ABSTRACT: Transcription profiling of bone marrow hematopoietic stem cells and myeloid progenitors from mice with conditional deletion of Forkhead O (FoxO) family of transcription factors
Project description:<p>We are studying the natural history, pathogenesis and treatment of patients with WHIM syndrome, an immunodeficiency disorder characterized by warts, hypogammaglobulinemia, recurrent infections and neutropenia usually due to autosomal dominant gain-of-function mutations in chemokine receptor <i>CXCR4</i>. We have identified a patient born with WHIM syndrome and the WHIM mutation <i>CXCR4<sup>R334X</sup></i> who has been disease-free for 20 years and who lacks <i>CXCR4<sup>R334X</sup></i> in myeloid cells, the cells that drive disease manifestations. She is a genetic and hematopoietic mosaic, since she still has the mutation in lymphoid cells and non-hematopoietic cells. Cytogenetics and microarray analysis revealed that the mechanism of loss of the mutation was deletion of the mutant allele from one copy of chromosome 2. Whole genome sequencing of patient neutrophil and skin fibroblast genomic DNA revealed that the mechanism of deletion was chromothripsis, a process of chromosome shattering resulting in deletions and rearrangements of the non-deleted chromosomal segments. In the patient, this process evidently occurred in a single hematopoietic stem cell (HSC), resulting in deletion of the disease allele <i>CXCR4<sup>R334X</sup></i> and one copy of 163 other genes on chromosome 2. This HSC evidently acquired a growth advantage and repopulated the HSC population and the myeloid lineage. Consistent with this, studies using gene targeted mice in competitive bone marrow transplantation experiments revealed that selective <i>Cxcr4</i> haploinsufficiency (inactivation of one copy of <i>Cxcr4</i> and not of any other genes) was sufficient to confer a strong engraftment advantage over bone marrow cells from wild type mice as well as bone marrow cells from a mouse model of WHIM syndrome. These results suggest that <i>CXCR4</i> knockdown may be a useful strategy to enhance bone marrow engraftment in the absence of toxic bone marrow conditioning regimens.</p>
Project description:To investigate the role of FoxO transcription factors as mediators of hematopoietic stem cell resistance to oxidative stress. Experiment Overall Design: Study the effect of the deficiency of FoxO1, FoxO3, and FoxO4 in murine bone marrow hematopoietic stem cells and myeloid progenitors on expression of genes involved in reactive oxygen species (ROS) metabolism.
Project description:Ubiquitination is a post-translational mechanism of control of diverse cellular processes. We focus here on the ubiquitin ligase Fbw7, a recently identified hematopoietic tumor suppressor that can target for degradation several important oncogenes including Notch1, c-Myc and cyclin E. We have generated conditional Fbw7 knock-out animals and inactivated the gene in hematopoietic stem cells (HSC) and their differentiated progeny. Deletion of Fbw7 specifically and rapidly affects the HSC compartment in a cell-autonomous manner. Fbw7-/- HSCs show defective maintenance of quiescence, leading to impaired self-renewal and a severe loss of competitive repopulating capacity. Furthermore, Fbw7-/- HSC are unable to colonize the thymus leading to a profound depletion of T cell progenitors. Deletion of Fbw7 in bone marrow stem cells and progenitors leads to the stabilization of c-Myc, a transcription factor previously implicated in HSC self-renewal. On the other hand, neither Notch1 nor cyclin E are stabilized in the bone marrow of Fbw7 deficient mice. Genome-wide transcriptome studies of Fbw7-/- HSC and hematopoietic progenitors indicate that Fbw7 controls, through the regulation of HSC cell cycle entry, the global transcriptional âsignatureâ that is associated with the quiescent, self-renewing HSC phenotype. Transcriptional consequences of inactivating Fbw7 in LKS cells. Experiment Overall Design: Four samples were analyzed: wild-type (WT) control and Fbw7-deficient (FBW7) Lin-ckit+Sca1+ (LSK) cells, as well as Lin-ckit+Sca1- myeloid progenitor (MP) cells, which served as a control for LSK-enriched/specific genes. Total bone marrow cells were pooled from three WT and three FBW7 mice before sorting LSK and MP populations.
Project description:Expression data from FACS-purified hematopoietic stem cells, common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors from human bone marrow samples of elderly anemic patients
Project description:Within the bone marrow, hematopoietic stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with the in vivo potential or gene regulatory mechanisms. Here we comprehensively identify myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups showing unexpected transcriptional priming towards seven differentiation fates, but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting the process is tightly regulated. Histone maps and knockout assays are consistent with the transcriptional states while traditional transplantation experiments are only partially overlapping myeloid transcriptional priming. Our analyses uncover the function of the underlying regulatory mechanisms for several sub groups and establishes a general framework for dissecting hematopoiesis. Bone marrow common myeloid progenitor H3K4me2 profiles were generated by deep sequencing of iChIP libraries on an Illumina NextSeq
Project description:Collombet2016 - Lymphoid and myeloid cell
specification and transdifferentiation
This model is described in the article:
Logical modeling of lymphoid
and myeloid cell specification and transdifferentiation
Samuel Collombet, Chris van Oevelen,
Jose Luis Sardina Ortega, Wassim Abou-Jaoudé, Bruno Di
Stefano, Morgane Thomas-Chollier, Thomas Graf, and Denis
Thieffry
Proceedings of the National Academy of
Sciences of the United States of America
Abstract:
Blood cells are derived from a common set of hematopoietic
stem cells, which differentiate into more specific progenitors
of the myeloid and lymphoid lineages, ultimately leading to
differentiated cells. This developmental process is controlled
by a complex regulatory network involving cytokines and their
receptors, transcription factors, and chromatin remodelers.
