Publications by authors named "Peter N Cockerill"

50 Publications

Stable Epigenetic Programming of Effector and Central Memory CD4 T Cells Occurs Within 7 Days of Antigen Exposure .

Front Immunol 2021 24;12:642807. Epub 2021 May 24.

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

T cell immunological memory is established within days of an infection, but little is known about the changes in gene regulatory networks accounting for their ability to respond more efficiently to secondary infections. To decipher the timing and nature of immunological memory we performed genome-wide analyses of epigenetic and transcriptional changes in a mouse model generating antigen-specific T cells. Epigenetic reprogramming for Th differentiation and memory T cell formation was already established by the peak of the T cell response after 7 days. The Th memory T cell program was associated with a gain of open chromatin regions, enriched for RUNX, ETS and T-bet motifs, which remained stable for 56 days. The epigenetic programs for both effector memory, associated with T-bet, and central memory, associated with TCF-1, were established in parallel. Memory T cell-specific regulatory elements were associated with greatly enhanced inducible Th1-biased responses during secondary exposures to antigen. Furthermore, memory T cells responded to re-exposure to antigen by rapidly reprograming the entire ETS factor gene regulatory network, by suppressing and activating expression. These data show that gene regulatory networks are epigenetically reprogrammed towards memory during infection, and undergo substantial changes upon re-stimulation.
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http://dx.doi.org/10.3389/fimmu.2021.642807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8181421PMC
May 2021

Isoform-specific and signaling-dependent propagation of acute myeloid leukemia by Wilms tumor 1.

Cell Rep 2021 Apr;35(3):109010

Institute of Cancer and Genomic Sciences, College of Medicine and Dentistry, University of Birmingham, Edgbaston, Birmingham B152TT, UK. Electronic address:

Acute myeloid leukemia (AML) is caused by recurrent mutations in members of the gene regulatory and signaling machinery that control hematopoietic progenitor cell growth and differentiation. Here, we show that the transcription factor WT1 forms a major node in the rewired mutation-specific gene regulatory networks of multiple AML subtypes. WT1 is frequently either mutated or upregulated in AML, and its expression is predictive for relapse. The WT1 protein exists as multiple isoforms. For two main AML subtypes, we demonstrate that these isoforms exhibit differential patterns of binding and support contrasting biological activities, including enhanced proliferation. We also show that WT1 responds to oncogenic signaling and is part of a signaling-responsive transcription factor hub that controls AML growth. WT1 therefore plays a central and widespread role in AML biology.
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http://dx.doi.org/10.1016/j.celrep.2021.109010DOI Listing
April 2021

RUNX1/ETO and mutant KIT both contribute to programming the transcriptional and chromatin landscape in t(8;21) acute myeloid leukemia.

Exp Hematol 2020 12 2;92:62-74. Epub 2020 Nov 2.

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK. Electronic address:

Acute myeloid leukemia development occurs in a stepwise fashion whereby an original driver mutation is followed by additional mutations. The first type of mutations tends to be in genes encoding members of the epigenetic/transcription regulatory machinery (i.e., RUNX1, DNMT3A, TET2), while the secondary mutations often involve genes encoding members of signaling pathways that cause uncontrolled growth of such cells such as the growth factor receptors c-KIT of FLT3. Patients usually present with both types of mutations, but it is currently unclear how both mutational events shape the epigenome in developing AML cells. To this end we generated an in vitro model of t(8;21) AML by expressing its driver oncoprotein RUNX1-ETO with or without a mutated (N822K) KIT protein. Expression of N822K-c-KIT strongly increases the self-renewal capacity of RUNX1-ETO-expressing cells. Global analysis of gene expression changes and alterations in the epigenome revealed that N822K-c-KIT expression profoundly influences the open chromatin landscape and transcription factor binding. However, our experiments also revealed that double mutant cells still differ from their patient-derived counterparts, highlighting the importance of studying patient cells to obtain a true picture of how gene regulatory networks have been reprogrammed during tumorigenesis.
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http://dx.doi.org/10.1016/j.exphem.2020.10.005DOI Listing
December 2020

IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors.

EMBO J 2020 11 15;39(22):e105220. Epub 2020 Sep 15.

