Publications by authors named "Muhammad Z H Fadlullah"

12 Publications

  • Page 1 of 1

Investigating the importance of B cells and antibodies during Trichuris muris infection using the IgMi mouse.

J Mol Med (Berl) 2020 09 10;98(9):1301-1317. Epub 2020 Aug 10.

Division of Infection, Immunity and Respiratory Medicine, Lydia Becker Institute for Immunology, The University of Manchester, Manchester, UK.

The IgMi mouse has normal B cell development; its B cells express an IgM B cell receptor but cannot class switch or secrete antibody. Thus, the IgMi mouse offers a model system by which to dissect out antibody-dependent and antibody-independent B cell function. Here, we provide the first detailed characterisation of the IgMi mouse post-Trichuris muris (T. muris) infection, describing expulsion phenotype, cytokine production, gut pathology and changes in T regulatory cells, T follicular helper cells and germinal centre B cells, in addition to RNA sequencing (RNA seq) analyses of wild-type littermates (WT) and mutant B cells prior to and post infection. IgMi mice were susceptible to a high-dose infection, with reduced Th2 cytokines and elevated B cell-derived IL-10 in mesenteric lymph nodes (MLN) compared to controls. A low-dose infection regime revealed IgMi mice to have significantly more apoptotic cells in the gut compared to WT mice, but no change in intestinal inflammation. IL-10 levels were again elevated. Collectively, this study showcases the potential of the IgMi mouse as a tool for understanding B cell biology and suggests that the B cell plays both antibody-dependent and antibody-independent roles post high- and low-dose T. muris infection. KEY MESSAGES: During a high-dose T. muris infection, B cells are important in maintaining the Th1/Th2 balance in the MLN through an antibody-independent mechanism. High levels of IL-10 in the MLN early post-infection, and the presence of IL-10-producing B cells, correlates with susceptibility to T. muris infection. B cells maintain gut homeostasis during chronic T. muris infection via an antibody-dependent mechanism.
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http://dx.doi.org/10.1007/s00109-020-01954-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447682PMC
September 2020

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

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

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

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

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

The Hemogenic Competence of Endothelial Progenitors Is Restricted by Runx1 Silencing during Embryonic Development.

Cell Rep 2016 06 26;15(10):2185-2199. Epub 2016 May 26.

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

It is now well-established that hematopoietic stem cells (HSCs) and progenitor cells originate from a specialized subset of endothelium, termed hemogenic endothelium (HE), via an endothelial-to-hematopoietic transition. However, the molecular mechanisms determining which endothelial progenitors possess this hemogenic potential are currently unknown. Here, we investigated the changes in hemogenic potential in endothelial progenitors at the early stages of embryonic development. Using an ETV2::GFP reporter mouse to isolate emerging endothelial progenitors, we observed a dramatic decrease in hemogenic potential between embryonic day (E)7.5 and E8.5. At the molecular level, Runx1 is expressed at much lower levels in E8.5 intra-embryonic progenitors, while Bmi1 expression is increased. Remarkably, the ectopic expression of Runx1 in these progenitors fully restores their hemogenic potential, as does the suppression of BMI1 function. Altogether, our data demonstrate that hemogenic competency in recently specified endothelial progenitors is restrained through the active silencing of Runx1 expression.
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http://dx.doi.org/10.1016/j.celrep.2016.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906370PMC
June 2016

RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis.

PLoS Genet 2016 Jan 25;12(1):e1005814. Epub 2016 Jan 25.

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

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into "pro-erythroid" and "pro-megakaryocyte" populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment.
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http://dx.doi.org/10.1371/journal.pgen.1005814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726605PMC
January 2016

Whole genomes redefine the mutational landscape of pancreatic cancer.

Nature 2015 Feb;518(7540):495-501

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.
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http://dx.doi.org/10.1038/nature14169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523082PMC
February 2015

A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing.

Biotechniques 2014 Jul 1;57(1):31-8. Epub 2014 Jul 1.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia; Wolfson Wohl Cancer Research Centre, Institute for Cancer Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.

Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.
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http://dx.doi.org/10.2144/000114189DOI Listing
July 2014
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