Publications by authors named "Andrew D Sharrocks"

93 Publications

Correction to article "The forkhead transcription factor FOXK2 premarks lineage-specific genes in human embryonic stem cells for activation during differentiation''.

Nucleic Acids Res 2021 Mar 22. Epub 2021 Mar 22.

Faculty of Biology, Medicine and Health, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.

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http://dx.doi.org/10.1093/nar/gkab221DOI Listing
March 2021

The forkhead transcription factor FOXK2 premarks lineage-specific genes in human embryonic stem cells for activation during differentiation.

Nucleic Acids Res 2021 02;49(3):1345-1363

Faculty of Biology, Medicine and Health, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.

Enhancers play important roles in controlling gene expression in a choreographed spatial and temporal manner during development. However, it is unclear how these regulatory regions are established during differentiation. Here we investigated the genome-wide binding profile of the forkhead transcription factor FOXK2 in human embryonic stem cells (ESCs) and downstream cell types. This transcription factor is bound to thousands of regulatory regions in human ESCs, and binding at many sites is maintained as cells differentiate to mesendodermal and neural precursor cell (NPC) types, alongside the emergence of new binding regions. FOXK2 binding is generally associated with active histone marks in any given cell type. Furthermore newly acquired, or retained FOXK2 binding regions show elevated levels of activating histone marks following differentiation to NPCs. In keeping with this association with activating marks, we demonstrate a role for FOXK transcription factors in gene activation during NPC differentiation. FOXK2 occupancy in ESCs is therefore an early mark for delineating the regulatory regions, which become activated in later lineages.
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http://dx.doi.org/10.1093/nar/gkaa1281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897486PMC
February 2021

Genome-wide Interrogation of Protein-DNA Interactions in Mammalian Cells Using ChIPmentation.

STAR Protoc 2020 Dec 4;1(3):100187. Epub 2020 Dec 4.

Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China.

Mapping the genomic locations of chromatin-associated proteins, such as transcription factors and histone modifications, is key to understanding the mechanisms of transcriptional regulation. ChIPmentation offers a simple and robust way of investigating the genomic binding sites of a protein using relatively low-input material. Here, we present a detailed protocol for the key steps that lead to a successful ChIPmentation experiment, as well as a quick analysis pipeline to examine the data. For complete details on the use and execution of this protocol, please refer to Schmidl et al. (2015). For example data produced by this protocol, please refer to Henriksson et al. (2019) and Zhang et al. (2019).
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http://dx.doi.org/10.1016/j.xpro.2020.100187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757419PMC
December 2020

Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations.

Authors:
Dervla M Connaughton Rufeng Dai Danielle J Owen Jonathan Marquez Nina Mann Adda L Graham-Paquin Makiko Nakayama Etienne Coyaud Estelle M N Laurent Jonathan R St-Germain Lot Snijders Blok Arianna Vino Verena Klämbt Konstantin Deutsch Chen-Han Wilfred Wu Caroline M Kolvenbach Franziska Kause Isabel Ottlewski Ronen Schneider Thomas M Kitzler Amar J Majmundar Florian Buerger Ana C Onuchic-Whitford Mao Youying Amy Kolb Daanya Salmanullah Evan Chen Amelie T van der Ven Jia Rao Hadas Ityel Steve Seltzsam Johanna M Rieke Jing Chen Asaf Vivante Daw-Yang Hwang Stefan Kohl Gabriel C Dworschak Tobias Hermle Mariëlle Alders Tobias Bartolomaeus Stuart B Bauer Michelle A Baum Eva H Brilstra Thomas D Challman Jacob Zyskind Carrie E Costin Katrina M Dipple Floor A Duijkers Marcia Ferguson David R Fitzpatrick Roger Fick Ian A Glass Peter J Hulick Antonie D Kline Ilona Krey Selvin Kumar Weining Lu Elysa J Marco Ingrid M Wentzensen Heather C Mefford Konrad Platzer Inna S Povolotskaya Juliann M Savatt Natalia V Shcherbakova Prabha Senguttuvan Audrey E Squire Deborah R Stein Isabelle Thiffault Victoria Y Voinova Michael J G Somers Michael A Ferguson Avram Z Traum Ghaleb H Daouk Ankana Daga Nancy M Rodig Paulien A Terhal Ellen van Binsbergen Loai A Eid Velibor Tasic Hila Milo Rasouly Tze Y Lim Dina F Ahram Ali G Gharavi Heiko M Reutter Heidi L Rehm Daniel G MacArthur Monkol Lek Kristen M Laricchia Richard P Lifton Hong Xu Shrikant M Mane Simone Sanna-Cherchi Andrew D Sharrocks Brian Raught Simon E Fisher Maxime Bouchard Mustafa K Khokha Shirlee Shril Friedhelm Hildebrandt