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

When dormant naïve T cells first become activated by antigen-presenting cells, they express the autocrine growth factor IL-2 which transforms them into rapidly dividing effector T cells. During this process, hundreds of genes undergo epigenetic reprogramming for efficient activation, and also for potential reactivation after they return to quiescence as memory T cells. However, the relative contributions of IL-2 and T cell receptor signaling to this process are unknown. Here, we show that IL-2 signaling is required to maintain open chromatin at hundreds of gene regulatory elements, many of which control subsequent stimulus-dependent alternative pathways of T cell differentiation. We demonstrate that IL-2 activates binding of AP-1 and STAT5 at sites that can subsequently bind lineage-determining transcription factors, depending upon what other external factors exist in the local T cell environment. Once established, priming can also be maintained by the stroma-derived homeostatic cytokine IL-7, and priming diminishes if Il7r is subsequently deleted in vivo. Hence, IL-2 is not just a growth factor; it lays the foundation for T cell differentiation and immunological memory.
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http://dx.doi.org/10.15252/embj.2020105220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667885PMC
November 2020

Chromatin Priming Renders T Cell Tolerance-Associated Genes Sensitive to Activation below the Signaling Threshold for Immune Response Genes.

Cell Rep 2020 06;31(10):107748

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK. Electronic address:

Immunological homeostasis in T cells is maintained by a tightly regulated signaling and transcriptional network. Full engagement of effector T cells occurs only when signaling exceeds a critical threshold that enables induction of immune response genes carrying an epigenetic memory of prior activation. Here we investigate the underlying mechanisms causing the suppression of normal immune responses when T cells are rendered anergic by tolerance induction. By performing an integrated analysis of signaling, epigenetic modifications, and gene expression, we demonstrate that immunological tolerance is established when both signaling to and chromatin priming of immune response genes are weakened. In parallel, chromatin priming of immune-repressive genes becomes boosted, rendering them sensitive to low levels of signaling below the threshold needed to activate immune response genes. Our study reveals how repeated exposure to antigens causes an altered epigenetic state leading to T cell anergy and tolerance, representing a basis for treating auto-immune diseases.
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http://dx.doi.org/10.1016/j.celrep.2020.107748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296351PMC
June 2020

RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction.

Cell Rep 2019 Sep;28(12):3022-3031.e7

Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B152TT, UK. Electronic address:

Acute myeloid leukemia (AML) is associated with mutations in transcriptional and epigenetic regulator genes impairing myeloid differentiation. The t(8;21)(q22;q22) translocation generates the RUNX1-ETO fusion protein, which interferes with the hematopoietic master regulator RUNX1. We previously showed that the maintenance of t(8;21) AML is dependent on RUNX1-ETO expression. Its depletion causes extensive changes in transcription factor binding, as well as gene expression, and initiates myeloid differentiation. However, how these processes are connected within a gene regulatory network is unclear. To address this question, we performed Promoter-Capture Hi-C assays, with or without RUNX1-ETO depletion and assigned interacting cis-regulatory elements to their respective genes. To construct a RUNX1-ETO-dependent gene regulatory network maintaining AML, we integrated cis-regulatory element interactions with gene expression and transcription factor binding data. This analysis shows that RUNX1-ETO participates in cis-regulatory element interactions. However, differential interactions following RUNX1-ETO depletion are driven by alterations in the binding of RUNX1-ETO-regulated transcription factors.
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http://dx.doi.org/10.1016/j.celrep.2019.08.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899442PMC
September 2019

Rewiring of the Transcription Factor Network in Acute Myeloid Leukemia.

Cancer Inform 2019 25;18:1176935119859863. Epub 2019 Jun 25.

Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.

Acute myeloid leukemia (AML) is a highly heterogeneous cancer associated with different patterns of gene expression determined by the nature of their DNA mutations. These mutations mostly act to deregulate gene expression by various mechanisms at the level of the nucleus. By performing genome-wide epigenetic profiling of cis-regulatory elements, we found that AML encompasses different mutation-specific subclasses associated with the rewiring of the gene regulatory networks that drive differentiation into different directions away from normal myeloid development. By integrating epigenetic profiles with gene expression and chromatin conformation data, we defined pathways within gene regulation networks that were differentially rewired within each mutation-specific subclass of AML. This analysis revealed 2 major classes of AML: one class defined by mutations in signaling molecules that activate AP-1 via the mitogen-activated protein (MAP) kinase pathway and a second class defined by mutations within genes encoding transcription factors such as RUNX1/CBFβ and C/EBPα. By identifying specific DNA motifs protected from DNase I digestion at cis-regulatory elements, we were able to infer candidate transcription factors bound to these motifs. These integrated analyses allowed the identification of AML subtype-specific core regulatory networks that are required for AML development and maintenance, which could now be targeted in personalized therapies.
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http://dx.doi.org/10.1177/1176935119859863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6595639PMC
June 2019

