Publications by authors named "Michael Lie-A-Ling"

21 Publications

  • Page 1 of 1

Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia.

J Hematol Oncol 2021 Mar 20;14(1):47. Epub 2021 Mar 20.

Univ Rennes 1, CNRS, IGDR (Institut de génétique et développement de Rennes) - UMR 6290, 35000, Rennes, France.

Background: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia.

Methods: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models.

Results: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation.

Conclusions: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.
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http://dx.doi.org/10.1186/s13045-021-01051-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7981807PMC
March 2021

Contributions of Embryonic HSC-Independent Hematopoiesis to Organogenesis and the Adult Hematopoietic System.

Front Cell Dev Biol 2021 18;9:631699. Epub 2021 Feb 18.

Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Macclesfield, United Kingdom.

During ontogeny, the establishment of the hematopoietic system takes place in several phases, separated both in time and location. The process is initiated extra-embryonically in the yolk sac (YS) and concludes in the main arteries of the embryo with the formation of hematopoietic stem cells (HSC). Initially, it was thought that HSC-independent hematopoietic YS cells were transient, and only required to bridge the gap to HSC activity. However, in recent years it has become clear that these cells also contribute to embryonic organogenesis, including the emergence of HSCs. Furthermore, some of these early HSC-independent YS cells persist into adulthood as distinct hematopoietic populations. These previously unrecognized abilities of embryonic HSC-independent hematopoietic cells constitute a new field of interest. Here, we aim to provide a succinct overview of the current knowledge regarding the contribution of YS-derived hematopoietic cells to the development of the embryo and the adult hematopoietic system.
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http://dx.doi.org/10.3389/fcell.2021.631699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930747PMC
February 2021

CUL2 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.

EMBO Rep 2021 Mar 15;22(3):e52164. Epub 2021 Feb 15.

The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK.

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.
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http://dx.doi.org/10.15252/embr.202052164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926238PMC
March 2021

RUNX1 Dosage in Development and Cancer.

Mol Cells 2020 Feb;43(2):126-138

Cancer Research UK Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Macclesfield, SK10 4TG, UK.

The transcription factor RUNX1 first came to prominence due to its involvement in the t(8;21) translocation in acute myeloid leukemia (AML). Since this discovery, RUNX1 has been shown to play important roles not only in leukemia but also in the ontogeny of the normal hematopoietic system. Although it is currently still challenging to fully assess the different parameters regulating RUNX1 dosage, it has become clear that the dose of RUNX1 can greatly affect both leukemia and normal hematopoietic development. It is also becoming evident that varying levels of RUNX1 expression can be used as markers of tumor progression not only in the hematopoietic system, but also in non-hematopoietic cancers. Here, we provide an overview of the current knowledge of the effects of RUNX1 dosage in normal development of both hematopoietic and epithelial tissues and their associated cancers.
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http://dx.doi.org/10.14348/molcells.2019.0301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057845PMC
February 2020

Identification of gene specific cis-regulatory elements during differentiation of mouse embryonic stem cells: An integrative approach using high-throughput datasets.

PLoS Comput Biol 2019 11 4;15(11):e1007337. Epub 2019 Nov 4.

School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.

Gene expression governs cell fate, and is regulated via a complex interplay of transcription factors and molecules that change chromatin structure. Advances in sequencing-based assays have enabled investigation of these processes genome-wide, leading to large datasets that combine information on the dynamics of gene expression, transcription factor binding and chromatin structure as cells differentiate. While numerous studies focus on the effects of these features on broader gene regulation, less work has been done on the mechanisms of gene-specific transcriptional control. In this study, we have focussed on the latter by integrating gene expression data for the in vitro differentiation of murine ES cells to macrophages and cardiomyocytes, with dynamic data on chromatin structure, epigenetics and transcription factor binding. Combining a novel strategy to identify communities of related control elements with a penalized regression approach, we developed individual models to identify the potential control elements predictive of the expression of each gene. Our models were compared to an existing method and evaluated using the existing literature and new experimental data from embryonic stem cell differentiation reporter assays. Our method is able to identify transcriptional control elements in a gene specific manner that reflect known regulatory relationships and to generate useful hypotheses for further testing.
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http://dx.doi.org/10.1371/journal.pcbi.1007337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855567PMC
November 2019

RUNX transcription factors: orchestrators of development.

