Publications by authors named "Hashem Koohy"

19 Publications

  • Page 1 of 1

Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation.

Cell Rep 2021 May;35(6):109101

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPβ binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPβ, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPβ binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.
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http://dx.doi.org/10.1016/j.celrep.2021.109101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131582PMC
May 2021

HLA binding of self-peptides is biased towards proteins with specific molecular functions.

bioRxiv 2021 Feb 17. Epub 2021 Feb 17.

Human leukocyte antigen (HLA) is highly polymorphic and plays a key role in guiding adaptive immune responses by presenting foreign and self peptides to T cells. Each HLA variant selects a minor fraction of peptides that match a certain motif required for optimal interaction with the peptide-binding groove. These restriction rules define the landscape of peptides presented to T cells. Given these limitations, one might suggest that the choice of peptides presented by HLA is non-random and there is preferential presentation of an array of peptides that is optimal for distinguishing self and foreign proteins. In this study we explore these preferences with a comparative analysis of self peptides enriched and depleted in HLA ligands. We show that HLAs exhibit preferences towards presenting peptides from certain proteins while disfavoring others with specific functions, and highlight differences between various HLA genes and alleles in those preferences. We link those differences to HLA anchor residue propensities and amino acid composition of preferentially presented proteins. The set of proteins that peptides presented by a given HLA are most likely to be derived from can be used to distinguish between class I and class II HLAs and HLA alleles. Our observations can be extrapolated to explain the protective effect of certain HLA alleles in infectious diseases, and we hypothesize that they can also explain susceptibility to certain autoimmune diseases and cancers. We demonstrate that these differences lead to differential presentation of HIV, influenza virus, SARS-CoV-1 and SARS-CoV-2 proteins by various HLA alleles. Finally, we show that the reported self peptidome preferences of distinct HLA variants can be compensated by combinations of HLA-A/HLA-B and HLA-A/HLA-C alleles in frequent haplotypes.
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http://dx.doi.org/10.1101/2021.02.16.431395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899460PMC
February 2021

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Cell 2021 Feb 5;184(3):810-826.e23. Epub 2021 Jan 5.

Medical Research Council (MRC) Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK; Translational Gastroenterology Unit, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address:

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.
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http://dx.doi.org/10.1016/j.cell.2020.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864098PMC
February 2021

Potential CD8+ T Cell Cross-Reactivity Against SARS-CoV-2 Conferred by Other Coronavirus Strains.

Front Immunol 2020 5;11:579480. Epub 2020 Nov 5.

MRC Human Immunology Unit, Medical Research Council (MRC) Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.

While individuals infected with coronavirus disease 2019 (COVID-19) manifested a broad range in susceptibility and severity to the disease, the pre-existing immune memory to related pathogens cross-reactive against SARS-CoV-2 can influence the disease outcome in COVID-19. Here, we investigated the potential extent of T cell cross-reactivity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can be conferred by other coronaviruses and influenza virus, and generated an map of public and private CD8+ T cell epitopes between coronaviruses. We observed 794 predicted SARS-CoV-2 epitopes of which 52% were private and 48% were public. Ninety-nine percent of the public epitopes were shared with SARS-CoV and 5.4% were shared with either one of four common coronaviruses, 229E, HKU1, NL63, and OC43. Moreover, to assess the potential risk of self-reactivity and/or diminished T cell response for peptides identical or highly similar to the host, we identified predicted epitopes with high sequence similarity with human proteome. Lastly, we compared predicted epitopes from coronaviruses with epitopes from influenza virus deposited in IEDB, and found only a small number of peptides with limited potential for cross-reactivity between the two virus families. We believe our comprehensive profile of private and public epitopes across coronaviruses would facilitate design of vaccines, and provide insights into the presence of pre-existing coronavirus-specific memory CD8+ T cells that may influence immune responses against SARS-CoV-2.
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http://dx.doi.org/10.3389/fimmu.2020.579480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676914PMC
December 2020

Predicting Cross-Reactivity and Antigen Specificity of T Cell Receptors.

Front Immunol 2020 22;11:565096. Epub 2020 Oct 22.

MRC Human Immunology Unit, Medical Research Council (MRC) Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.