Global long terminal repeat activation participates in establishing the unique gene expression programme of classical Hodgkin lymphoma.

Leukemia 2019 06 13;33(6):1463-1474. Epub 2018 Dec 13.

Institute for Cancer and Genomic Sciences, University of Birmingham, College of Medical and Dental Sciences, Birmingham, B152TT, UK.

Long terminal repeat (LTR) elements are wide-spread in the human genome and have the potential to act as promoters and enhancers. Their expression is therefore under tight epigenetic control. We previously reported in classical Hodgkin Lymphoma (cHL) that a member of the THE1B class of LTR elements acted as a promoter for the proto-oncogene and growth factor receptor gene CSF1R and that expression of this gene is required for cHL tumour survival. However, to which extent and how such elements participate in globally shaping the unique cHL gene expression programme is unknown. To address this question we mapped the genome-wide activation of THE1-LTRs in cHL cells using a targeted next generation sequencing approach (RACE-Seq). Integration of these data with global gene expression data from cHL and control B cell lines showed a unique pattern of LTR activation impacting on gene expression, including genes associated with the cHL phenotype. We also show that global LTR activation is induced by strong inflammatory stimuli. Together these results demonstrate that LTR activation provides an additional layer of gene deregulation in classical Hodgkin lymphoma and highlight the potential impact of genome-wide LTR activation in other inflammatory diseases.
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http://dx.doi.org/10.1038/s41375-018-0311-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558280PMC
June 2019

Subtype-specific regulatory network rewiring in acute myeloid leukemia.

Nat Genet 2019 01 12;51(1):151-162. Epub 2018 Nov 12.

Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by a variety of alterations in transcription factors, epigenetic regulators and signaling molecules. To determine how different mutant regulators establish AML subtype-specific transcriptional networks, we performed a comprehensive global analysis of cis-regulatory element activity and interaction, transcription factor occupancy and gene expression patterns in purified leukemic blast cells. Here, we focused on specific subgroups of subjects carrying mutations in genes encoding transcription factors (RUNX1, CEBPα), signaling molecules (FTL3-ITD, RAS) and the nuclear protein NPM1). Integrated analysis of these data demonstrates that each mutant regulator establishes a specific transcriptional and signaling network unrelated to that seen in normal cells, sustaining the expression of unique sets of genes required for AML growth and maintenance.
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http://dx.doi.org/10.1038/s41588-018-0270-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330064PMC
January 2019

The Oncogenic Transcription Factor RUNX1/ETO Corrupts Cell Cycle Regulation to Drive Leukemic Transformation.

Cancer Cell 2018 10;34(4):626-642.e8

Institute for Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK. Electronic address:

Oncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML.
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http://dx.doi.org/10.1016/j.ccell.2018.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179967PMC
October 2018

Prospective Isolation and Characterization of Genetically and Functionally Distinct AML Subclones.

Cancer Cell 2018 10 20;34(4):674-689.e8. Epub 2018 Sep 20.

Department of Experimental Hematology, Cancer Research Centre Groningen (CRCG), University Medical Centre Groningen, University of Groningen, Hanzeplein 1, DA13, 9700 RB Groningen, the Netherlands. Electronic address:

Intra-tumor heterogeneity caused by clonal evolution is a major problem in cancer treatment. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth, and engraftment behavior, as determined by RNA sequencing, DNase I hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture, and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.
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http://dx.doi.org/10.1016/j.ccell.2018.08.014DOI Listing
October 2018

C/EBPα overrides epigenetic reprogramming by oncogenic transcription factors in acute myeloid leukemia.

