Publications by authors named "Georges Lacaud"

84 Publications

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 marks a luminal castration-resistant lineage established at the onset of prostate development.

Elife 2020 10 7;9. Epub 2020 Oct 7.

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

The characterization of prostate epithelial hierarchy and lineage heterogeneity is critical to understand its regenerative properties and malignancies. Here, we report that the transcription factor RUNX1 marks a specific subpopulation of proximal luminal cells (PLCs), enriched in the periurethral region of the developing and adult mouse prostate, and distinct from the previously identified NKX3.1 luminal castration-resistant cells. Using scRNA-seq profiling and genetic lineage tracing, we show that RUNX1 PLCs are unaffected by androgen deprivation, and do not contribute to the regeneration of the distal luminal compartments. Furthermore, we demonstrate that a transcriptionally similar RUNX1 population emerges at the onset of embryonic prostate specification to populate the proximal region of the ducts. Collectively, our results reveal that RUNX1 PLCs is an intrinsic castration-resistant and self-sustained lineage that emerges early during prostate development and provide new insights into the lineage relationships of the prostate epithelium.
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http://dx.doi.org/10.7554/eLife.60225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644213PMC
October 2020

Alternative Enhancer Usage and Targeted Polycomb Marking Hallmark Promoter Choice during T Cell Differentiation.

Cell Rep 2020 08;32(7):108048

Institut de Génétique Moléculaire de Montpellier, Université de Montpellier, CNRS, 1919 Route de Mende, Montpellier 34293, France. Electronic address:

During thymic development and upon peripheral activation, T cells undergo extensive phenotypic and functional changes coordinated by lineage-specific developmental programs. To characterize the regulatory landscape controlling T cell identity, we perform a wide epigenomic and transcriptional analysis of mouse thymocytes and naive CD4 differentiated T helper cells. Our investigations reveal a dynamic putative enhancer landscape, and we could validate many of the enhancers using the high-throughput CapStarr sequencing (CapStarr-seq) approach. We find that genes using multiple promoters display increased enhancer usage, suggesting that apparent "enhancer redundancy" might relate to isoform selection. Furthermore, we can show that two Runx3 promoters display long-range interactions with specific enhancers. Finally, our analyses suggest a novel function for the PRC2 complex in the control of alternative promoter usage. Altogether, our study has allowed for the mapping of an exhaustive set of active enhancers and provides new insights into their function and that of PRC2 in controlling promoter choice during T cell differentiation.
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http://dx.doi.org/10.1016/j.celrep.2020.108048DOI Listing
August 2020

Decoding hematopoietic stem cells' birth.

Authors:
Georges Lacaud

Blood 2020 08;136(7):775-776

University of Manchester.

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http://dx.doi.org/10.1182/blood.2020006612DOI Listing
August 2020

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

Transcriptional control of blood cell emergence.

FEBS Lett 2019 12 31;593(23):3304-3315. Epub 2019 Aug 31.

Developmental Haematopoiesis Group, Faculty of Biology, Medicine and Health, the University of Manchester, UK.

The haematopoietic system is established during embryonic life through a series of developmental steps that culminates with the generation of haematopoietic stem cells. Characterisation of the transcriptional network that regulates blood cell emergence has led to the identification of transcription factors essential for this process. Among the many factors wired within this complex regulatory network, ETV2, SCL and RUNX1 are the central components. All three factors are absolutely required for blood cell generation, each one controlling a precise step of specification from the mesoderm germ layer to fully functional blood progenitors. Insight into the transcriptional control of blood cell emergence has been used for devising protocols to generate blood cells de novo, either through reprogramming of somatic cells or through forward programming of pluripotent stem cells. Interestingly, the physiological process of blood cell generation and its laboratory-engineered counterpart have very little in common.
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http://dx.doi.org/10.1002/1873-3468.13585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916194PMC
December 2019

Author Correction: Runx/Cbfβ complexes protect group 2 innate lymphoid cells from exhausted-like hyporesponsiveness during allergic airway inflammation.