Adaptive immune recognition is mediated by specific interactions between heterodimeric T cell receptors (TCRs) and their cognate peptide-MHC (pMHC) ligands, and the methods to accurately predict TCR:pMHC interaction would have profound clinical, therapeutic and pharmaceutical applications. Herein, we review recent developments in predicting cross-reactivity and antigen specificity of TCR recognition. We discuss current experimental and computational approaches to investigate cross-reactivity and antigen-specificity of TCRs and highlight how integrating kinetic, biophysical and structural features may offer valuable insights in modeling immunogenicity. We further underscore the close inter-relationship of these two interconnected notions and the need to investigate each in the light of the other for a better understanding of T cell responsiveness for the effective clinical applications.
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http://dx.doi.org/10.3389/fimmu.2020.565096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642207PMC
May 2021

Single-cell atlas of colonic CD8 T cells in ulcerative colitis.

Nat Med 2020 09 3;26(9):1480-1490. Epub 2020 Aug 3.

Medical Research Council (MRC) Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford, UK.

Colonic antigen-experienced lymphocytes such as tissue-resident memory CD8 T cells can respond rapidly to repeated antigen exposure. However, their cellular phenotypes and the mechanisms by which they drive immune regulation and inflammation remain unclear. Here we compiled an unbiased atlas of human colonic CD8 T cells in health and ulcerative colitis (UC) using single-cell transcriptomics with T-cell receptor repertoire analysis and mass cytometry. We reveal extensive heterogeneity in CD8 T-cell composition, including expanded effector and post-effector terminally differentiated CD8 T cells. While UC-associated CD8 effector T cells can trigger tissue destruction and produce tumor necrosis factor (TNF)-α, post-effector cells acquire innate signatures to adopt regulatory functions that may mitigate excessive inflammation. Thus, we identify colonic CD8 T-cell phenotypes in health and UC, define their clonal relationships and characterize terminally differentiated dysfunctional UC CD8 T cells expressing IL-26, which attenuate acute colitis in a humanized IL-26 transgenic mouse model.
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http://dx.doi.org/10.1038/s41591-020-1003-4DOI Listing
September 2020

identification of vaccine targets for 2019-nCoV.

F1000Res 2020 25;9:145. Epub 2020 Feb 25.

MRC Human Immunology Unit, Medical Research Council (MRC) Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford, UK, Oxford, UK.

The newly identified coronavirus known as 2019-nCoV has posed a serious global health threat. According to the latest report (18-February-2020), it has infected more than 72,000 people globally and led to deaths of more than 1,016 people in China. The 2019 novel coronavirus proteome was aligned to a curated database of viral immunogenic peptides. The immunogenicity of detected peptides and their binding potential to HLA alleles was predicted by immunogenicity predictive models and NetMHCpan 4.0. We report identification of a comprehensive list of immunogenic peptides that can be used as potential targets for 2019 novel coronavirus (2019-nCoV) vaccine development. First, we found 28 nCoV peptides identical to Severe acute respiratory syndrome-related coronavirus (SARS CoV) that have previously been characterized immunogenic by T cell assays. Second, we identified 48 nCoV peptides having a high degree of similarity with immunogenic peptides deposited in The Immune Epitope Database (IEDB). Lastly, we conducted a search of 2019-nCoV 9-mer peptides that i) bind to common HLA alleles in Chinese and European population and ii) have T Cell Receptor (TCR) recognition potential by positional weight matrices and a recently developed immunogenicity algorithm, iPred, and identified in total 63 peptides with a high immunogenicity potential. Given the limited time and resources to develop vaccine and treatments for 2019-nCoV, our work provides a shortlist of candidates for experimental validation and thus can accelerate development pipeline.
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http://dx.doi.org/10.12688/f1000research.22507.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111504PMC
May 2020

Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogeny-related developmental programs.

Blood 2019 09 5;134(13):1059-1071. Epub 2019 Aug 5.

Department of Paediatrics and.

Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19 B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid-restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid-restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.
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http://dx.doi.org/10.1182/blood.2019001289DOI Listing
September 2019

Colonic epithelial cell diversity in health and inflammatory bowel disease.

Nature 2019 03 27;567(7746):49-55. Epub 2019 Feb 27.