Blood Adv 2018 02;2(3):271-284

Institute for Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by recurrent mutations in the transcription regulatory machinery, resulting in abnormal growth and a block in differentiation. One type of recurrent mutations affects , which is subject to mutations and translocations, the latter giving rise to fusion proteins with aberrant transcriptional activities. We recently compared the mechanism by which the products of the t(8;21) and the t(3;21) translocation RUNX1-ETO and RUNX1-EVI1 reprogram the epigenome. We demonstrated that a main component of the block in differentiation in both types of AML is direct repression of the gene encoding the myeloid regulator C/EBPα by both fusion proteins. Here, we examined at the global level whether C/EBPα is able to reverse aberrant chromatin programming in t(8;21) and t(3;21) AML. C/EBPα overexpression does not change oncoprotein expression or globally displace these proteins from their binding sites. Instead, it upregulates a core set of common target genes important for myeloid differentiation and represses genes regulating leukemia maintenance. This study, therefore, identifies common -regulated pathways as targets for therapeutic intervention.
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http://dx.doi.org/10.1182/bloodadvances.2017012781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812331PMC
February 2018

Integration of Kinase and Calcium Signaling at the Level of Chromatin Underlies Inducible Gene Activation in T Cells.

J Immunol 2017 10 13;199(8):2652-2667. Epub 2017 Sep 13.

Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, United Kingdom;

TCR signaling pathways cooperate to activate the inducible transcription factors NF-κB, NFAT, and AP-1. In this study, using the calcium ionophore ionomycin and/or PMA on Jurkat T cells, we show that the gene expression program associated with activation of TCR signaling is closely related to specific chromatin landscapes. We find that calcium and kinase signaling cooperate to induce chromatin remodeling at ∼2100 chromatin regions, which demonstrate enriched binding motifs for inducible factors and correlate with target gene expression. We found that these regions typically function as inducible enhancers. Many of these elements contain composite NFAT/AP-1 sites, which typically support cooperative binding, thus further reinforcing the need for cooperation between calcium and kinase signaling in the activation of genes in T cells. In contrast, treatment with PMA or ionomycin alone induces chromatin remodeling at far fewer regions (∼600 and ∼350, respectively), which mostly represent a subset of those induced by costimulation. This suggests that the integration of TCR signaling largely occurs at the level of chromatin, which we propose plays a crucial role in regulating T cell activation.
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http://dx.doi.org/10.4049/jimmunol.1602033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632840PMC
October 2017

Prognostic significance of high GFI1 expression in AML of normal karyotype and its association with a FLT3-ITD signature.

Sci Rep 2017 09 11;7(1):11148. Epub 2017 Sep 11.

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK.

Growth Factor Independence 1 (GFI1) is a transcriptional repressor that plays a critical role during both myeloid and lymphoid haematopoietic lineage commitment. Several studies have demonstrated the involvement of GFI1 in haematological malignancies and have suggested that low expression of GFI1 is a negative indicator of disease progression for both myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). In this study, we have stratified AML patients into those defined as having a normal karyotype (CN-AML). Unlike the overall pattern in AML, those patients with CN-AML have a poorer survival rate when GFI1 expression is high. In this group, high GFI1 expression is paralleled by higher FLT3 expression, and, even when the FLT3 gene is not mutated, exhibit a FLT3-ITD signature of gene expression. Knock-down of GFI1 expression in the human AML Fujioka cell line led to a decrease in the level of FLT3 RNA and protein and to the down regulation of FLT3-ITD signature genes, thus linking two major prognostic indicators for AML.
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http://dx.doi.org/10.1038/s41598-017-11718-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593973PMC
September 2017

RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21) and t(3;21) AML.

Cell Rep 2017 05;19(8):1654-1668

Institute for Cancer and Genomic Sciences, College of Medicine and Dentistry, University of Birmingham, B15 2TT Birmingham, UK. Electronic address:

