Publications by authors named "Daniel Hebenstreit"

36 Publications

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe.

Nucleic Acids Res 2022 Jan;50(1):350-367

School of Biosciences and Birmingham Centre of Genome Biology (BCGB), University of Birmingham, UK.

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive, correlates with Pol II transcription and mRNA expression levels. Changes in Pol II occupancy were detected in a Upf1 deficient (upf1Δ) strain, prevalently at genes showing a high Upf1 relative to Pol II association in wild-type. Additionally, an increased Ser2 Pol II signal was detected at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1Δ cells are hypersensitive to the transcription elongation inhibitor 6-azauracil. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1Δ. These data envisage that by operating on the nascent transcript, Upf1 might influence Pol II phosphorylation and transcription.
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http://dx.doi.org/10.1093/nar/gkab1249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8754637PMC
January 2022

Timing RNA polymerase pausing with TV-PRO-seq.

Cell Rep Methods 2021 Oct 25;1(6):None. Epub 2021 Oct 25.

School of Life Sciences, Gibbet Hill Campus, the University of Warwick, CV4 7AL Coventry, UK.

Transcription of many genes in metazoans is subject to polymerase pausing, which is the transient stop of transcriptionally engaged polymerases. This is known to mainly occur in promoter-proximal regions but it is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking. We present here the time-variant precision nuclear run-on and sequencing (TV-PRO-seq) assay, an extension of the standard PRO-seq that allows us to estimate genome-wide pausing times at single-base resolution. Its application to human cells demonstrates that, proximal to promoters, polymerases pause more frequently but for shorter times than in other genomic regions. Comparison with single-cell gene expression data reveals that the polymerase pausing times are longer in highly expressed genes, while transcriptionally noisier genes have higher pausing frequencies and slightly longer pausing times. Analyses of histone modifications suggest that the marker H3K36me3 is related to the polymerase pausing.
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http://dx.doi.org/10.1016/j.crmeth.2021.100083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8547241PMC
October 2021

Anti-bias training for (sc)RNA-seq: experimental and computational approaches to improve precision.

Brief Bioinform 2021 11;22(6)

University of Warwick, CV4 7AL Coventry, UK.

RNA-seq, including single cell RNA-seq (scRNA-seq), is plagued by insufficient sensitivity and lack of precision. As a result, the full potential of (sc)RNA-seq is limited. Major factors in this respect are the presence of global bias in most datasets, which affects detection and quantitation of RNA in a length-dependent fashion. In particular, scRNA-seq is affected by technical noise and a high rate of dropouts, where the vast majority of original transcripts is not converted into sequencing reads. We discuss these biases origins and implications, bioinformatics approaches to correct for them, and how biases can be exploited to infer characteristics of the sample preparation process, which in turn can be used to improve library preparation.
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http://dx.doi.org/10.1093/bib/bbab148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8574610PMC
November 2021

Systemic and adipocyte transcriptional and metabolic dysregulation in idiopathic intracranial hypertension.

JCI Insight 2021 05 24;6(10). Epub 2021 May 24.

Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

BACKGROUNDIdiopathic intracranial hypertension (IIH) is a condition predominantly affecting obese women of reproductive age. Recent evidence suggests that IIH is a disease of metabolic dysregulation, androgen excess, and an increased risk of cardiovascular morbidity. Here we evaluate systemic and adipose specific metabolic determinants of the IIH phenotype.METHODSIn fasted, matched IIH (n = 97) and control (n = 43) patients, we assessed glucose and insulin homeostasis and leptin levels. Body composition was assessed along with an interrogation of adipose tissue function via nuclear magnetic resonance metabolomics and RNA sequencing in paired omental and subcutaneous biopsies in a case-control study.RESULTSWe demonstrate an insulin- and leptin-resistant phenotype in IIH in excess of that driven by obesity. Adiposity in IIH is preferentially centripetal and is associated with increased disease activity and insulin resistance. IIH adipocytes appear transcriptionally and metabolically primed toward depot-specific lipogenesis.CONCLUSIONThese data show that IIH is a metabolic disorder in which adipose tissue dysfunction is a feature of the disease. Managing IIH as a metabolic disease could reduce disease morbidity and improve cardiovascular outcomes.FUNDINGThis study was supported by the UK NIHR (NIHR-CS-011-028), the UK Medical Research Council (MR/K015184/1), Diabetes UK, Wellcome Trust (104612/Z/14/Z), the Sir Jules Thorn Award, and the Midlands Neuroscience Teaching and Research Fund.
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http://dx.doi.org/10.1172/jci.insight.145346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262372PMC
May 2021

3 -5 crosstalk contributes to transcriptional bursting.