Am J Hum Genet 2020 10 4;107(4):727-742. Epub 2020 Sep 4.

Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.
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http://dx.doi.org/10.1016/j.ajhg.2020.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536580PMC
October 2020

Repurposing of KLF5 activates a cell cycle signature during the progression from a precursor state to oesophageal adenocarcinoma.

Elife 2020 09 3;9. Epub 2020 Sep 3.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.

Oesophageal adenocarcinoma (OAC) is one of the most common causes of cancer deaths. Barrett's oesophagus (BO) is the only known precancerous precursor to OAC, but our understanding about the molecular events leading to OAC development is limited. Here, we have integrated gene expression and chromatin accessibility profiles of human biopsies and identified a strong cell cycle gene expression signature in OAC compared to BO. Through analysing associated chromatin accessibility changes, we have implicated the transcription factor KLF5 in the transition from BO to OAC. Importantly, we show that KLF5 expression is unchanged during this transition, but instead, KLF5 is redistributed across chromatin to directly regulate cell cycle genes specifically in OAC cells. This new KLF5 target gene programme has potential prognostic significance as high levels correlate with poorer patient survival. Thus, the repurposing of KLF5 for novel regulatory activity in OAC provides new insights into the mechanisms behind disease progression.
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http://dx.doi.org/10.7554/eLife.57189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544504PMC
September 2020

Dynamic changes in the epigenomic landscape regulate human organogenesis and link to developmental disorders.

Nat Commun 2020 08 6;11(1):3920. Epub 2020 Aug 6.

Faculty of Biology, Medicine & Health, Manchester Academic Health Sciences Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development.
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http://dx.doi.org/10.1038/s41467-020-17305-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413392PMC
August 2020

Cooperative behaviour and phenotype plasticity evolve during melanoma progression.

Pigment Cell Melanoma Res 2020 09 20;33(5):695-708. Epub 2020 Mar 20.

Manchester Cancer Research Centre, Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.

A major challenge for managing melanoma is its tumour heterogeneity based on individual co-existing melanoma cell phenotypes. These phenotypes display variable responses to standard therapies, and they drive individual steps of melanoma progression; hence, understanding their behaviour is imperative. Melanoma phenotypes are defined by distinct transcriptional states, which relate to different melanocyte lineage development phases, ranging from a mesenchymal, neural crest-like to a proliferative, melanocytic phenotype. It is thought that adaptive phenotype plasticity based on transcriptional reprogramming drives melanoma progression, but at which stage individual phenotypes dominate and moreover, how they interact is poorly understood. We monitored melanocytic and mesenchymal phenotypes throughout melanoma progression and detected transcriptional reprogramming at different stages, with a gain in mesenchymal traits in circulating melanoma cells (CTCs) and proliferative features in metastatic tumours. Intriguingly, we found that distinct phenotype populations interact in a cooperative manner, which generates tumours of greater "fitness," supports CTCs and expands organotropic cues in metastases. Fibronectin, expressed in mesenchymal cells, acts as key player in cooperativity and promotes survival of melanocytic cells. Our data reveal an important role for inter-phenotype communications at various stages of disease progression, suggesting these communications could act as therapeutic target.
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http://dx.doi.org/10.1111/pcmr.12873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496243PMC
September 2020

ELK1 has a dual activating and repressive role in human embryonic stem cells.