Development 2019 09 5;146(17). Epub 2019 Sep 5.

Cancer Research UK Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park, Alderley Edge, Macclesfield SK10 4TG, UK

RUNX transcription factors orchestrate many different aspects of biology, including basic cellular and developmental processes, stem cell biology and tumorigenesis. In this Primer, we introduce the molecular hallmarks of the three mammalian RUNX genes, RUNX1, RUNX2 and RUNX3, and discuss the regulation of their activities and their mechanisms of action. We then review their crucial roles in the specification and maintenance of a wide array of tissues during embryonic development and adult homeostasis.
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http://dx.doi.org/10.1242/dev.148296DOI Listing
September 2019

Regulation of RUNX1 dosage is crucial for efficient blood formation from hemogenic endothelium.

Development 2018 03 12;145(5). Epub 2018 Mar 12.

Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK

During ontogeny, hematopoietic stem and progenitor cells arise from hemogenic endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in hemogenic endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects hemogenic endothelium differentiation. The use of inducible expression combined with alterations in the expression of the RUNX1 co-factor CBFβ allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in hemogenic endothelium maturation and the establishment of the hematopoietic system.
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http://dx.doi.org/10.1242/dev.149419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868988PMC
March 2018

Interplay between SOX7 and RUNX1 regulates hemogenic endothelial fate in the yolk sac.

Development 2016 12 17;143(23):4341-4351. Epub 2016 Oct 17.

Stem Cell Hematopoiesis, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX, UK

Endothelial to hematopoietic transition (EHT) is a dynamic process involving the shutting down of endothelial gene expression and switching on of hematopoietic gene transcription. Although the factors regulating EHT in hemogenic endothelium (HE) of the dorsal aorta have been relatively well studied, the molecular regulation of yolk sac HE remains poorly understood. Here, we show that SOX7 inhibits the expression of RUNX1 target genes in HE, while having no effect on RUNX1 expression itself. We establish that SOX7 directly interacts with RUNX1 and inhibits its transcriptional activity. Through this interaction we demonstrate that SOX7 hinders RUNX1 DNA binding as well as the interaction between RUNX1 and its co-factor CBFβ. Finally, we show by single-cell expression profiling and immunofluorescence that SOX7 is broadly expressed across the RUNX1 yolk sac HE population compared with SOX17. Collectively, these data demonstrate for the first time how direct protein-protein interactions between endothelial and hematopoietic transcription factors regulate contrasting transcriptional programs during HE differentiation and EHT.
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http://dx.doi.org/10.1242/dev.140970DOI Listing
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

New insights into the regulation by RUNX1 and GFI1(s) proteins of the endothelial to hematopoietic transition generating primordial hematopoietic cells.

Cell Cycle 2016 Aug 11;15(16):2108-2114. Epub 2016 Jul 11.

a CRUK Stem Cell Biology, Cancer Research UK Manchester Institute , Manchester , UK.

The first hematopoietic cells are generated very early in ontogeny to support the growth of the embryo and to provide the foundation to the adult hematopoietic system. There is a considerable therapeutic interest in understanding how these first blood cells are generated in order to try to reproduce this process in vitro. This would allow generating blood products, or hematopoietic cell populations from embryonic stem (ES) cells, induced pluripotent stem cells or through directed reprogramming. Recent studies have clearly established that the first hematopoietic cells originate from a hemogenic endothelium (HE) through an endothelial to hematopoietic transition (EHT). The molecular mechanisms underlining this transition remain largely unknown with the exception that the transcription factor RUNX1 is critical for this process. In this Extra Views report, we discuss our recent studies demonstrating that the transcriptional repressors GFI1 and GFI1B have a critical role in the EHT. We established that these RUNX1 transcriptional targets are actively implicated in the downregulation of the endothelial program and the loss of endothelial identity during the formation of the first blood cells. In addition, our results suggest that GFI1 expression provides an ideal novel marker to identify, isolate and study the HE cell population.
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http://dx.doi.org/10.1080/15384101.2016.1203491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993433PMC
August 2016

Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation.