Genome Biol 2021 02 4;22(1):56. Epub 2021 Feb 4.

School of Life Sciences, University of Warwick, Coventry, UK.

Background: Transcription in mammalian cells is a complex stochastic process involving shuttling of polymerase between genes and phase-separated liquid condensates. It occurs in bursts, which results in vastly different numbers of an mRNA species in isogenic cell populations. Several factors contributing to transcriptional bursting have been identified, usually classified as intrinsic, in other words local to single genes, or extrinsic, relating to the macroscopic state of the cell. However, some possible contributors have not been explored yet. Here, we focus on processes at the 3 and 5 ends of a gene that enable reinitiation of transcription upon termination.

Results: Using Bayesian methodology, we measure the transcriptional bursting in inducible transgenes, showing that perturbation of polymerase shuttling typically reduces burst size, increases burst frequency, and thus limits transcriptional noise. Analysis based on paired-end tag sequencing (PolII ChIA-PET) suggests that this effect is genome wide. The observed noise patterns are also reproduced by a generative model that captures major characteristics of the polymerase flux between the ends of a gene and a phase-separated compartment.

Conclusions: Interactions between the 3 and 5 ends of a gene, which facilitate polymerase recycling, are major contributors to transcriptional noise.
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http://dx.doi.org/10.1186/s13059-020-02227-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860045PMC
February 2021

RNA editing contributes to epitranscriptome diversity in chronic lymphocytic leukemia.

Leukemia 2021 04 30;35(4):1053-1063. Epub 2020 Jul 30.

Department of Internal Medicine III with Haematology, Medical Oncology, Haemostaseology, Infectiology and Rheumatology, Oncologic Center, Salzburg Cancer Research Institute-Laboratory for Immunological and Molecular Cancer Research (SCRI-LIMCR), Paracelsus Medical University, Salzburg, Austria.

RNA editing-primarily conversion of adenosine to inosine (A > I)-is a widespread posttranscriptional mechanism, mediated by Adenosine Deaminases acting on RNA (ADAR) enzymes to alter the RNA sequence of primary transcripts. Hence, in addition to somatic mutations and alternative RNA splicing, RNA editing can be a further source for recoding events. Although RNA editing has been detected in many solid cancers and normal tissue, RNA editing in chronic lymphocytic leukemia (CLL) has not been addressed so far. We determined global RNA editing and recurrent, recoding RNA editing events from matched RNA-sequencing and whole exome sequencing data in CLL samples from 45 untreated patients. RNA editing was verified in a validation cohort of 98 CLL patients and revealed substantially altered RNA editing profiles in CLL compared with normal B cells. We further found that RNA editing patterns were prognostically relevant. Finally, we showed that ADAR knockout decreased steady state viability of MEC1 cells and made them more susceptible to treatment with fludarabine and ibrutinib in vitro. We propose that RNA editing contributes to the pathophysiology of CLL and targeting the RNA editing machinery could be a future strategy to maximize treatment efficacy.
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http://dx.doi.org/10.1038/s41375-020-0995-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024191PMC
April 2021

Experience Recruits MSK1 to Expand the Dynamic Range of Synapses and Enhance Cognition.

J Neurosci 2020 06 6;40(24):4644-4660. Epub 2020 May 6.