Wellcome Open Res 2019 5;4:41. Epub 2019 Jul 5.

Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PT, UK.

The ERK MAPK pathway plays a pivotal role in regulating numerous cellular processes during normal development and in the adult but is often deregulated in disease scenarios. One of its key nuclear targets is the transcription factor ELK1, which has been shown to play an important role in controlling gene expression in human embryonic stem cells (hESCs). ELK1 is known to act as a transcriptional activator in response to ERK pathway activation but repressive roles have also been uncovered, including a putative interaction with the PRC2 complex. Here we probe the activity of ELK1 in hESCs by using a combination of gene expression analysis in hESCs and during differentiation following ELK1 depletion and also analysis of chromatin occupancy of transcriptional regulators and histone mark deposition that accompany changes in gene expression. We find that ELK1 can exert its canonical activating activity downstream from the ERK pathway but also possesses additional repressive activities. Despite its co-binding to PRC2 occupied regions, we could not detect any ELK1-mediated repression at these regions. Instead, we find that ELK1 has a repressive role at a subset of co-occupied SRF binding regions. ELK1 should therefore be viewed as a dichotomous transcriptional regulator that can act through SRF to generate both activating and repressing properties at different genomic loci.
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http://dx.doi.org/10.12688/wellcomeopenres.15091.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619381PMC
July 2019

Geno2proteo, a Tool for Batch Retrieval of DNA and Protein Sequences from Any Genomic or Protein Regions.

J Integr Bioinform 2019 Jul 13;16(3). Epub 2019 Jul 13.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK.

The interconversion of sequences that constitute the genome and the proteome is becoming increasingly important due to the generation of large amounts of DNA sequence data. Following mapping of DNA segments to the genome, one fundamentally important task is to find the amino acid sequences which are coded within a list of genomic sections. Conversely, given a series of protein segments, an important task is to find the genomic loci which code for a list of protein regions. To perform these tasks on a region by region basis is extremely laborious when a large number of regions are being studied. We have therefore implemented an R package geno2proteo which performs the two mapping tasks and subsequent sequence retrieval in a batch fashion. In order to make the tool more accessible to users, we have created a web interface of the R package which allows the users to perform the mapping tasks by going to the web page http://sharrocksresources.manchester.ac.uk/tofigaps and using the web service.
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http://dx.doi.org/10.1515/jib-2018-0090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798850PMC
July 2019

ZIC3 Controls the Transition from Naive to Primed Pluripotency.

Cell Rep 2019 06;27(11):3215-3227.e6

Faculty of Biology, Medicine and Health, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. Electronic address:

Embryonic stem cells (ESCs) must transition through a series of intermediate cell states before becoming terminally differentiated. Here, we investigated the early events in this transition by determining the changes in the open chromatin landscape as naive mouse ESCs transition to epiblast-like cells (EpiLCs). Motif enrichment analysis of the newly opening regions coupled with expression analysis identified ZIC3 as a potential regulator of this cell fate transition. Chromatin binding and genome-wide transcriptional profiling following Zic3 depletion confirmed ZIC3 as an important regulatory transcription factor, and among its targets are genes encoding a number of transcription factors. Among these is GRHL2, which acts through enhancer switching to maintain the expression of a subset of genes from the ESC state. Our data therefore place ZIC3 upstream of a set of pro-differentiation transcriptional regulators and provide an important advance in our understanding of the regulatory factors governing the early steps in ESC differentiation.
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http://dx.doi.org/10.1016/j.celrep.2019.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581693PMC
June 2019

Identification of a primitive intestinal transcription factor network shared between esophageal adenocarcinoma and its precancerous precursor state.

Genome Res 2019 05 8;29(5):723-736. Epub 2019 Apr 8.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, United Kingdom.