Acute myeloid leukemia (AML) is a heterogeneous disease caused by mutations in transcriptional regulator genes, but how different mutant regulators shape the chromatin landscape is unclear. Here, we compared the transcriptional networks of two types of AML with chromosomal translocations of the RUNX1 locus that fuse the RUNX1 DNA-binding domain to different regulators, the t(8;21) expressing RUNX1-ETO and the t(3;21) expressing RUNX1-EVI1. Despite containing the same DNA-binding domain, the two fusion proteins display distinct binding patterns, show differences in gene expression and chromatin landscape, and are dependent on different transcription factors. RUNX1-EVI1 directs a stem cell-like transcriptional network reliant on GATA2, whereas that of RUNX1-ETO-expressing cells is more mature and depends on RUNX1. However, both types of AML are dependent on the continuous expression of the fusion proteins. Our data provide a molecular explanation for the differences in clinical prognosis for these types of AML.
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http://dx.doi.org/10.1016/j.celrep.2017.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457485PMC
May 2017

T Cell Receptor and Cytokine Signaling Can Function at Different Stages to Establish and Maintain Transcriptional Memory and Enable T Helper Cell Differentiation.

Front Immunol 2017 3;8:204. Epub 2017 Mar 3.

Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, University of Birmingham , Birmingham , UK.

Experienced T cells exhibit immunological memory a rapid recall response, responding to restimulation much faster than naïve T cells. The formation of immunological memory starts during an initial slow response, when naïve T cells become transformed to proliferating T blast cells, and inducible immune response genes are reprogrammed as active chromatin domains. We demonstrated that these active domains are supported by thousands of priming elements which cooperate with inducible transcriptional enhancers to enable efficient responses to stimuli. At the conclusion of this response, a small proportion of these cells return to the quiescent state as long-term memory T cells. We proposed that priming elements can be established in a hit-and-run process dependent on the inducible factor AP-1, but then maintained by the constitutive factors RUNX1 and ETS-1. This priming mechanism may also function to render genes receptive to additional differentiation-inducing factors such as GATA3 and TBX21 that are encountered under polarizing conditions. The proliferation of recently activated T cells and the maintenance of immunological memory in quiescent memory T cells are also dependent on various cytokine signaling pathways upstream of AP-1. We suggest that immunological memory is established by T cell receptor signaling, but maintained by cytokine signaling.
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http://dx.doi.org/10.3389/fimmu.2017.00204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5334638PMC
March 2017

Chromatin priming of genes in development: Concepts, mechanisms and consequences.

Exp Hematol 2017 05 7;49:1-8. Epub 2017 Feb 7.

Institute of Cancer and Genomic Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham, UK. Electronic address:

During ontogeny, cells progress through multiple alternate differentiation states by activating distinct gene regulatory networks. In this review, we highlight the important role of chromatin priming in facilitating gene activation during lineage specification and in maintaining an epigenetic memory of previous gene activation. We show that chromatin priming is part of a hugely diverse repertoire of regulatory mechanisms that genes use to ensure that they are expressed at the correct time, in the correct cell type, and at the correct level, but also that they react to signals. We also emphasize how increasing our knowledge of these principles could inform our understanding of developmental failure and disease.
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http://dx.doi.org/10.1016/j.exphem.2017.01.003DOI Listing
May 2017

Chromatin priming elements establish immunological memory in T cells without activating transcription: T cell memory is maintained by DNA elements which stably prime inducible genes without activating steady state transcription.

Bioessays 2017 02 27;39(2). Epub 2016 Dec 27.

Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham, West Midlands, UK.

We have identified a simple epigenetic mechanism underlying the establishment and maintenance of immunological memory in T cells. By studying the transcriptional regulation of inducible genes we found that a single cycle of activation of inducible factors is sufficient to initiate stable binding of pre-existing transcription factors to thousands of newly activated distal regulatory elements within inducible genes. These events lead to the creation of islands of active chromatin encompassing nearby enhancers, thereby supporting the accelerated activation of inducible genes, without changing steady state levels of transcription in memory T cells. These studies also highlighted the need for more sophisticated definitions of gene regulatory elements. The chromatin priming elements defined here are distinct from classical enhancers because they function by maintaining chromatin accessibility rather than directly activating transcription. We propose that these priming elements are members of a wider class of genomic elements that support correct developmentally regulated gene expression.
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http://dx.doi.org/10.1002/bies.201600184DOI Listing
February 2017

Receptor Signaling Directs Global Recruitment of Pre-existing Transcription Factors to Inducible Elements.

Yale J Biol Med 2016 12 23;89(4):591-596. Epub 2016 Dec 23.

Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, University of Birmingham, U.K.