Using public data and data from our own molecular genetic
experiments (quantitative PCR, Western blot, EMSA) or
genome-wide assays (RNA-sequencing, ChIP-sequencing), we have
assembled a comprehensive regulatory network encompassing the
main transcription factors and signaling components involved in
myeloid and lymphoid development. Focusing on B-cell and
macrophage development, we defined a qualitative dynamical
model recapitulating cytokine-induced differentiation of common
progenitors, the effect of various reported gene knockdowns,
and the reprogramming of pre-B cells into macrophages induced
by the ectopic expression of specific transcription factors.
The resulting network model can be used as a template for the
integration of new hematopoietic differentiation and
transdifferentiation data to foster our understanding of
lymphoid/myeloid cell-fate decisions.
This model is hosted on
BioModels Database
and identified by:
MODEL1610240000.
To cite BioModels Database, please use:
BioModels Database:
An enhanced, curated and annotated resource for published
quantitative kinetic models.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.
Project description:The hematopoietic system is maintained throughout life by hematopoietic stem cells that are capable of differentiation to all hematopoietic lineages. An intimate balance between self-renewal, differentiation, and quiescence is required to maintain hematopoiesis. Disruption of this balance can result in hematopoietic malignancy, including acute myeloid leukemia (AML). FBXO9, from the F-box ubiquitin E3 ligases, is down-regulated in patients with AML compared to normal bone marrow. FBXO9 is a substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex. FBXO9 is highly expressed in hematopoietic stem and progenitor populations, which contain the tumor-initiating population in AML. In AML patients, decrease in FBXO9 expression is most pronounced in patients with the inversion of chromosome 16 (Inv(16)), a rearrangement that generates the transcription factor fusion gene, CBFB-MYH11. To study FBXO9 in malignant hematopoiesis, we generated a conditional knockout mouse model using a novel CRISPR/Cas9 strategy. Our data shows that deletion of Fbxo9 in mice expressing Cbfb-MYH11 leads to markedly accelerated and aggressive leukemia development. In addition, we find loss of FBXO9 leads to increased proteasome expression and tumors are more sensitive to bortezomib suggesting that FBXO9 expression may predict patient response to bortezomib treatment.
Project description:It has been shown previously that endothelial cells and LepR+ stromal cells are the main sources of SCF in vivo in the mouse bone marrow. We tested whether SCF from endothelial cells and/or LepR+ stromal cells is important for the maintenance of hematopoietic progenitors and erythroid progenitors in mouse bone marrow by conditional deletion of Scf from these two cell types. We discovered that Scf deletion from LepR+ stromal cells, but not endothelial cells, reduced the numbers of hematopoietic progenitors and erythroid progenitors in mice. We performed RNA-Seq on PreCFU-E and CFU-E progenitors from control mice and from mice with Scf deletion from LepR+ stromal cells. We discovered that lack of SCF from LepR+ cells induces a premature differentiation of PreCFU-E and CFU-E progenitors.
Project description:Within the bone marrow, hematopoietic stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with the in vivo potential or gene regulatory mechanisms. Here we comprehensively identify myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups showing unexpected transcriptional priming towards seven differentiation fates, but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting the process is tightly regulated. Histone maps and knockout assays are consistent with the transcriptional states while traditional transplantation experiments are only partially overlapping myeloid transcriptional priming. Our analyses uncover the function of the underlying regulatory mechanisms for several sub groups and establishes a general framework for dissecting hematopoiesis. Bone marrow Lin- cKit+ Sca1- myeloid progenitors mRNA profiles from single cells were generated by deep sequencing of thousands of single cells, sequenced in several batches in an Illumina NextSeq Please note that [1] raw data files were processed as single-ended file since second read (mate) files contain only cell/molecule barcodes and therefore, not provided. This information was appended to the fastq entry header [2] The 'experimental_design.txt' file explains the correspondence of each single cell (WXXXX) in the 'umitab.txt' to a sample (ABXXXX).
Project description:DNA methylation is essential for mammalian development and plays crucial roles in a variety of biological processes. The DNA methyltransferase Dnmt1 serves to maintain parental cell methylation patterns on daughter DNA strands in mitotic cells, however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we conditionally disrupted Dnmt1 in the hematopoietic system. We used microarrays to profile the global gene expression program in hematopoietic stem and progenitor cells following deletion of Dnmt1. Dnmt1 was conditionally deleted in the hematopoietic system by injections of poly(I)poly(C) to induce Cre expression from the Mx-Cre transgene. Control mice were also injected with poly(I)poly(C) but do not carry the Mx-Cre transgene. Four days after completion of poly(I)poly(C) injections, bone marrow was harvested from the mice, antibody-mediated magnetic bead selection was used to remove cells expressing mature lineage markers, and the resulting lineage-depleted cells were stained with fluorochrome-conjugated antibodies against the surface receptors c-Kit, Sca-1 and CD34. Populations of LT-HSCs, MPPs and myeloid progenitors were FACS sorted, RNA was extracted and amplified from these sorted populations and hybridized to Affymetrix microarrays to compare changes in gene expression induced by conditional knockout of Dnmt1 compared to control in each of the three cell populations. There are 2 biological replicates for the LT-HSCs and MPPs and 3 biological replicates for the myeloid progenitors.