Nat Commun 2019 03 1;10(1):1075. Epub 2019 Mar 1.

Laboratory for Transcriptional Regulation, RIKEN Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.

The original version of this Article contained an error in the author affiliations. Affiliation 5 incorrectly read 'Laboratory for Prediction of Cell Systems Dynamics, RIKEN Center for Biosystems Dynamics Research (BDR), Suite, Hyogo 565-0874, Japan.' This has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-019-08932-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397247PMC
March 2019

Runx/Cbfβ complexes protect group 2 innate lymphoid cells from exhausted-like hyporesponsiveness during allergic airway inflammation.

Nat Commun 2019 01 25;10(1):447. Epub 2019 Jan 25.

Laboratory for Transcriptional Regulation, RIKEN Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.

Group 2 innate lymphoid cells (ILC2s) have tissue-resident competence and contribute to the pathogenesis of allergic diseases. However, the mechanisms regulating prolonged ILC2-mediated T2 cytokine production under chronic inflammatory conditions are unclear. Here we show that, at homeostasis, Runx deficiency induces excessive ILC2 activation due to overly active GATA-3 functions. By contrast, during allergic inflammation, the absence of Runx impairs the ability of ILC2s to proliferate and produce effector T2 cytokines and chemokines. Instead, functional deletion of Runx induces the expression of exhaustion markers, such as IL-10 and TIGIT, on ILC2s. Finally, these 'exhausted-like' ILC2s are unable to induce type 2 immune responses to repeated allergen exposures. Thus, Runx confers competence for sustained ILC2 activity at the mucosa, and contributes to allergic pathogenesis.
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http://dx.doi.org/10.1038/s41467-019-08365-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347616PMC
January 2019

Early Human Hemogenic Endothelium Generates Primitive and Definitive Hematopoiesis In Vitro.

Stem Cell Reports 2018 11 25;11(5):1061-1074. Epub 2018 Oct 25.

Developmental Haematopoiesis Group, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, UK. Electronic address:

The differentiation of human embryonic stem cells (hESCs) to hematopoietic lineages initiates with the specification of hemogenic endothelium, a transient specialized endothelial precursor of all blood cells. This in vitro system provides an invaluable model to dissect the emergence of hematopoiesis in humans. However, the study of hematopoiesis specification is hampered by a lack of consensus in the timing of hemogenic endothelium analysis and the full hematopoietic potential of this population. Here, our data reveal a sharp decline in the hemogenic potential of endothelium populations isolated over the course of hESC differentiation. Furthermore, by tracking the dynamic expression of CD31 and CD235a at the onset of hematopoiesis, we identified three populations of hematopoietic progenitors, representing primitive and definitive subsets that all emerge from the earliest specified hemogenic endothelium. Our data establish that hemogenic endothelium populations endowed with primitive and definitive hematopoietic potential are specified simultaneously from the mesoderm in differentiating hESCs.
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http://dx.doi.org/10.1016/j.stemcr.2018.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234921PMC
November 2018

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

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

Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University, Brewery Lane, Newcastle upon Tyne NE1 7RU, UK; Princess Maxima Center for Pediatric Oncology, Utrecht 3584CS, the Netherlands. 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

Single-cell transcriptomics reveal the dynamic of haematopoietic stem cell production in the aorta.

Nat Commun 2018 06 28;9(1):2517. Epub 2018 Jun 28.

Hubrecht Institute-KNAW, University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.