School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK

Experience powerfully influences neuronal function and cognitive performance, but the cellular and molecular events underlying the experience-dependent enhancement of mental ability have remained elusive. In particular, the mechanisms that couple the external environment to the genomic changes underpinning this improvement are unknown. To address this, we have used male mice harboring an inactivating mutation of mitogen- and stress-activated protein kinase 1 (MSK1), a brain-derived neurotrophic factor (BDNF)-activated enzyme downstream of the mitogen-activated protein kinase (MAPK) pathway. We show that MSK1 is required for the full extent of experience-induced improvement of spatial memory, for the expansion of the dynamic range of synapses, exemplified by the enhancement of hippocampal long-term potentiation (LTP) and long-term depression (LTD), and for the regulation of the majority of genes influenced by enrichment. In addition, and unexpectedly, we show that experience is associated with an MSK1-dependent downregulation of key MAPK and plasticity-related genes, notably of EGR1/Zif268 and Arc/Arg3.1, suggesting the establishment of a novel genomic landscape adapted to experience. By coupling experience to homeostatic changes in gene expression MSK1, represents a prime mechanism through which the external environment has an enduring influence on gene expression, synaptic function, and cognition. Our everyday experiences strongly influence the structure and function of the brain. Positive experiences encourage the growth and development of the brain and support enhanced learning and memory and resistance to mood disorders such as anxiety. While this has been known for many years, how this occurs is not clear. Here, we show that many of the positive aspects of experience depend on an enzyme called mitogen- and stress-activated protein kinase 1 (MSK1). Using male mice with a mutation in MSK1, we show that MSK1 is necessary for the majority of gene expression changes associated with experience, extending the range over which the communication between neurons occurs, and for both the persistence of memory and the ability to learn new task rules.
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http://dx.doi.org/10.1523/JNEUROSCI.2765-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294801PMC
June 2020

The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from gene loci.

Elife 2019 03 25;8. Epub 2019 Mar 25.

School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

UPF1 is an RNA helicase that is required for nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3'UTRs of mRNAs that are directed for NMD in the cytoplasm. Here we offer evidence, obtained from , that UPF1 constantly moves between the nucleus and cytoplasm by a mechanism that requires its RNA helicase activity. UPF1 is associated, genome-wide, with nascent RNAs at most of the active Pol II transcription sites and at some Pol III-transcribed genes, as demonstrated microscopically on the polytene chromosomes of salivary glands and by ChIP-seq analysis in S2 cells. Intron recognition seems to interfere with association and translocation of UPF1 on nascent pre-mRNAs, and cells depleted of UPF1 show defects in the release of mRNAs from transcription sites and their export from the nucleus.
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http://dx.doi.org/10.7554/eLife.41444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447362PMC
March 2019

LiBiNorm: an htseq-count analogue with improved normalisation of Smart-seq2 data and library preparation diagnostics.

PeerJ 2019 4;7:e6222. Epub 2019 Feb 4.

School of Life Sciences, University of Warwick, Coventry, UK.

Protocols for preparing RNA sequencing (RNA-seq) libraries, most prominently "Smart-seq" variations, introduce global biases that can have a significant impact on the quantification of gene expression levels. This global bias can lead to drastic over- or under-representation of RNA in non-linear length-dependent fashion due to enzymatic reactions during cDNA production. It is currently not corrected by any RNA-seq software, which mostly focus on local bias in coverage along RNAs. This paper describes LiBiNorm, a simple command line program that mimics the popular htseq-count software and allows diagnostics, quantification, and global bias removal. LiBiNorm outputs gene expression data that has been normalized to correct for global bias introduced by the Smart-seq2 protocol. In addition, it produces data and several plots that allow insights into the experimental history underlying library preparation. The LiBiNorm package includes an R script that allows visualization of the main results. LiBiNorm is the first software application to correct for the global bias that is introduced by the Smart-seq2 protocol. It is freely downloadable at http://www2.warwick.ac.uk/fac/sci/lifesci/research/libinorm.
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http://dx.doi.org/10.7717/peerj.6222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366399PMC
February 2019

Bayesian inference on stochastic gene transcription from flow cytometry data.

Bioinformatics 2018 09;34(17):i647-i655

Department of Statistics, University of Warwick, Coventry, UK.

Motivation: Transcription in single cells is an inherently stochastic process as mRNA levels vary greatly between cells, even for genetically identical cells under the same experimental and environmental conditions. We present a stochastic two-state switch model for the population of mRNA molecules in single cells where genes stochastically alternate between a more active ON state and a less active OFF state. We prove that the stationary solution of such a model can be written as a mixture of a Poisson and a Poisson-beta probability distribution. This finding facilitates inference for single cell expression data, observed at a single time point, from flow cytometry experiments such as FACS or fluorescence in situ hybridization (FISH) as it allows one to sample directly from the equilibrium distribution of the mRNA population. We hence propose a Bayesian inferential methodology using a pseudo-marginal approach and a recent approximation to integrate over unobserved states associated with measurement error.