Dev Cell 2016 Mar 25;36(5):572-87. Epub 2016 Feb 25.

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

Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.
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http://dx.doi.org/10.1016/j.devcel.2016.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780867PMC
March 2016

Expression of the MOZ-TIF2 oncoprotein in mice represses senescence.

Exp Hematol 2016 Apr 5;44(4):231-7.e4. Epub 2016 Feb 5.

Cancer Research UK Stem Cell Biology Group, CR-UK Manchester Institute, University of Manchester, Manchester, UK. Electronic address:

The MOZ-TIF2 translocation, which fuses monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase (HAT) with the nuclear co-activator TIF2, is associated with the development of acute myeloid leukemia. We recently found that in the absence of MOZ HAT activity, p16(INK4a) transcriptional levels are significantly increased, triggering an early entrance into replicative senescence. Because oncogenic fusion proteins must bypass cellular safeguard mechanisms, such as senescence and apoptosis, to induce leukemia, we hypothesized that this repressive activity of MOZ over p16(INK4a) transcription could be preserved, or even reinforced, in MOZ leukemogenic fusion proteins, such as MOZ-TIF2. We describe here that, indeed, MOZ-TIF2 silences expression of the CDKN2A locus (p16(INK4a) and p19(ARF)), inhibits the triggering of senescence and enhances proliferation, providing conditions favorable to the development of leukemia. Furthermore, we describe that abolishing the MOZ HAT activity of the fusion protein leads to a significant increase in expression of the CDKN2A locus and the number of hematopoietic progenitors undergoing senescence. Finally, we report that inhibition of senescence by MOZ-TIF2 is associated with increased apoptosis, suggesting a role for the fusion protein in p53 apoptosis-versus-senescence balance. Our results underscore the importance of the HAT activity of MOZ, preserved in the fusion protein, for repression of the CDKN2A locus transcription and the subsequent block of senescence, a necessary step for the survival of leukemic cells.
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http://dx.doi.org/10.1016/j.exphem.2015.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819447PMC
April 2016

RUNX1 positively regulates a cell adhesion and migration program in murine hemogenic endothelium prior to blood emergence.

Blood 2014 Sep 31;124(11):e11-20. Epub 2014 Jul 31.

Cancer Research UK Stem Cell Biology Group, and.

During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called hemogenic endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic program and downregulation of the endothelial program. However, due to the rare and transient nature of the HE, little is known about the initial role of RUNX1 within this population. We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE. This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development.
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http://dx.doi.org/10.1182/blood-2014-04-572958DOI Listing
September 2014

MOZ-mediated repression of p16(INK) (4) (a) is critical for the self-renewal of neural and hematopoietic stem cells.

Stem Cells 2014 Jun;32(6):1591-601

Cancer Research UK Stem Cell Biology Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, United Kingdom.

Although inhibition of p16(INK4a) expression is critical to preserve the proliferative capacity of stem cells, the molecular mechanisms responsible for silencing p16(INK4a) expression remain poorly characterized. Here, we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16(INK4a) . In the absence of the HAT activity of MOZ, expression of p16(INK4a) is upregulated in progenitor and stem cells, inducing an early entrance into replicative senescence. Genetic deletion of p16(INK4a) reverses the proliferative defect in both Moz(HAT) (-) (/) (-) hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16(INK4a) expression and to protect stem cells from early entrance into replicative senescence.
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http://dx.doi.org/10.1002/stem.1606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237135PMC
June 2014

Smooth muscle cells largely develop independently of functional hemogenic endothelium.

Stem Cell Res 2014 Jan 5;12(1):222-32. Epub 2013 Nov 5.