Haematopoietic stem cells (HSCs) are generated from haemogenic endothelial (HE) cells via the formation of intra-aortic haematopoietic clusters (IAHCs) in vertebrate embryos. The molecular events controlling endothelial specification, endothelial-to-haematopoietic transition (EHT) and IAHC formation, as it occurs in vivo inside the aorta, are still poorly understood. To gain insight in these processes, we performed single-cell RNA-sequencing of non-HE cells, HE cells, cells undergoing EHT, IAHC cells, and whole IAHCs isolated from mouse embryo aortas. Our analysis identified the genes and transcription factor networks activated during the endothelial-to-haematopoietic switch and IAHC cell maturation toward an HSC fate. Our study provides an unprecedented complete resource to study in depth HSC generation in vivo. It will pave the way for improving HSC production in vitro to address the growing need for tailor-made HSCs to treat patients with blood-related disorders.
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http://dx.doi.org/10.1038/s41467-018-04893-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023921PMC
June 2018

HDAC1 and HDAC2 Modulate TGF-β Signaling during Endothelial-to-Hematopoietic Transition.

Stem Cell Reports 2018 04;10(4):1369-1383

CRUK Stem Cell Biology Group, CRUK Manchester Institute, 555 Wilmslow Road, Manchester M20 4GJ, UK. Electronic address:

The first hematopoietic stem and progenitor cells are generated during development from hemogenic endothelium (HE) through trans-differentiation. The molecular mechanisms underlying this endothelial-to-hematopoietic transition (EHT) remain poorly understood. Here, we explored the role of the epigenetic regulators HDAC1 and HDAC2 in the emergence of these first blood cells in vitro and in vivo. Loss of either of these epigenetic silencers through conditional genetic deletion reduced hematopoietic transition from HE, while combined deletion was incompatible with blood generation. We investigated the molecular basis of HDAC1 and HDAC2 requirement and identified TGF-β signaling as one of the pathways controlled by HDAC1 and HDAC2. Accordingly, we experimentally demonstrated that activation of this pathway in HE cells reinforces hematopoietic development. Altogether, our results establish that HDAC1 and HDAC2 modulate TGF-β signaling and suggest that stimulation of this pathway in HE cells would be beneficial for production of hematopoietic cells for regenerative therapies.
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http://dx.doi.org/10.1016/j.stemcr.2018.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998800PMC
April 2018

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

HOXB4 Promotes Hemogenic Endothelium Formation without Perturbing Endothelial Cell Development.

Stem Cell Reports 2018 03 15;10(3):875-889. Epub 2018 Feb 15.

Institute for Transfusion Medicine, University Hospital Essen, Virchowstraße 179, 45147 Essen, Germany. Electronic address:

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro, is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1 ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of hemogenic endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression.
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http://dx.doi.org/10.1016/j.stemcr.2018.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919293PMC
March 2018

A novel prospective isolation of murine fetal liver progenitors to study in utero hematopoietic defects.

PLoS Genet 2018 01 4;14(1):e1007127. Epub 2018 Jan 4.

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

In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers' expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible.
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http://dx.doi.org/10.1371/journal.pgen.1007127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754050PMC
January 2018

Primitive erythrocytes are generated from hemogenic endothelial cells.

Sci Rep 2017 07 25;7(1):6401. Epub 2017 Jul 25.

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

Primitive erythroblasts are the first blood cells generated during embryonic hematopoiesis. Tracking their emergence both in vivo and in vitro has remained challenging due to the lack of specific cell surface markers. To selectively investigate primitive erythropoiesis, we have engineered a new transgenic embryonic stem (ES) cell line, where eGFP expression is driven by the regulatory sequences of the embryonic βH1 hemoglobin gene expressed specifically in primitive erythroid cells. Using this ES cell line, we observed that the first primitive erythroblasts are detected in vitro around day 1.5 of blast colony differentiation, within the cell population positive for the early hematopoietic progenitor marker CD41. Moreover, we establish that these eGFP cells emerge from a hemogenic endothelial cell population similarly to their definitive hematopoietic counterparts. We further generated a corresponding βH1-eGFP transgenic mouse model and demonstrated the presence of a primitive erythroid primed hemogenic endothelial cell population in the developing embryo. Taken together, our findings demonstrate that both in vivo and in vitro primitive erythrocytes are generated from hemogenic endothelial cells.
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http://dx.doi.org/10.1038/s41598-017-06627-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526883PMC
July 2017

SOX7 expression is critically required in FLK1-expressing cells for vasculogenesis and angiogenesis during mouse embryonic development.