Medical Research Council (MRC) Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford, UK.

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.
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http://dx.doi.org/10.1038/s41586-019-0992-yDOI Listing
March 2019

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Cell 2018 10 27;175(2):372-386.e17. Epub 2018 Sep 27.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK; Translational Gastroenterology Unit, John Radcliffe Hospital, Oxford, UK. Electronic address:

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.
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http://dx.doi.org/10.1016/j.cell.2018.08.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176871PMC
October 2018

Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells.

Genome Biol 2018 09 5;19(1):126. Epub 2018 Sep 5.

Nuclear Dynamics Programme, Babraham Institute, Cambridge, UK.

Background: Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice.

Results: Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging.

Conclusions: Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.
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http://dx.doi.org/10.1186/s13059-018-1489-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124017PMC
September 2018

The rise and fall of machine learning methods in biomedical research.

Authors:
Hashem Koohy

F1000Res 2017 10;6:2012. Epub 2017 Nov 10.

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine , University of Oxford, Oxford, UK.

In the era of explosion in biological data, machine learning techniques are becoming more popular in life sciences, including biology and medicine. This research note examines the rise and fall of the most commonly used machine learning techniques in life sciences over the past three decades.
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http://dx.doi.org/10.12688/f1000research.13016.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760972PMC
November 2017

Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(D)J Recombination.

Front Immunol 2017 17;8:1550. Epub 2017 Nov 17.

Nuclear Dynamics Programme, Babraham Institute, Cambridge, United Kingdom.

V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa () light chain recombination follows immunoglobulin heavy chain () recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during recombination. Here we adapt VDJ-seq to profile the VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for , as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(D)J recombination and provide avenues for further investigation of chromatin signatures that may underpin V(D)J-mediated chromosomal translocations.
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http://dx.doi.org/10.3389/fimmu.2017.01550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698286PMC
November 2017

Two Mutually Exclusive Local Chromatin States Drive Efficient V(D)J Recombination.

Cell Rep 2016 06 2;15(11):2475-87. Epub 2016 Jun 2.

Nuclear Dynamics Programme, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK. Electronic address:

Variable (V), diversity (D), and joining (J) (V(D)J) recombination is the first determinant of antigen receptor diversity. Understanding how recombination is regulated requires a comprehensive, unbiased readout of V gene usage. We have developed VDJ sequencing (VDJ-seq), a DNA-based next-generation-sequencing technique that quantitatively profiles recombination products. We reveal a 200-fold range of recombination efficiency among recombining V genes in the primary mouse Igh repertoire. We used machine learning to integrate these data with local chromatin profiles to identify combinatorial patterns of epigenetic features that associate with active VH gene recombination. These features localize downstream of VH genes and are excised by recombination, revealing a class of cis-regulatory element that governs recombination, distinct from expression. We detect two mutually exclusive chromatin signatures at these elements, characterized by CTCF/RAD21 and PAX5/IRF4, which segregate with the evolutionary history of associated VH genes. Thus, local chromatin signatures downstream of VH genes provide an essential layer of regulation that determines recombination efficiency.
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http://dx.doi.org/10.1016/j.celrep.2016.05.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914699PMC
June 2016

A new phosphate-starvation response in fission yeast requires the endocytic function of myosin I.

J Cell Sci 2015 Oct 7;128(20):3707-13. Epub 2015 Sep 7.

Nuclear Dynamics, Babraham Institute, Cambridge CB22 3AT, USA

Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here, we show a new role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of myosin I (Myo1), Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low-phosphate medium compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1. Transcriptomic analysis revealed a wide perturbation of gene expression with induction of stress-regulated genes upon phosphate starvation in wild-type but not Δmyo1 cells. Thus, endocytosis plays a pivotal role in mediating the cellular response to nutrients, bridging the external environment and internal molecular functions of the cell.
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http://dx.doi.org/10.1242/jcs.171314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631780PMC
October 2015

A comparison of peak callers used for DNase-Seq data.

PLoS One 2014 8;9(5):e96303. Epub 2014 May 8.

The Babraham Institute, Babraham Research Campus, Cambridge, United Kingdom.