Cancer Research UK Stem Cell Biology Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK. Electronic address:

Vascular smooth muscle cells represent a major component of the cardiovascular system. In vitro studies have shown that FLK1(+) cells derived from embryonic stem (ES) cells can differentiate into both endothelial and smooth muscle cells. These FLK1(+) cells also contain a mesodermal precursor, the hemangioblast, able to produce endothelial, blood and smooth muscle cells. The generation of blood precursors from the hemangioblast was recently shown to occur through a transient cell population of specialised endothelium, a hemogenic endothelium. To date, the lineage relationship between this cell population and smooth muscle cell progenitors has not been investigated. In this study, we generated a reporter ES cell line in which expression of the fluorescent protein H2B-VENUS is driven by the α-smooth muscle actin (α-SMA) regulatory sequences. We demonstrated that this reporter cell line efficiently trace smooth muscle development during ES cell differentiation. Although some smooth muscle cells are associated with broad endothelial development, we established that smooth muscle cells are mostly generated independently from a specialised functional hemogenic endothelium. This study provides new and important insights into hematopoietic and vascular development, which may help in driving further progress towards the development of bioengineered vascular grafts for regenerative medicine.
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http://dx.doi.org/10.1016/j.scr.2013.10.009DOI Listing
January 2014

The MYSTerious MOZ, a histone acetyltransferase with a key role in haematopoiesis.

Immunology 2013 Jun;139(2):161-5

Cancer Research UK Stem Cell Biology Group, Paterson Institute for Cancer Research, The University of Manchester, Manchester, UK.

The MOnocytic leukaemia Zing finger (MOZ; MYST3 or KAT6A(1)) gene is frequently found translocated in acute myeloid leukaemia. MOZ encodes a large multidomain protein that contains, besides others, a histone acetyl transferase catalytic domain. Several studies have now established the critical function of MOZ in haematopoiesis. In this review we summarize the recent findings that underscore the relevance of the different biological activities of MOZ in the regulation of haematopoiesis.
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http://dx.doi.org/10.1111/imm.12072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647182PMC
June 2013

RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis.

EMBO J 2012 Nov 12;31(22):4318-33. Epub 2012 Oct 12.

Section of Experimental Haematology, Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK.

Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a Runx1-/- embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPβ and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies.
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http://dx.doi.org/10.1038/emboj.2012.275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501222PMC
November 2012

Identification and characterization of a novel transcriptional target of RUNX1/AML1 at the onset of hematopoietic development.

Blood 2011 Jul 15;118(3):594-7. Epub 2011 Apr 15.

Cancer Research UK Stem Cell Biology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom.

Although the critical requirement for the transcription factor RUNX1/AML1 at the onset of hematopoietic development is well established, little is known about its transcriptional targets at this pivotal stage of blood development. Using microarrays, we identified the uncharacterized gene AI467606 as a gene whose expression level is dramatically reduced in the absence of RUNX1. We further demonstrated by chromatin immunoprecipitation and promoter assay a direct regulation of its transcription by RUNX1. Using a bacterial artificial chromosome transgenic approach, we established that AI467606 is expressed during the development of the hematopoietic system in vivo and in vitro and that its expression is detected within the CD41(+) population and marks definitive hematopoietic potential. Similarly, in the adult mouse, all hematopoietic cell lineages, except mature erythrocytes, express AI467606. Taken together, these findings indicate that AI467606 is a novel transcriptional target of RUNX1/AML1 at the onset of hematopoietic development that is extensively expressed within the hematopoietic system.
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http://dx.doi.org/10.1182/blood-2010-06-294124DOI Listing
July 2011

Cytokine stimulation and the choice of promoter are critical factors for the efficient transduction of mouse T cells with HIV-1 vectors.

J Gene Med 2010 Feb;12(2):129-36

Cell Therapy Group, Cancer Research UK Department of Medical Oncology, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.

Background: HIV-1 fails to successfully infect mouse T cells as a result of several blocks in the viral replication cycle. We investigated whether this also impacted on the use of HIV-1 derived lentiviral vectors for stable gene transfer into mouse T cells.

