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      Growth Factor Independence 1b ( Gfi1b) Is Important for the Maturation of Erythroid Cells and the Regulation of Embryonic Globin Expression

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          Abstract

          Growth factor independence 1b (GFI1B) is a DNA binding repressor of transcription with vital functions in hematopoiesis. Gfi1b-null embryos die at midgestation very likely due to defects in erythro- and megakaryopoiesis. To analyze the full functionality of Gfi1b, we used conditionally deficient mice that harbor floxed Gfi1b alleles and inducible ( Mx-Cre, Cre-ERT) or erythroid specific ( EpoR-Cre) Cre expressing transgenes. In contrast to the germline knockout, EpoR-Cre mediated erythroid specific ablation of Gfi1b allows full gestation, but causes perinatal lethality with very few mice surviving to adulthood. Both the embryonic deletion of Gfi1b by EpoR-Cre and the deletion in adult mice by Mx-Cre or Cre-ERT leads to reduced numbers of erythroid precursors, perturbed and delayed erythroid maturation, anemia and extramedullary erythropoiesis. Global expression analyses showed that the Hba-x, Hbb-bh1 and Hbb-y embryonic globin genes were upregulated in Gfi1b deficient TER119 + fetal liver cells over the gestation period from day 12.5–17.5 p.c. and an increased level of Hbb-bh1 and Hbb-y embryonic globin gene expression was even maintained in adult Gfi1b deficient mice. While the expression of Bcl11a, a regulator of embryonic globin expression was not affected by Gfi1b deficiency, the expression of Gata1 was reduced and the expression of Sox6, also involved in globin switch, was almost entirely lost when Gfi1b was absent. These findings establish Gfi1b as a regulator of embryonic globin expression and embryonic and adult erythroid maturation.

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          Most cited references30

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          Inducible gene targeting in mice.

          A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an interferon-responsive promoter to control the expression of Cre recombinase. Here, Cre was used to delete a segment of the DNA polymerase beta gene flanked by IoxP recombinase recognition sites. Deletion was complete in liver and nearly complete in lymphocytes within a few days, whereas partial deletion was obtained in other tissues. This method can be used for the inducible inactivation of any other gene in vivo.
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            Transcriptional silencing of {gamma}-globin by BCL11A involves long-range interactions and cooperation with SOX6.

            The developmental switch from human fetal (gamma) to adult (beta) hemoglobin represents a clinically important example of developmental gene regulation. The transcription factor BCL11A is a central mediator of gamma-globin silencing and hemoglobin switching. Here we determine chromatin occupancy of BCL11A at the human beta-globin locus and other genomic regions in vivo by high-resolution chromatin immunoprecipitation (ChIP)-chip analysis. BCL11A binds the upstream locus control region (LCR), epsilon-globin, and the intergenic regions between gamma-globin and delta-globin genes. A chromosome conformation capture (3C) assay shows that BCL11A reconfigures the beta-globin cluster by modulating chromosomal loop formation. We also show that BCL11A and the HMG-box-containing transcription factor SOX6 interact physically and functionally during erythroid maturation. BCL11A and SOX6 co-occupy the human beta-globin cluster along with GATA1, and cooperate in silencing gamma-globin transcription in adult human erythroid progenitors. These findings collectively demonstrate that transcriptional silencing of gamma-globin genes by BCL11A involves long-range interactions and cooperation with SOX6. Our findings provide insight into the mechanism of BCL11A action and new clues for the developmental gene regulatory programs that function at the beta-globin locus.
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              Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1.

              The transcriptional repressor Gfi1 is a nuclear zinc-finger protein expressed in T-cell precursors in the thymus and in activated mature T lymphocytes. Previous experiments have shown that Gfi1 is involved in T-cell lymphomagenesis and in the development of T-cell progenitors. Here we show that Gfi1 is also expressed outside the lymphoid system in granulocytes and activated macrophages, cells that mediate innate immunity (that is, non-specific immunity). We have generated Gfi1-deficient mice (Gfi1-/-) and show that these animals are severely neutropenic and accumulate immature monocytic cells in blood and bone marrow. Their myeloid precursor cells are unable to differentiate into granulocytes upon stimulation with granulocyte colony-stimulating factor (G-CSF) but can develop into mature macrophages. We found that Gfi1-/- macrophages produce enhanced levels of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-10 (IL-10) and IL-1beta, when stimulated with bacterial lipopolysaccharide (LPS) and that Gfi1-/- mice succumb to low doses of this endotoxin that are tolerated by wildtype mice. We conclude that Gfi1 influences the differentiation of myeloid precursors into granulocytes or monocytes and acts in limiting the inflammatory immune response.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                6 May 2014
                : 9
                : 5
                : e96636
                Affiliations
                [1 ]Institut de Recherches Cliniques de Montréal, IRCM, Montréal, Québec, Canada
                [2 ]Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montréal, Québec, Canada
                [3 ]Division of Experimental Medicine, McGill University, Montréal, Québec, Canada
                Southern Illinois University School of Medicine, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: LV HB TM MT. Performed the experiments: LV HB WL JK. Analyzed the data: LV HB. Contributed reagents/materials/analysis tools: TM MT. Wrote the paper: LV HB TM.

                Article
                PONE-D-14-02982
                10.1371/journal.pone.0096636
                4011847
                24800817
                267a7a6e-dd94-42de-97d8-cde5a4a97ddd
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 27 January 2014
                : 9 April 2014
                Page count
                Pages: 14
                Funding
                This work was supported by grants from the Canadian Institutes of Health Research (MOP – 111247) and the Canadian Foundation for Innovation and the Canadian Blood Services/Canadian Institutes of Health Research (210399) to M.T. T.M. holds the Canada Research Chair (Tier 1) in Hematopoiesis and Immune Cell Differentiation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Stem Cells
                Hematopoietic Stem Cells
                Blood Cells
                Bone Marrow Cells
                Cytometry
                Molecular Cell Biology
                Computational Biology
                Genome Analysis
                Transcriptome Analysis
                Genome Expression Analysis
                Genetics
                Gene expression
                DNA transcription
                Research and Analysis Methods
                Spectrum Analysis Techniques
                Spectrophotometry
                Cytophotometry
                Flow Cytometry
                Model Organisms
                Animal Models
                Mouse Models

                Uncategorized
                Uncategorized

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