Esophageal adenocarcinoma (EAC) is one of the most frequent causes of cancer death, and yet compared to other common cancers, we know relatively little about the molecular composition of this tumor type. To further our understanding of this cancer, we have used open chromatin profiling to decipher the transcriptional regulatory networks that are operational in EAC. We have uncovered a transcription factor network that is usually found in primitive intestinal cells during embryonic development, centered on HNF4A and GATA6. These transcription factors work together to control the EAC transcriptome. We show that this network is activated in Barrett's esophagus, the putative precursor state to EAC, thereby providing novel molecular evidence in support of stepwise malignant transition. Furthermore, we show that HNF4A alone is sufficient to drive chromatin opening and activation of a Barrett's-like chromatin signature when expressed in normal human epithelial cells. Collectively, these data provide a new way to categorize EAC at a genome scale and implicate HNF4A activation as a potential pivotal event in its malignant transition from healthy cells.
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http://dx.doi.org/10.1101/gr.243345.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499311PMC
May 2019

Author Correction: Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

Sci Rep 2019 Jan 22;9(1):318. Epub 2019 Jan 22.

Faculty of Medicine, University of Southampton, Southampton General Hospital, Mailpoint 801, South Academic Block, Tremona Road, Southampton, SO16 6YD, United Kingdom.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-37591-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342942PMC
January 2019

Classifying cells with Scasat, a single-cell ATAC-seq analysis tool.

Nucleic Acids Res 2019 01;47(2):e10

Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PL, UK.

ATAC-seq is a recently developed method to identify the areas of open chromatin in a cell. These regions usually correspond to active regulatory elements and their location profile is unique to a given cell type. When done at single-cell resolution, ATAC-seq provides an insight into the cell-to-cell variability that emerges from otherwise identical DNA sequences by identifying the variability in the genomic location of open chromatin sites in each of the cells. This paper presents Scasat (single-cell ATAC-seq analysis tool), a complete pipeline to process scATAC-seq data with simple steps. Scasat treats the data as binary and applies statistical methods that are especially suitable for binary data. The pipeline is developed in a Jupyter notebook environment that holds the executable code along with the necessary description and results. It is robust, flexible, interactive and easy to extend. Within Scasat we developed a novel differential accessibility analysis method based on information gain to identify the peaks that are unique to a cell. The results from Scasat showed that open chromatin locations corresponding to potential regulatory elements can account for cellular heterogeneity and can identify regulatory regions that separates cells from a complex population.
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http://dx.doi.org/10.1093/nar/gky950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344856PMC
January 2019

SUMOylation modulates FOXK2-mediated paclitaxel sensitivity in breast cancer cells.

Oncogenesis 2018 Mar 13;7(3):29. Epub 2018 Mar 13.

Department of Surgery and Cancer, Imperial College London, Imperial Centre for Translational and Experimental Medicine (ICTEM), Du Cane Road, London, W12 0NN, UK.

The forkhead transcription factor FOXK2 plays a critical role in suppressing tumorigenesis and mediating cytotoxic drug action in breast cancer. However, the mechanism by which the biological function of FOXK2 is regulated remains poorly understood. Here, we investigated the role of SUMOylation in modulating FOXK2-mediated drug sensitivity. We identified SUMOylation consensus motifs within the FOXK2 sequence and constructed two SUMOylation-defective double mutants by converting lysine 527 and 633 to arginines and glutamic acid 529 and 635 to alanines, respectively. We found that both the FOXK2 SUMOylation-deficient (K527/633 R) and (E529/635 A) mutants were ineffective in mediating the cytotoxic function of paclitaxel when compared to the wild-type (WT) FOXK2. When overexpressed, unlike the wild-type (WT) FOXK2, the K527/633 R mutant had little effect on the sensitivity of MCF-7 and MDA-MB-231 cells to paclitaxel, as examined by cell viability and clonogenic assays. Our results also showed that MCF-7 cells overexpressing the K527/633 R mutant form of FOXK2 or the empty expression vector have lower protein and mRNA levels of its tumour suppressive transcriptional target FOXO3 compared to the wild-type FOXK2. Consistently, ChIP assays revealed that unlike wild-type FOXK2, the SUMOylation-defective (K527/633 R) mutant is unable to bind to the FOXO3 promoter, despite expressing comparable levels of protein and having the same subcellular localization as the wild-type FOXK2 in MCF-7 cells. Interestingly, expression of neither the wild-type nor the K527/633 R mutant FOXK2 had any effect on the proliferation and paclitaxel sensitivity of the MCF-7 Tax paclitaxel-resistant cells. In agreement, both the wild-type and the (K527/633 R) mutant FOXK2 failed to bind to the endogenous FOXO3 promoter in these cells. Collectively, our results suggest that SUMOylation positively regulates FOXK2 transcriptional activity and has a role in mediating the cytotoxic response to paclitaxel through the tumour suppressor FOXO3.
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http://dx.doi.org/10.1038/s41389-018-0038-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852961PMC
March 2018