Results: We provide a general inferential framework which can be widely used to study transcription in single cells from the kind of data arising in flow cytometry experiments. The approach allows us to separate between the intrinsic stochasticity of the molecular dynamics and the measurement noise. The methodology is tested in simulation studies and results are obtained for experimental multiple single cell expression data from FISH flow cytometry experiments.

Availability And Implementation: All analyses were implemented in R. Source code and the experimental data are available at https://github.com/SimoneTiberi/Bayesian-inference-on-stochastic-gene-transcription-from-flow-cytometry-data.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129284PMC
September 2018

Exome sequencing of the TCL1 mouse model for CLL reveals genetic heterogeneity and dynamics during disease development.

Leukemia 2019 04 27;33(4):957-968. Epub 2018 Sep 27.

Department of Internal Medicine III with Haematology, Medical Oncology, Haemostaseology, Infectiology and Rheumatology, Oncologic Center, Paracelsus Medical University, Salzburg, Austria.

The TCL1 mouse model is widely used to study pathophysiology, clonal evolution, and drug sensitivity or resistance of chronic lymphocytic leukemia (CLL). By performing whole exome sequencing, we present the genetic landscape of primary tumors from TCL1 mice and of TCL1 tumors serially transplanted into wild-type recipients to mimic clonal evolution. We show that similar to CLL patients, mutations in mice are frequently subclonal and heterogenous among different primary TCL1 mice. We further describe that this molecular heterogeneity mirrors heterogenous disease characteristics such as organ infiltration or CLL dependent T cell skewing. Similar to human CLL, we further observed the occurrence of novel mutations and structural variations during clonal evolution and found plasticity in the expansion of B cell receptor specific subclones. Thus, our results uncover that the genetic complexity, pathway dependence and clonal dynamics in mouse CLL are in relevant agreement to human CLL, and they are important to consider in future research using the TCL1 mouse for studying CLL.
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http://dx.doi.org/10.1038/s41375-018-0260-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477797PMC
April 2019

Nicotinamide Nucleotide Transhydrogenase as a Novel Treatment Target in Adrenocortical Carcinoma.

Endocrinology 2018 08;159(8):2836-2849

Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (NADPH) generator nicotinamide nucleotide transhydrogenase (NNT). NNT has a central role within mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating human NNT mutations result in congenital adrenal insufficiency. We hypothesized that NNT silencing in ACC cells will induce toxic levels of oxidative stress. To explore this, we transiently knocked down NNT in NCI-H295R ACC cells. As predicted, this manipulation increased intracellular levels of oxidative stress; this resulted in a pronounced suppression of cell proliferation and higher apoptotic rates, as well as sensitization of cells to chemically induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by mass spectrometry-based steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line to investigate the longer lasting effects of NNT silencing. After long-term culture, cells adapted metabolically to chronic NNT knockdown, restoring their redox balance and resilience to oxidative stress, although their proliferation remained suppressed. This was associated with higher rates of oxygen consumption. The molecular pathways underpinning these responses were explored in detail by RNA sequencing and nontargeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding, and polyamine metabolism. This study provides preclinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.
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http://dx.doi.org/10.1210/en.2018-00014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093335PMC
August 2018

Imprecision and DNA Break Repair Biased towards Incompatible End Joining in Leukemia.

Mol Cancer Res 2018 03 8;16(3):428-438. Epub 2017 Dec 8.

Department of Internal Medicine III with Haematology, Medical Oncology, Haemostaseology, Infectious Disease, Rheumatology, Oncologic Center, Laboratory for Immunological and Molecular Cancer Research, Paracelsus Medical University Salzburg, Austria.

