homeostasis, while their dysfunction constitutes a cardinal feature of autoimmunity. Under steady-state conditions, mitochondrial metabolism is critical for Treg function; however, the metabolic adaptations of Tregs during autoimmunity are ill-defined. Herein, we report that elevated mitochondrial oxidative stress and a robust DNA damage response (DDR) associated with cell death occur in Tregs in individuals with autoimmunity. In an experimental autoimmune encephalitis (EAE) mouse model of autoimmunity, we found a Treg dysfunction recapitulating the features of autoimmune Tregs with a prominent mtROS signature. Scavenging of mtROS in Tregs of EAE mice reversed the DDR and prevented Treg death, while attenuating the Th1 and Th17 autoimmune responses. These findings highlight an unrecognized role of mitochondrial oxidative stress in defining Treg fate during autoimmunity, which may facilitate the design of novel immunotherapies for diseases with disturbed immune tolerance.

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Cell metabolism, 1, 1,

PMID: 32738205

Establishing a Unified COVID-19 "Immunome": Integrating Coronavirus Pathogenesis and Host Immunopathology.
Wauters E, Thevissen K, Wouters C, Bosisio FM, De Smet F, Gunst J, Humblet-Baron S, Lambrechts D, Liston A, Matthys P, Neyts J, Proost P, Weynand B, Wauters J, Tejpar S, Garg AD

No abstract available

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Frontiers in immunology, 11, 1,

PMID: 32719686

Open Access

DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance.
D'Anna F, Van Dyck L, Xiong J, Zhao H, Berrens RV, Qian J, Bieniasz-Krzywiec P, Chandra V, Schoonjans L, Matthews J, De Smedt J, Minnoye L, Amorim R, Khorasanizadeh S, Yu Q, Zhao L, De Borre M, Savvides SN, Simon MC, Carmeliet P, Reik W, Rastinejad F, Mazzone M, Thienpont B, Lambrechts D

Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia.

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Genome Biology, 21, 1,

PMID: 32718321

Open Access

Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.
Pasciuto E, Burton OT, Roca CP, Lagou V, Rajan WD, Theys T, Mancuso R, Tito RY, Kouser L, Callaerts-Vegh Z, de la Fuente AG, Prezzemolo T, Mascali LG, Brajic A, Whyte CE, Yshii L, Martinez-Muriana A, Naughton M, Young A, Moudra A, Lemaitre P, Poovathingal S, Raes J, De Strooper B, Fitzgerald DC, Dooley J, Liston A

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69 CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

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Cell, 1, 1,

PMID: 32702313

Open Access

Glypicans shield the Wnt lipid moiety to enable signalling at a distance.
McGough IJ, Vecchia L, Bishop B, Malinauskas T, Beckett K, Joshi D, O'Reilly N, Siebold C, Jones EY, Vincent JP

A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.

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Nature, 585, 7823,

PMID: 32699409

Open Access

Transition to naïve human pluripotency mirrors pan-cancer DNA hypermethylation.
Patani H, Rushton MD, Higham J, Teijeiro SA, Oxley D, Cutillas P, Sproul D, Ficz G

Epigenetic reprogramming is a cancer hallmark, but how it unfolds during early neoplastic events and its role in carcinogenesis and cancer progression is not fully understood. Here we show that resetting from primed to naïve human pluripotency results in acquisition of a DNA methylation landscape mirroring the cancer DNA methylome, with gradual hypermethylation of bivalent developmental genes. We identify a dichotomy between bivalent genes that do and do not become hypermethylated, which is also mirrored in cancer. We find that loss of H3K4me3 at bivalent regions is associated with gain of methylation. Additionally, we observe that promoter CpG island hypermethylation is not restricted solely to emerging naïve cells, suggesting that it is a feature of a heterogeneous intermediate population during resetting. These results indicate that transition to naïve pluripotency and oncogenic transformation share common epigenetic trajectories, which implicates reprogramming and the pluripotency network as a central hub in cancer formation.

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Nature communications, 11, 1,

PMID: 32699299

Open Access

Cohesin-Dependent and -Independent Mechanisms Mediate Chromosomal Contacts between Promoters and Enhancers.
Thiecke MJ, Wutz G, Muhar M, Tang W, Bevan S, Malysheva V, Stocsits R, Neumann T, Zuber J, Fraser P, Schoenfelder S, Peters JM, Spivakov M

It is currently assumed that 3D chromosomal organization plays a central role in transcriptional control. However, depletion of cohesin and CTCF affects the steady-state levels of only a minority of transcripts. Here, we use high-resolution Capture Hi-C to interrogate the dynamics of chromosomal contacts of all annotated human gene promoters upon degradation of cohesin and CTCF. We show that a majority of promoter-anchored contacts are lost in these conditions, but many contacts with distinct properties are maintained, and some new ones are gained. The rewiring of contacts between promoters and active enhancers upon cohesin degradation associates with rapid changes in target gene transcription as detected by SLAM sequencing (SLAM-seq). These results provide a mechanistic explanation for the limited, but consistent, effects of cohesin and CTCF depletion on steady-state transcription and suggest the existence of both cohesin-dependent and -independent mechanisms of enhancer-promoter pairing.

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Cell reports, 32, 3,

PMID: 32698000

Proximity RNA-seq: A Sequencing Method to Identify Co-localization of RNA.
Morf J, Wingett SW

RNA localization is an important regulatory layer of gene expression and cell functioning. The protocol guides through the Proximity RNA-seq method, in which RNA molecules are sequenced in their spatial, cellular context to derive RNA co-localization and transcriptome organization. Transcripts in individual subcellular particles from chemically crosslinked cells are tagged with the same, unique DNA barcode in water-in-oil emulsion droplets. First, single DNA barcodes are PCR amplified and immobilized on single, small magnetic beads in droplets. Subsequently, 3' ends of bead-bound barcode copies are tailed with random pentadecamers. Then beads are encapsulated again into droplets together with crosslinked subcellular particles containing RNA. Reverse transcription using random pentadecamers as primers is performed in droplets, which optimally contain one bead and one particle, in order to tag RNAs co-localized to the same particle. Sequencing such cDNA molecules identifies the RNA molecule and the barcode. Subsequent analysis of transcripts that share the same barcode, i.e., co-barcoding, reveals RNA co-localization and interactions. The technique is not restricted to pairs of RNAs but can as well detect groups of transcripts and estimates local RNA density or connectivity for individual transcripts. We provide here a detailed protocol to perform and analyze Proximity RNA-seq on cell nuclei to study spatial, nuclear RNA organization.

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Methods in molecular biology (Clifton, N.J.), 2161, 1,

PMID: 32681513