Open chromatin profiling identifies AP1 as a transcriptional regulator in oesophageal adenocarcinoma.

PLoS Genet 2017 Aug 31;13(8):e1006879. Epub 2017 Aug 31.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.

Oesophageal adenocarcinoma (OAC) is one of the ten most prevalent forms of cancer and is showing a rapid increase in incidence and yet exhibits poor survival rates. Compared to many other common cancers, the molecular changes that occur in this disease are relatively poorly understood. However, genes encoding chromatin remodeling enzymes are frequently mutated in OAC. This is consistent with the emerging concept that cancer cells exhibit reprogramming of their chromatin environment which leads to subsequent changes in their transcriptional profile. Here, we have used ATAC-seq to interrogate the chromatin changes that occur in OAC using both cell lines and patient-derived material. We demonstrate that there are substantial changes in the regulatory chromatin environment in the cancer cells and using this data we have uncovered an important role for ETS and AP1 transcription factors in driving the changes in gene expression found in OAC cells.
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http://dx.doi.org/10.1371/journal.pgen.1006879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578490PMC
August 2017

EINCR1 is an EGF inducible lincRNA overexpressed in lung adenocarcinomas.

PLoS One 2017 21;12(7):e0181902. Epub 2017 Jul 21.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom.

Long non-coding RNAs are being increasingly recognised as important molecules involved in regulating a diverse array of biological functions. For example, many long non-coding RNAs have been associated with tumourigenesis and in this context their molecular functions often involves impacting on chromatin and transcriptional control processes. One important cellular control system that is often deregulated in cancer cells is the ERK MAP kinase pathway. Here we have investigated whether ERK pathway signaling in response to EGF stimulation, leads to changes in the production of long non-coding RNAs. We identify several different classes of EGF pathway-regulated lncRNAs. We focus on one of the inducible lincRNAs, EGF inducible long intergenic non-coding RNA 1 (EINCR1). EINCR1 is predominantly nuclear and shows delayed activation kinetics compared to other immediate-early EGF-inducible genes. In humans it is expressed in a tissue-specific manner and is mainly confined to the heart but it exhibits little evolutionary conservation. Importantly, in several cancers EINCR1 shows elevated expression levels which correlate with poor survival in lung adenocarcinoma patients. In the context of lung adenocarcinomas, EINCR1 expression is anti-correlated with the expression of several protein coding EGF-regulated genes. A potential functional connection is demonstrated as EINCR1 overexpression is shown to reduce the expression of EGF-regulated protein coding genes including FOS and FOSB.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181902PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5521836PMC
September 2017

RNF4 interacts with multiSUMOylated ETV4.

Wellcome Open Res 2016 17;1. Epub 2017 Feb 17.

Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PT, UK.