Cancer is a genetic disease caused by mutations and chromosomal abnormalities that contribute to uncontrolled cell growth. In addition, cancer cells can rapidly respond to conventional and targeted therapies by accumulating novel and often specific genetic lesions leading to acquired drug resistance and relapsing disease. In chronic lymphocytic leukemia (CLL), however, diverse chromosomal aberrations often occur. In many cases, improper repair of DNA double-strand breaks (DSB) is a major source for genomic abnormalities. Therefore, this study examined the repair of DNA DSBs by nonhomologous end joining (NHEJ) in CLL by performing plasmid-based repair assays in primary CLL cells and normal B cells, isolated from patients, as well as TALEN/Cas9-induced chromosomal deletions in the CLL cell line Mec1. It is demonstrated that DNA repair is aberrant in CLL cells, featuring perturbed DNA break structure preference with efficient joining of noncohesive ends and more deletions at repair junctions. In addition, increased microhomology-mediated end joining (MMEJ) of DNA substrates was observed in CLL together with increased expression of MMEJ-specific repair factors. In summary, these data identify major differences in DNA repair efficiency between CLL cells and normal B cells isolated from patients. This study suggests inherently aberrant DNA DSB repair in the acquisition of subclonal genomic structural variations important for clonal evolution and treatment resistance in CLL. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837005PMC
March 2018

AKR1C3-Mediated Adipose Androgen Generation Drives Lipotoxicity in Women With Polycystic Ovary Syndrome.

J Clin Endocrinol Metab 2017 09;102(9):3327-3339

Institute of Metabolism and Systems Research, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.

Context: Polycystic ovary syndrome (PCOS) is a prevalent metabolic disorder occurring in up to 10% of women of reproductive age. PCOS is associated with insulin resistance and cardiovascular risk. Androgen excess is a defining feature of PCOS and has been suggested as causally associated with insulin resistance; however, mechanistic evidence linking both is lacking. We hypothesized that adipose tissue is an important site linking androgen activation and metabolic dysfunction in PCOS.

Methods: We performed a human deep metabolic in vivo phenotyping study examining the systemic and intra-adipose effects of acute and chronic androgen exposure in 10 PCOS women, in comparison with 10 body mass index-matched healthy controls, complemented by in vitro experiments.

Results: PCOS women had increased intra-adipose concentrations of testosterone (P = 0.0006) and dihydrotestosterone (P = 0.01), with increased expression of the androgen-activating enzyme aldo-ketoreductase type 1 C3 (AKR1C3) (P = 0.04) in subcutaneous adipose tissue. Adipose glycerol levels in subcutaneous adipose tissue microdialysate supported in vivo suppression of lipolysis after acute androgen exposure in PCOS (P = 0.04). Mirroring this, nontargeted serum metabolomics revealed prolipogenic effects of androgens in PCOS women only. In vitro studies showed that insulin increased adipose AKR1C3 expression and activity, whereas androgen exposure increased adipocyte de novo lipid synthesis. Pharmacologic AKR1C3 inhibition in vitro decreased de novo lipogenesis.

Conclusions: These findings define an intra-adipose mechanism of androgen activation that contributes to adipose remodeling and a systemic lipotoxic metabolome, with intra-adipose androgens driving lipid accumulation and insulin resistance in PCOS. AKR1C3 represents a promising therapeutic target in PCOS.
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http://dx.doi.org/10.1210/jc.2017-00947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587066PMC
September 2017

Modeling Enzyme Processivity Reveals that RNA-Seq Libraries Are Biased in Characteristic and Correctable Ways.

Cell Syst 2016 11 10;3(5):467-479.e12. Epub 2016 Nov 10.

School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK. Electronic address:

Experimental procedures for preparing RNA-seq and single-cell (sc) RNA-seq libraries are based on assumptions regarding their underlying enzymatic reactions. Here, we show that the fairness of these assumptions varies within libraries: coverage by sequencing reads along and between transcripts exhibits characteristic, protocol-dependent biases. To understand the mechanistic basis of this bias, we present an integrated modeling framework that infers the relationship between enzyme reactions during library preparation and the characteristic coverage patterns observed for different protocols. Analysis of new and existing (sc)RNA-seq data from six different library preparation protocols reveals that polymerase processivity is the mechanistic origin of coverage biases. We apply our framework to demonstrate that lowering incubation temperature increases processivity, yield, and (sc)RNA-seq sensitivity in all protocols. We also provide correction factors based on our model for increasing accuracy of transcript quantification in existing samples prepared at standard temperatures. In total, our findings improve our ability to accurately reflect in vivo transcript abundances in (sc)RNA-seq libraries.
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http://dx.doi.org/10.1016/j.cels.2016.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167349PMC
November 2016

Funding: Would Mendel have won it?