Methods: Freshly isolated primary mouse T cells were immediately mixed with lentiviral vectors encoding an enhanced green fluorescent protein marker gene and transduction frequency was determined after 5 days of culture.

Results: Optimal transduction required both mouse T cell activation and cytokine support. Furthermore, transduction was also dependent upon the promoter chosen, with the rank order of potency being PGK > EF1 > SFFV > CMV. HIV-1 lentiviral vectors also efficiently transduced cytokine-stimulated T cells (in the absence of antibody driven T cell activation), albeit with a lower level of transgene expression compared to fully-activated T cells.

Conclusions: The present study demonstrates that primary mouse T cells can be efficiently transduced with HIV-1 lentiviral vectors, opening up prospects for their use in mouse models of gene-modified adoptive cellular therapy.
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http://dx.doi.org/10.1002/jgm.1421DOI Listing
February 2010

Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification.

Blood 2009 Nov 2;114(23):4813-22. Epub 2009 Oct 2.

Cancer Research UK, Stem Cell Haematopoiesis Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, United Kingdom.

The molecular mechanisms that regulate the balance between proliferation and differentiation of precursors at the onset of hematopoiesis specification are poorly understood. By using a global gene expression profiling approach during the course of embryonic stem cell differentiation, we identified Sox7 as a potential candidate gene involved in the regulation of blood lineage formation from the mesoderm germ layer. In the present study, we show that Sox7 is transiently expressed in mesodermal precursors as they undergo specification to the hematopoietic program. Sox7 knockdown in vitro significantly decreases the formation of both primitive erythroid and definitive hematopoietic progenitors as well as endothelial progenitors. In contrast, Sox7-sustained expression in the earliest committed hematopoietic precursors promotes the maintenance of their multipotent and self-renewing status. Removal of this differentiation block driven by Sox7-enforced expression leads to the efficient differentiation of hematopoietic progenitors to all erythroid and myeloid lineages. This study identifies Sox7 as a novel and important player in the molecular regulation of the first committed blood precursors. Furthermore, our data demonstrate that the mere sustained expression of Sox7 is sufficient to completely alter the balance between proliferation and differentiation at the onset of hematopoiesis.
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http://dx.doi.org/10.1182/blood-2009-06-226290DOI Listing
November 2009

Selection of tumour specific promoters for adenoviral gene therapy of cholangiocarcinoma.

J Hepatol 2006 Jan 11;44(1):126-33. Epub 2005 Jul 11.

Academic Medical Center, AMC Liver Center, Meibergdreef 69-71 (S1-168), 1105 BK Amsterdam, The Netherlands.

Background/aims: The majority of cholangiocarcinoma patients present with advanced incurable disease. Therefore development of new therapeutic modalities including adenoviral gene therapy is of paramount importance. We set out to identify tumour specific promoters which have low activity in human liver cells and retain their specificity in an adenoviral vector.

Methods: mRNA levels of cyclo-oxygenase-2, cytokeratin-19, mucin-1, midkine and telomerase reverse transcriptase (TERT) were determined in human liver, cholangiocarcinoma (resection specimens and cell lines), primary human hepatocytes, cholangiocytes and endothelial cells by Reverse Transcriptase-quantitative PCR. The activity of candidate promoters in adenoviral vectors was then determined in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes.

Results: mRNA levels of all tested tumour markers were significantly higher in cholangiocarcinoma than in normal liver. Based on low expression in hepatocytes, either in combination with low expression in primary cholangiocytes or endothelial cells, the cytokeratin-19, mucin-1 and TERT promoters were selected for further analyses. In an adenoviral vector, the activity of the TERT or cytokeratin-19 promoters were low in normal human hepatocytes and cholangiocytes, and high in cholangiocarcinoma cell lines.

Conclusions: The TERT and Cytokeratin-19 promoters are highly expressed in cholangiocarcinoma and seem suitable to restrict adenoviral gene therapy to these intra-hepatic tumours.
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http://dx.doi.org/10.1016/j.jhep.2005.06.007DOI Listing
January 2006