Mech Dev 2017 08 31;146:31-41. Epub 2017 May 31.

Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow road, M20 4BX, UK; Division of Developmental Biology and Medicine, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. Electronic address:

The transcriptional program that regulates the differentiation of endothelial precursor cells into a highly organized vascular network is still poorly understood. Here we explore the role of SOX7 during this process, performing a detailed analysis of the vascular defects resulting from either a complete deficiency in Sox7 expression or from the conditional deletion of Sox7 in FLK1-expressing cells. We analysed the consequence of Sox7 deficiency from E7.5 onward to determine from which stage of development the effect of Sox7 deficiency can be observed. We show that while Sox7 is expressed at the onset of endothelial specification from mesoderm, Sox7 deficiency does not impact the emergence of the first endothelial progenitors. However, by E8.5, clear signs of defective vascular development are already observed with the presence of highly unorganised endothelial cords rather than distinct paired dorsal aorta. By E10.5, both Sox7 complete knockout and FLK1-specific deletion of Sox7 lead to widespread vascular defects. In contrast, while SOX7 is expressed in the earliest specified blood progenitors, the VAV-specific deletion of Sox7 does not affect the hematopoietic system. Together, our data reveal the unique role of SOX7 in vasculogenesis and angiogenesis during embryonic development.
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http://dx.doi.org/10.1016/j.mod.2017.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496588PMC
August 2017

Mouse RUNX1C regulates premegakaryocytic/erythroid output and maintains survival of megakaryocyte progenitors.

Blood 2017 07 10;130(3):271-284. Epub 2017 May 10.

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

RUNX1 is crucial for the regulation of megakaryocyte specification, maturation, and thrombopoiesis. possesses 2 promoters: the distal and proximal promoters. The major protein isoforms generated by and are RUNX1C and RUNX1B, respectively, which differ solely in their -terminal amino acid sequences. RUNX1C is the most abundantly expressed isoform in adult hematopoiesis, present in all RUNX1-expressing populations, including the cKit hematopoietic stem and progenitor cells. RUNX1B expression is more restricted, being highly expressed in the megakaryocyte lineage but downregulated during erythropoiesis. We generated a knock-in of RUNX1B, termed This mouse line lacks RUNX1C expression but has normal total RUNX1 levels, solely comprising RUNX1B. Using this mouse line, we establish a specific requirement for the RUNX1C isoform in megakaryopoiesis, which cannot be entirely compensated for by RUNX1B overexpression. knock-in megakaryocyte progenitors have reduced proliferative capacity and undergo increased cell death, resulting in thrombocytopenia. knock-in premegakaryocyte/erythroid progenitors demonstrate an erythroid-specification bias, evident from increased erythroid colony-forming ability and decreased megakaryocyte output. At a transcriptional level, multiple erythroid-specific genes are upregulated and megakaryocyte-specific transcripts are downregulated. In addition, proapoptotic pathways are activated in knock-in premegakaryocyte/erythroid progenitors, presumably accounting for the increased cell death in the megakaryocyte progenitor compartment. Unlike in the conditional adult null models, megakaryocytic maturation is not affected in the knock-in mice, suggesting that RUNX1B can regulate endomitosis and thrombopoiesis. Therefore, despite the high degree of structural similarity, RUNX1B and RUNX1C isoforms have distinct and specific roles in adult megakaryopoiesis.
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http://dx.doi.org/10.1182/blood-2016-06-723635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833261PMC
July 2017

TIAM1 Antagonizes TAZ/YAP Both in the Destruction Complex in the Cytoplasm and in the Nucleus to Inhibit Invasion of Intestinal Epithelial Cells.