Nature 2016 08;536(7615):148

University of Warwick, Coventry, UK.

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http://dx.doi.org/10.1038/536148cDOI Listing
August 2016

The Regulatory T Cell Lineage Factor Foxp3 Regulates Gene Expression through Several Distinct Mechanisms Mostly Independent of Direct DNA Binding.

PLoS Genet 2015 Jun 24;11(6):e1005251. Epub 2015 Jun 24.

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3's function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression.
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http://dx.doi.org/10.1371/journal.pgen.1005251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480970PMC
June 2015

AID induces intraclonal diversity and genomic damage in CD86(+) chronic lymphocytic leukemia cells.

Eur J Immunol 2014 Dec 18;44(12):3747-57. Epub 2014 Oct 18.

Laboratory for Immunological and Molecular Cancer Research, Department of Internal Medicine III with Hematology, Medical Oncology, Hemostaseology, Infectious Diseases, Rheumatology, Oncologic Center, Paracelsus Medical University, Salzburg, Austria.

The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sμ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.
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http://dx.doi.org/10.1002/eji.201344421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276288PMC
December 2014

Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis.

Cell Rep 2014 May 10;7(4):1130-42. Epub 2014 May 10.

EMBL-European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. Electronic address:

T helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a "lymphosteroid," a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.
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http://dx.doi.org/10.1016/j.celrep.2014.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4039991PMC
May 2014

Supply and demand: Apply market forces to peer review.

Nature 2014 Feb;506(7488):295

University of Warwick, UK.

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http://dx.doi.org/10.1038/506295bDOI Listing
February 2014

Are gene loops the cause of transcriptional noise?

Trends Genet 2013 Jun 9;29(6):333-8. Epub 2013 May 9.

School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry, CV4 7AL, UK.

Expression levels of the same mRNA or protein vary significantly among the cells of an otherwise identical population. Such biological noise has great functional implications and is largely due to transcriptional bursting, the episodic production of mRNAs in short, intense bursts, interspersed by periods of transcriptional inactivity. Bursting has been demonstrated in a wide range of pro- and eukaryotic species, attesting to its universal importance. However, the mechanistic origins of bursting remain elusive. A different type of phenomenon, which has also been suggested to be widespread, is the physical interaction between the promoter and 3' end of a gene. Several functional roles have been proposed for such gene loops, including the facilitation of transcriptional reinitiation. Here, I discuss the most recent findings related to these subjects and argue that gene loops are a likely cause of transcriptional bursting and, thus, biological noise.
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http://dx.doi.org/10.1016/j.tig.2013.04.001DOI Listing
June 2013

Duel of the fates: the role of transcriptional circuits and noise in CD4+ cells.

Curr Opin Cell Biol 2012 Jun 11;24(3):350-8. Epub 2012 Apr 11.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

CD4+ T cells play key roles in orchestrating adaptive immune responses, and are a popular model for mammalian cell differentiation. While immune regulation would seem to require exactly adjusted mRNA and protein expression levels of key factors, there is little evidence that this is strictly the case. Stochastic gene expression and plasticity of cell types contrast the apparent need for precision. Recent work has provided insight into the magnitude of molecular noise, as well as the relationship between noise, transcriptional circuits and epigenetic modifications in a variety of cell types. These processes and their interplay will also govern gene expression patterns in the different CD4+ cell types, and the determination of their cellular fates.
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http://dx.doi.org/10.1016/j.ceb.2012.03.007DOI Listing
June 2012

Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs.