Gene expression programs are largely regulated by the tissue-specific expression of lineage-defining transcription factors or by the inducible expression of transcription factors in response to specific stimuli. Here I will review our own work over the last 20 years to show how specific activation signals also lead to the wide-spread re-distribution of pre-existing constitutive transcription factors to sites undergoing chromatin reorganization. I will summarize studies showing that activation of kinase signaling pathways creates open chromatin regions that recruit pre-existing factors which were previously unable to bind to closed chromatin. As models I will draw upon genes activated or primed by receptor signaling in memory T cells, and genes activated by cytokine receptor mutations in acute myeloid leukemia. I also summarize a hit-and-run model of stable epigenetic reprograming in memory T cells, mediated by transient Activator Protein 1 (AP-1) binding, which enables the accelerated activation of inducible enhancers.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5168834PMC
December 2016

Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate.

Development 2016 12 17;143(23):4324-4340. Epub 2016 Oct 17.

Institute of Biomedical Research, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK

The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.
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http://dx.doi.org/10.1242/dev.139857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201045PMC
December 2016

Inducible chromatin priming is associated with the establishment of immunological memory in T cells.

EMBO J 2016 Mar 21;35(5):515-35. Epub 2016 Jan 21.

Institute of Biomedical Research, College of Medicine and Dentistry, University of Birmingham, Birmingham, UK

Immunological memory is a defining feature of vertebrate physiology, allowing rapid responses to repeat infections. However, the molecular mechanisms required for its establishment and maintenance remain poorly understood. Here, we demonstrated that the first steps in the acquisition of T-cell memory occurred during the initial activation phase of naïve T cells by an antigenic stimulus. This event initiated extensive chromatin remodeling that reprogrammed immune response genes toward a stably maintained primed state, prior to terminal differentiation. Activation induced the transcription factors NFAT and AP-1 which created thousands of new DNase I-hypersensitive sites (DHSs), enabling ETS-1 and RUNX1 recruitment to previously inaccessible sites. Significantly, these DHSs remained stable long after activation ceased, were preserved following replication, and were maintained in memory-phenotype cells. We show that primed DHSs maintain regions of active chromatin in the vicinity of inducible genes and enhancers that regulate immune responses. We suggest that this priming mechanism may contribute to immunological memory in T cells by facilitating the induction of nearby inducible regulatory elements in previously activated T cells.
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http://dx.doi.org/10.15252/embj.201592534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772849PMC
March 2016

Wellington-bootstrap: differential DNase-seq footprinting identifies cell-type determining transcription factors.

BMC Genomics 2015 Nov 25;16:1000. Epub 2015 Nov 25.

Warwick Systems Biology Centre, University of Warwick, Coventry, CV4 7AL, UK.

Background: The analysis of differential gene expression is a fundamental tool to relate gene regulation with specific biological processes. Differential binding of transcription factors (TFs) can drive differential gene expression. While DNase-seq data can provide global snapshots of TF binding, tools for detecting differential binding from pairs of DNase-seq data sets are lacking.

Results: In order to link expression changes with changes in TF binding we introduce the concept of differential footprinting alongside a computational tool. We demonstrate that differential footprinting is associated with differential gene expression and can be used to define cell types by their specific TF occupancy patterns.

Conclusions: Our new tool, Wellington-bootstrap, will enable the detection of differential TF binding facilitating the study of gene regulatory systems.
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http://dx.doi.org/10.1186/s12864-015-2081-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658755PMC
November 2015

Chromatin Structure Profiling Identifies Crucial Regulators of Tumor Maintenance.

Trends Cancer 2015 Nov 5;1(3):157-160. Epub 2015 Nov 5.

Institute of Cancer and Genomic Medicine, Institute for Biomedical Research, University of Birmingham, Birmingham B15 2TT, UK. Electronic address:

Cancer is primarily caused by mutations in genes encoding transcriptional regulators and signaling molecules. These mutations cooperate to deregulate the tight control over gene expression that is otherwise seen in normal cells. One consequence of this process is deregulated transcription factor (TF) activity. This forum article highlights novel strategies that use genome-wide chromatin structure profiling to identify the deregulated factors on which cancer cells depend, with the ultimate aim of targeting them.
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http://dx.doi.org/10.1016/j.trecan.2015.10.003DOI Listing
November 2015

Chronic FLT3-ITD Signaling in Acute Myeloid Leukemia Is Connected to a Specific Chromatin Signature.