Cancer Cell 2017 05 13;31(5):621-634.e6. Epub 2017 Apr 13.

Cell Signalling Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX, UK. Electronic address:

Aberrant WNT signaling drives colorectal cancer (CRC). Here, we identify TIAM1 as a critical antagonist of CRC progression through inhibiting TAZ and YAP, effectors of WNT signaling. We demonstrate that TIAM1 shuttles between the cytoplasm and nucleus antagonizing TAZ/YAP by distinct mechanisms in the two compartments. In the cytoplasm, TIAM1 localizes to the destruction complex and promotes TAZ degradation by enhancing its interaction with βTrCP. Nuclear TIAM1 suppresses TAZ/YAP interaction with TEADs, inhibiting expression of TAZ/YAP target genes implicated in epithelial-mesenchymal transition, cell migration, and invasion, and consequently suppresses CRC cell migration and invasion. Importantly, high nuclear TIAM1 in clinical specimens associates with increased CRC patient survival. Together, our findings suggest that in CRC TIAM1 suppresses tumor progression by regulating YAP/TAZ activity.
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http://dx.doi.org/10.1016/j.ccell.2017.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425402PMC
May 2017

Runx1 Structure and Function in Blood Cell Development.

Adv Exp Med Biol 2017 ;962:65-81

Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK.

RUNX transcription factors belong to a highly conserved class of transcriptional regulators which play various roles in the development of the majority of metazoans. In this review we focus on the founding member of the family, RUNX1, and its role in the transcriptional control of blood cell development in mammals. We summarize data showing that RUNX1 functions both as activator and repressor within a chromatin environment, a feature that requires its interaction with multiple other transcription factors and co-factors. Furthermore, we outline how RUNX1 works together with other factors to reshape the epigenetic landscape and the three-dimensional structure of gene loci within the nucleus. Finally, we review how aberrant forms of RUNX1 deregulate blood cell development and cause hematopoietic malignancies.
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http://dx.doi.org/10.1007/978-981-10-3233-2_5DOI Listing
September 2017

Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis.

Exp Hematol 2017 05 30;49:19-24. Epub 2016 Dec 30.

Division of Developmental Biology and Medicine, The University of Manchester, Manchester, United Kingdom. Electronic address:

The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however, can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived in vitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from hemogenic endothelium? Is the hemangioblast an in vitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.
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http://dx.doi.org/10.1016/j.exphem.2016.12.009DOI Listing
May 2017

Shedding light on hematopoietic stem cells: formation, regulation, and utilization.

FEBS Lett 2016 11;590(22):3963-3964

Hubrecht Institute-KNAW & University Medical Center, Utrecht, The Netherlands.

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http://dx.doi.org/10.1002/1873-3468.12481DOI Listing
November 2016

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

SOXF transcription factors in cardiovascular development.

Semin Cell Dev Biol 2017 03 25;63:50-57. Epub 2016 Jul 25.

Cancer Research UK, Stem Cell Hematopoiesis, The University of Manchester, Wilmslow road, M20 4BX, UK. Electronic address:

Cardiovascular development during embryogenesis involves complex changes in gene regulatory networks regulated by a variety of transcription factors. In this review we discuss the various reported roles of the SOXF factors: SOX7, SOX17 and SOX18 in cardiac, vascular and lymphatic development. SOXF factors have pleiotropic roles during these processes, and there is significant redundancy and functional compensation between SOXF family members. Despite this, evidence suggests that there is some specificity in the transcriptional programs they regulate which is necessary to control the differentiation and behaviour of endothelial subpopulations. Furthermore, SOXF factors appear to have an indirect role in regulating cardiac mesoderm specification and differentiation. Understanding how SOXF factors are regulated, as well as their downstream transcriptional target genes, will be important for unravelling their roles in cardiovascular development and related diseases.
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http://dx.doi.org/10.1016/j.semcdb.2016.07.021DOI Listing
March 2017