Cell Stem Cell 2012 Feb;10(2):157-70

Genome Function Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

Polycomb repressor complexes (PRCs) are important chromatin modifiers fundamentally implicated in pluripotency and cancer. Polycomb silencing in embryonic stem cells (ESCs) can be accompanied by active chromatin and primed RNA polymerase II (RNAPII), but the relationship between PRCs and RNAPII remains unclear genome-wide. We mapped PRC repression markers and four RNAPII states in ESCs using ChIP-seq, and found that PRC targets exhibit a range of RNAPII variants. First, developmental PRC targets are bound by unproductive RNAPII (S5p(+)S7p(-)S2p(-)) genome-wide. Sequential ChIP, Ring1B depletion, and genome-wide correlations show that PRCs and RNAPII-S5p physically bind to the same chromatin and functionally synergize. Second, we identify a cohort of genes marked by PRC and elongating RNAPII (S5p(+)S7p(+)S2p(+)); they produce mRNA and protein, and their expression increases upon PRC1 knockdown. We show that this group of PRC targets switches between active and PRC-repressed states within the ESC population, and that many have roles in metabolism.
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http://dx.doi.org/10.1016/j.stem.2011.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682187PMC
February 2012

Methods, Challenges and Potentials of Single Cell RNA-seq.

Biology (Basel) 2012 Nov 16;1(3):658-67. Epub 2012 Nov 16.

The University of Warwick, School of Life Sciences, Coventry CV4 7AL, UK.

RNA-sequencing (RNA-seq) has become the tool of choice for transcriptomics. Several recent studies demonstrate its successful adaption to single cell analysis. This allows new biological insights into cell differentiation, cell-to-cell variation and gene regulation, and how these aspects depend on each other. Here, I review the current single cell RNA-seq (scRNA-seq) efforts and discuss experimental protocols, challenges and potentials.
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http://dx.doi.org/10.3390/biology1030658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009822PMC
November 2012

RNA sequencing reveals two major classes of gene expression levels in metazoan cells.

Mol Syst Biol 2011 Jun 7;7:497. Epub 2011 Jun 7.

Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK.

The expression level of a gene is often used as a proxy for determining whether the protein or RNA product is functional in a cell or tissue. Therefore, it is of fundamental importance to understand the global distribution of gene expression levels, and to be able to interpret it mechanistically and functionally. Here we use RNA sequencing (RNA-seq) of mouse Th2 cells, coupled with a range of other techniques, to show that all genes can be separated, based on their expression abundance, into two distinct groups: one group comprised of lowly expressed and putatively non-functional mRNAs, and the other of highly expressed mRNAs with active chromatin marks at their promoters. These observations are confirmed in many other microarray and RNA-seq data sets of metazoan cell types.
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http://dx.doi.org/10.1038/msb.2011.28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159973PMC
June 2011

Analysis and simulation of gene expression profiles in pure and mixed cell populations.

Phys Biol 2011 Jun 13;8(3):035013. Epub 2011 May 13.

MRC Laboratory of Molecular Biology, Hills Rd, Cambridge CB2 0QH, UK.

For analysis and interpretation of data obtained from experimental readouts of gene expression, such as microarrays and RNA-sequencing, log transformation is routinely applied. This is because expression data, like many biological parameters, are strongly skewed. We show here that gene expression levels in multicellular organisms often deviate from simple (log) normal distributions and instead exhibit shouldered or bimodal distributions. Based on a mathematical model and numerical simulations, we demonstrate that many observed distributions can be explained as mixtures of bimodal two-component lognormal models. This is due to the fact that after log-transformation, the resulting distributions display reductions in the first peak rather than increasing overlaps over a wide range of parameter values. By comparing the theoretical results with biological datasets, our findings suggest that the distributions are generally bimodal for single cell types and get obscured by the different cell types that are present in tissue samples. Our analysis thus provides an initial explanation for the various types of expression level distributions that are found for different datasets. This will be important for the interpretation of next-generation sequencing data such as transcriptomics by mRNA-sequencing and ChIP-sequencing of epigenetic marks.
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http://dx.doi.org/10.1088/1478-3975/8/3/035013DOI Listing
June 2011

EpiChIP: gene-by-gene quantification of epigenetic modification levels.

Nucleic Acids Res 2011 Mar 3;39(5):e27. Epub 2010 Dec 3.

MRC Laboratory of Molecular Biology, Hills Rd, CB2 0QH Cambridge, UK.