Protein SUMOylation represents an important regulatory event that changes the activities of numerous proteins. Recent evidence demonstrates that polySUMO chains can act as a trigger to direct the ubiquitin ligase RNF4 to substrates to cause their turnover through the ubiquitin pathway. RNF4 uses multiple SUMO interaction motifs (SIMs) to bind to these chains. However, in addition to polySUMO chains, a multimeric binding surface created by the simultaneous SUMOylation of multiple residues on a protein or complex could also provide a platform for the recruitment of multi-SIM proteins like RNF4. Here we demonstrate that multiSUMOylated ETV4 can bind to RNF4 and that a unique combination of SIMs is required for RNF4 to interact with this multiSUMOylated platform. Thus RNF4 can bind to proteins that are either polySUMOylated through a single site or multiSUMOylated on several sites and raises the possibility that such multiSIM-multiSUMO interactions might be more widespread.
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http://dx.doi.org/10.12688/wellcomeopenres.9935.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445624PMC
February 2017

The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding Proteins.

Methods Mol Biol 2016 ;1475:195-204

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester, M13 9PT, UK.

The use of in vitro assays, such as glutathione S-transferase (GST) pull-downs, enables the study of complex cellular processes in a simplified form. Pull-down assays facilitate the discovery and detailed study of protein-protein interactions, which can then be extrapolated to the cellular environment. Here, we describe the expression, purification and use of a multi-SUMO platform to identify SUMO-interacting proteins. This SUMO-platform can be easily expressed and purified from bacterial cells for use as baits in pull-down assays. This methodology facilitates the discovery of novel SUMO-binding proteins or further characterization of SUMO with known binding partners.
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http://dx.doi.org/10.1007/978-1-4939-6358-4_14DOI Listing
January 2018

Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

Sci Rep 2016 09 7;6:32417. Epub 2016 Sep 7.

Faculty of Medicine, University of Southampton, Southampton General Hospital, Mailpoint 801, South Academic Block, Tremona Road, Southampton, SO16 6YD, United Kingdom.

New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.
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http://dx.doi.org/10.1038/srep32417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013399PMC
September 2016

Jun-Mediated Changes in Cell Adhesion Contribute to Mouse Embryonic Stem Cell Exit from Ground State Pluripotency.

Stem Cells 2016 05 13;34(5):1213-24. Epub 2016 Feb 13.

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

Embryonic stem cells (ESC) are able to give rise to any somatic cell type. A lot is known about how ESC pluripotency is maintained, but comparatively less is known about how differentiation is promoted. Cell fate decisions are regulated by interactions between signaling and transcriptional networks. Recent studies have shown that the overexpression or downregulation of the transcription factor Jun can affect the ESC fate. Here we have focussed on the role of the Jun in the exit of mouse ESCs from ground state pluripotency and the onset of early differentiation. Transcriptomic analysis of differentiating ESCs reveals that Jun is required to upregulate a programme of genes associated with cell adhesion as ESCs exit the pluripotent ground state. Several of these Jun-regulated genes are shown to be required for efficient adhesion. Importantly this adhesion is required for the timely regulated exit of ESCs from ground state pluripotency and the onset of early differentiation events. Stem Cells 2016;34:1213-1224.
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http://dx.doi.org/10.1002/stem.2294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864893PMC
May 2016

Genome-wide binding studies reveal DNA binding specificity mechanisms and functional interplay amongst Forkhead transcription factors.

Nucleic Acids Res 2016 Feb 17;44(4):1566-78. Epub 2015 Nov 17.

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK

Transcription factors belonging to the same transcription factor families contain very similar DNA binding domains and hence have the potential to bind to related DNA sequences. However, subtle differences in binding specificities can be detected in vitro with the potential to direct specific responses in vivo. Here, we have examined the binding properties of three Forkhead (FOX) transcription factors, FOXK2, FOXO3 and FOXJ3 in vivo. Extensive overlap in chromatin binding is observed, although underlying differential DNA binding specificity can dictate the recruitment of FOXK2 and FOXJ3 to chromatin. However, functionally, FOXO3-dependent gene regulation is generally mediated not through uniquely bound regions but through regions occupied by both FOXK2 and FOXO3 where both factors play a regulatory role. Our data point to a model whereby FOX transcription factors control gene expression through dynamically binding and generating partial occupancy of the same site rather than mutually exclusive binding derived by stable binding of individual FOX proteins.
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http://dx.doi.org/10.1093/nar/gkv1120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770209PMC
February 2016

Protein kinase C coordinates histone H3 phosphorylation and acetylation.