Genome-wide profiling of open chromatin regions using DNase I and high-throughput sequencing (DNase-seq) is an increasingly popular approach for finding and studying regulatory elements. A variety of algorithms have been developed to identify regions of open chromatin from raw sequence-tag data, which has motivated us to assess and compare their performance. In this study, four published, publicly available peak calling algorithms used for DNase-seq data analysis (F-seq, Hotspot, MACS and ZINBA) are assessed at a range of signal thresholds on two published DNase-seq datasets for three cell types. The results were benchmarked against an independent dataset of regulatory regions derived from ENCODE in vivo transcription factor binding data for each particular cell type. The level of overlap between peak regions reported by each algorithm and this ENCODE-derived reference set was used to assess sensitivity and specificity of the algorithms. Our study suggests that F-seq has a slightly higher sensitivity than the next best algorithms. Hotspot and the ChIP-seq oriented method, MACS, both perform competitively when used with their default parameters. However the generic peak finder ZINBA appears to be less sensitive than the other three. We also assess accuracy of each algorithm over a range of signal thresholds. In particular, we show that the accuracy of F-Seq can be considerably improved by using a threshold setting that is different from the default value.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096303PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014496PMC
October 2015

Chromatin accessibility data sets show bias due to sequence specificity of the DNase I enzyme.

PLoS One 2013 26;8(7):e69853. Epub 2013 Jul 26.

Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

Background: DNase I is an enzyme which cuts duplex DNA at a rate that depends strongly upon its chromatin environment. In combination with high-throughput sequencing (HTS) technology, it can be used to infer genome-wide landscapes of open chromatin regions. Using this technology, systematic identification of hundreds of thousands of DNase I hypersensitive sites (DHS) per cell type has been possible, and this in turn has helped to precisely delineate genomic regulatory compartments. However, to date there has been relatively little investigation into possible biases affecting this data.

Results: We report a significant degree of sequence preference spanning sites cut by DNase I in a number of published data sets. The two major protocols in current use each show a different pattern, but for a given protocol the pattern of sequence specificity seems to be quite consistent. The patterns are substantially different from biases seen in other types of HTS data sets, and in some cases the most constrained position lies outside the sequenced fragment, implying that this constraint must relate to the digestion process rather than events occurring during library preparation or sequencing.

Conclusions: DNase I is a sequence-specific enzyme, with a specificity that may depend on experimental conditions. This sequence specificity is not taken into account by existing pipelines for identifying open chromatin regions. Care must be taken when interpreting DNase I results, especially when looking at the precise locations of the reads. Future studies may be able to improve the sensitivity and precision of chromatin state measurement by compensating for sequence bias.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069853PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724795PMC
April 2014

An alignment-free model for comparison of regulatory sequences.

Bioinformatics 2010 Oct 9;26(19):2391-7. Epub 2010 Aug 9.

MOAC Doctoral Training Centre, Coventry House, University of Warwick, Coventry, CV4 7AL, UK.

Motivation: Some recent comparative studies have revealed that regulatory regions can retain function over large evolutionary distances, even though the DNA sequences are divergent and difficult to align. It is also known that such enhancers can drive very similar expression patterns. This poses a challenge for the in silico detection of biologically related sequences, as they can only be discovered using alignment-free methods.

Results: Here, we present a new computational framework called Regulatory Region Scoring (RRS) model for the detection of functional conservation of regulatory sequences using predicted occupancy levels of transcription factors of interest. We demonstrate that our model can detect the functional and/or evolutionary links between some non-alignable enhancers with a strong statistical significance. We also identify groups of enhancers that are likely to be similarly regulated. Our model is motivated by previous work on prediction of expression patterns and it can capture similarity by strong binding sites, weak binding sites and even the statistically significant absence of sites. Our results support the hypothesis that weak binding sites contribute to the functional similarity of sequences. Our model fills a gap between two families of models: detailed, data-intensive models for the prediction of precise spatio-temporal expression patterns on the one side, and crude, generally applicable models on the other side. Our model borrows some of the strengths of each group and addresses their drawbacks.

Availability: The RRS source code is freely available upon publication of this manuscript: http://www2.warwick.ac.uk/fac/sci/systemsbiology/staff/ott/tools_and_software/rrs.
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http://dx.doi.org/10.1093/bioinformatics/btq453DOI Listing
October 2010