The combination of chromatin immunoprecipitation with next-generation sequencing technology (ChIP-seq) is a powerful and increasingly popular method for mapping protein-DNA interactions in a genome-wide fashion. The conventional way of analyzing this data is to identify sequencing peaks along the chromosomes that are significantly higher than the read background. For histone modifications and other epigenetic marks, it is often preferable to find a characteristic region of enrichment in sequencing reads relative to gene annotations. For instance, many histone modifications are typically enriched around transcription start sites. Calculating the optimal window that describes this enrichment allows one to quantify modification levels for each individual gene. Using data sets for the H3K9/14ac histone modification in Th cells and an accompanying IgG control, we present an analysis strategy that alternates between single gene and global data distribution levels and allows a clear distinction between experimental background and signal. Curve fitting permits false discovery rate-based classification of genes as modified versus unmodified. We have developed a software package called EpiChIP that carries out this type of analysis, including integration with and visualization of gene expression data.
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http://dx.doi.org/10.1093/nar/gkq1226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061070PMC
March 2011

Inhibition of suppressive T cell factor 1 (TCF-1) isoforms in naive CD4+ T cells is mediated by IL-4/STAT6 signaling.

J Biol Chem 2011 Jan 27;286(2):919-28. Epub 2010 Oct 27.

Department of Molecular Biology, University of Salzburg, Hellbrunner Strasse 34, A-5020 Salzburg, Austria.

The Wnt pathway transcription factor T cell factor 1 (TCF-1) plays essential roles in the control of several developmental processes, including T cell development in the thymus. Although previously regarded as being required only during early T cell development, recent studies demonstrate an important role for TCF-1 in T helper 2 (Th2) cell polarization. TCF-1 was shown to activate expression of the Th2 transcription factor GATA-binding protein 3 (GATA3) and thus to promote the development of IL-4-producing Th2 cells independent of STAT6 signaling. In this study, we show that TCF-1 is down-regulated in human naive CD4(+) T cells cultured under Th2-polarizing conditions. The down-regulation is largely due to the polarizing cytokine IL-4 because IL-4 alone is sufficient to substantially inhibit TCF-1 expression. The IL-4-induced suppression of TCF-1 is mediated by STAT6, as shown by electrophoretic mobility shift assays, chromatin immunoprecipitation, and STAT6 knockdown experiments. Moreover, we found that IL-4/STAT6 predominantly inhibits the shorter, dominant-negative TCF-1 isoforms, which were reported to inhibit IL-4 transcription. Thus, this study provides a model for an IL-4/STAT6-dependent fine tuning mechanism of TCF-1-driven T helper cell polarization.
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http://dx.doi.org/10.1074/jbc.M110.144949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019123PMC
January 2011

LEF-1 negatively controls interleukin-4 expression through a proximal promoter regulatory element.

J Biol Chem 2008 Aug 25;283(33):22490-7. Epub 2008 Jun 25.

Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.

Lymphoid enhancer-binding factor 1 (LEF-1) and T cell factor (TCF-1) are downstream effectors of the Wnt signaling pathway and are involved in the regulation of T cell development in the thymus. LEF-1 and TCF-1 are also expressed in mature peripheral primary T cells, but their expression is down-regulated following T cell activation. Although the decisive roles of LEF-1 and TCF-1 in the early stages of T cell development are well documented, the functions of these factors in mature peripheral T cells are largely unknown. Recently, LEF-1 was shown to suppress Th2 cytokines interleukin-4 (IL-4), -5, and -13 expression from the developing Th2 cells that overexpress LEF-1 through retrovirus gene transduction. In this study, we further investigated the expression and functions of LEF-1 and TCF-1 in peripheral CD4+ T cells and revealed that LEF-1 is dominantly expressed in Th1 but not in Th2 cells. We identified a high affinity LEF-1-binding site in the negative regulatory element of the IL-4 promoter. Knockdown LEF-1 expression by LEF-1-specific small interfering RNA resulted in an increase in the IL-4 mRNA expression. This study further confirms a negative regulatory role of LEF-1 in mature peripheral T cells. Furthermore, we found that IL-4 stimulation possesses a negative effect on the expressions of LEF-1 and TCF-1 in primary T cells, suggesting a positive feedback effect of IL-4 on IL4 gene expression.
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http://dx.doi.org/10.1074/jbc.M804096200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995034PMC
August 2008
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