Elife 2015 Oct 15;4:e09886. Epub 2015 Oct 15.

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly.
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http://dx.doi.org/10.7554/eLife.09886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714974PMC
October 2015

JNK-associated Leucine Zipper Protein Functions as a Docking Platform for Polo-like Kinase 1 and Regulation of the Associating Transcription Factor Forkhead Box Protein K1.

J Biol Chem 2015 Dec 14;290(49):29617-28. Epub 2015 Oct 14.

From the Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029,

JLP (JNK-associated leucine zipper protein) is a scaffolding protein that interacts with various signaling proteins associated with coordinated regulation of cellular process such as endocytosis, motility, neurite outgrowth, cell proliferation, and apoptosis. Here we identified PLK1 (Polo-like kinase 1) as a novel interaction partner of JLP through mass spectrometric approaches. Our results indicate that JLP is phospho-primed by PLK1 on Thr-351, which is recognized by the Polo box domain of PLK1 leading to phosphorylation of JLP at additional sites. Stable isotope labeling by amino acids in cell culture and quantitative LC-MS/MS analysis was performed to identify PLK1-dependent JLP-interacting proteins. Treatment of cells with the PLK1 kinase inhibitor BI2536 suppressed binding of the Forkhead box protein K1 (FOXK1) transcriptional repressor to JLP. JLP was found to interact with PLK1 and FOXK1 during mitosis. Moreover, knockdown of PLK1 affected the interaction between JLP and FOXK1. FOXK1 is a known transcriptional repressor of the CDK inhibitor p21/WAF1, and knockdown of JLP resulted in increased FOXK1 protein levels and a reduction of p21 transcript levels. Our results suggest a novel mechanism by which FOXK1 protein levels and activity are regulated by associating with JLP and PLK1.
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http://dx.doi.org/10.1074/jbc.M115.664649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705960PMC
December 2015

Screen for multi-SUMO-binding proteins reveals a multi-SIM-binding mechanism for recruitment of the transcriptional regulator ZMYM2 to chromatin.

Proc Natl Acad Sci U S A 2015 Sep 17;112(35):E4854-63. Epub 2015 Aug 17.

Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom;

Protein SUMOylation has emerged as an important regulatory event, particularly in nuclear processes such as transcriptional control and DNA repair. In this context, small ubiquitin-like modifier (SUMO) often provides a binding platform for the recruitment of proteins via their SUMO-interacting motifs (SIMs). Recent discoveries point to an important role for multivalent SUMO binding through multiple SIMs in the binding partner as exemplified by poly-SUMOylation acting as a binding platform for ubiquitin E3 ligases such as ring finger protein 4. Here, we have investigated whether other types of protein are recruited through multivalent SUMO interactions. We have identified dozens of proteins that bind to multi-SUMO platforms, thereby uncovering a complex potential regulatory network. Multi-SUMO binding is mediated through multi-SIM modules, and the functional importance of these interactions is demonstrated for the transcriptional corepressor ZMYM2/ZNF198 where its multi-SUMO-binding activity is required for its recruitment to chromatin.
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http://dx.doi.org/10.1073/pnas.1509716112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568223PMC
September 2015

Deregulation of the FOXM1 target gene network and its coregulatory partners in oesophageal adenocarcinoma.

Mol Cancer 2015 Mar 26;14:69. Epub 2015 Mar 26.

Faculty of Medical and Human Sciences, University of Manchester, Oxford Road, Manchester, UK.

Background: Survival rates for oesophageal adenocarcinoma (OAC) remain disappointingly poor and current conventional treatment modalities have minimal impact on long-term survival. This is partly due to a lack of understanding of the molecular changes that occur in this disease. Previous studies have indicated that the transcription factor FOXM1 is commonly upregulated in this cancer type but the impact of this overexpression on gene expression in the context of OAC is largely unknown. FOXM1 does not function alone but works alongside the antagonistically-functioning co-regulatory MMB and DREAM complexes.