Cell Rep 2015 Aug 23;12(5):821-36. Epub 2015 Jul 23.

School of Immunity and Infection, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK. Electronic address:

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.
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http://dx.doi.org/10.1016/j.celrep.2015.06.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726916PMC
August 2015

Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma.

Proc Natl Acad Sci U S A 2014 Oct 6;111(42):E4513-22. Epub 2014 Oct 6.

Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany; Hematology, Oncology, and Tumor Immunology, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany;

Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.
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http://dx.doi.org/10.1073/pnas.1406985111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210307PMC
October 2014

Identification of a dynamic core transcriptional network in t(8;21) AML that regulates differentiation block and self-renewal.

Cell Rep 2014 Sep 18;8(6):1974-1988. Epub 2014 Sep 18.

School of Cancer Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK. Electronic address:

Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21), subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq) to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21) cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.
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http://dx.doi.org/10.1016/j.celrep.2014.08.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487811PMC
September 2014

Wellington: a novel method for the accurate identification of digital genomic footprints from DNase-seq data.

Nucleic Acids Res 2013 Nov 25;41(21):e201. Epub 2013 Sep 25.

Warwick Systems Biology Centre, University of Warwick, Coventry, CV4 7AL, United Kingdom, School of Cancer Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom, Department of Statistics, University of Warwick, Coventry, CV4 7AL, United Kingdom and School of Immunity and Infection, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom.

The expression of eukaryotic genes is regulated by cis-regulatory elements such as promoters and enhancers, which bind sequence-specific DNA-binding proteins. One of the great challenges in the gene regulation field is to characterise these elements. This involves the identification of transcription factor (TF) binding sites within regulatory elements that are occupied in a defined regulatory context. Digestion with DNase and the subsequent analysis of regions protected from cleavage (DNase footprinting) has for many years been used to identify specific binding sites occupied by TFs at individual cis-elements with high resolution. This methodology has recently been adapted for high-throughput sequencing (DNase-seq). In this study, we describe an imbalance in the DNA strand-specific alignment information of DNase-seq data surrounding protein-DNA interactions that allows accurate prediction of occupied TF binding sites. Our study introduces a novel algorithm, Wellington, which considers the imbalance in this strand-specific information to efficiently identify DNA footprints. This algorithm significantly enhances specificity by reducing the proportion of false positives and requires significantly fewer predictions than previously reported methods to recapitulate an equal amount of ChIP-seq data. We also provide an open-source software package, pyDNase, which implements the Wellington algorithm to interface with DNase-seq data and expedite analyses.
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http://dx.doi.org/10.1093/nar/gkt850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3834841PMC
November 2013

The inducible tissue-specific expression of the human IL-3/GM-CSF locus is controlled by a complex array of developmentally regulated enhancers.

J Immunol 2012 Nov 28;189(9):4459-69. Epub 2012 Sep 28.

Leeds Institute of Molecular Medicine, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, United Kingdom.

The closely linked human IL-3 and GM-CSF genes are tightly regulated and are expressed in activated T cells and mast cells. In this study, we used transgenic mice to study the developmental regulation of this locus and to identify DNA elements required for its correct activity in vivo. Because these two genes are separated by a CTCF-dependent insulator, and the GM-CSF gene is regulated primarily by its own upstream enhancer, the main objective in this study was to identify regions of the locus required for correct IL-3 gene expression. We initially found that the previously identified proximal upstream IL-3 enhancers were insufficient to account for the in vivo activity of the IL-3 gene. However, an extended analysis of DNase I-hypersensitive sites (DHSs) spanning the entire upstream IL-3 intergenic region revealed the existence of a complex cluster of both constitutive and inducible DHSs spanning the -34- to -40-kb region. The tissue specificity of these DHSs mirrored the activity of the IL-3 gene, and included a highly inducible cyclosporin A-sensitive enhancer at -37 kb that increased IL-3 promoter activity 40-fold. Significantly, inclusion of this region enabled correct in vivo regulation of IL-3 gene expression in T cells, mast cells, and myeloid progenitor cells.
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http://dx.doi.org/10.4049/jimmunol.1201915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480713PMC
November 2012