Methods: To establish how FOXM1 affects gene expression in OAC we have identified the FOXM1 target gene network in OAC-derived cells using ChIP-seq and determined the expression of both its coregulatory partners and members of this target gene network in OAC by digital transcript counting using the Nanostring gene expression assay.

Results: We find co-upregulation of FOXM1 with its target gene network in OAC. Furthermore, we find changes in the expression of its coregulatory partners, including co-upregulation of LIN9 and, surprisingly, reduced expression of LIN54. Mechanistically, we identify LIN9 as the direct binding partner for FOXM1 in the MMB complex. In the context of OAC, both coregulator (eg LIN54) and target gene (eg UHRF1) expression levels are predictive of disease stage.

Conclusions: Together our data demonstrate that there are global changes to the FOXM1 regulatory network in OAC and the expression of components of this network help predict cancer prognosis.
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http://dx.doi.org/10.1186/s12943-015-0339-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392876PMC
March 2015

The ubiquitin ligase UBE3A dampens ERK pathway signalling in HPV E6 transformed HeLa cells.

PLoS One 2015 27;10(3):e0119366. Epub 2015 Mar 27.

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, United Kingdom.

Signalling through the ERK MAP kinase pathway plays an important role in many biological processes and it is often deregulated in disease states such as cancer. One major effect of MAP kinase signalling is to promote gene expression through the phosphorylation and activation of transcription factors like ELK1. ELK1 in turn controls the activity of immediate-early genes such as FOS. Here we have used ELK1 activation in HeLa cells as a read out to conduct a genome-wide siRNA screen to identify negative regulators of ERK-mediated immediate-early gene activation. One of the candidates that we identified was the E3 ubiquitin ligase UBE3A/E6-AP. Reductions in UBE3A levels cause increased basal levels of ERK activity, a loss of growth factor-mediated ERK activation and concomitant defects in immediate-early gene expression. Thus, UBE3A acts to dampen down basal level ERK activation and to prime the pathway for growth factor-mediated activation. Mechanistically, we demonstrate that UBE3A functions in HeLa cells through its binding partner, HPV18 E6 protein and the E6 target protein p53. Loss of either E6 or p53 blocks the effect of UBE3A depletion on ERK pathway signalling, indicating that in the context of oncogenic viral protein expression, UBE3A plays an important role in negating the consequences of p53 activation on ERK pathway signalling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119366PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4376912PMC
February 2016

Changing partners: transcription factors form different complexes on and off chromatin.

Mol Syst Biol 2015 Jan 21;11(1):782. Epub 2015 Jan 21.

Faculty of Life Sciences, University of Manchester, Manchester, UK.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332156PMC
http://dx.doi.org/10.15252/msb.20145936DOI Listing
January 2015

Otx2 and Oct4 drive early enhancer activation during embryonic stem cell transition from naive pluripotency.

Cell Rep 2014 Jun 12;7(6):1968-81. Epub 2014 Jun 12.

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK. Electronic address:

Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.
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http://dx.doi.org/10.1016/j.celrep.2014.05.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074343PMC
June 2014

The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex.

Nucleic Acids Res 2014 Jun 19;42(10):6232-42. Epub 2014 Apr 19.

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK

There are numerous forkhead transcription factors in mammalian cells but we know little about the molecular functions of the majority of these. FOXK2 is a ubiquitously expressed family member suggesting an important function across multiple cell types. Here, we show that FOXK2 binds to the SIN3A and PR-DUB complexes. The PR-DUB complex contains the important tumour suppressor protein, the deubiquitinase BAP1. FOXK2 recruits BAP1 to DNA, promotes local histone deubiquitination and causes changes in target gene activity. Our results therefore provide an important link between BAP1 and the transcription factor FOXK2 and demonstrate how BAP1 can be recruited to specific regulatory loci.
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http://dx.doi.org/10.1093/nar/gku274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041447PMC
June 2014