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      PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development

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          Abstract

          Phosphoinositide-dependent kinase-1 (PDK1) controls the activation of a subset of AGC kinases. Using a conditional knockout of PDK1 in haematopoietic cells, we demonstrate that PDK1 is essential for B cell development. B-cell progenitors lacking PDK1 arrested at the transition of pro-B to pre-B cells, due to a cell autonomous defect. Loss of PDK1 decreased the expression of the IgH chain in pro-B cells due to impaired recombination of the IgH distal variable segments, a process coordinated by the transcription factor Pax5. The expression of Pax5 in pre-B cells was decreased in PDK1 knockouts, which correlated with reduced expression of the Pax5 target genes IRF4, IRF8 and Aiolos. As a result, Ccnd3 is upregulated in PDK1 knockout pre-B cells and they have an impaired ability to undergo cell-cycle arrest, a necessary event for Ig light chain rearrangement. Instead, these cells underwent apoptosis that correlated with diminished expression of the pro-survival gene Bcl2A1. Reintroduction of both Pax5 and Bcl2A1 together into PDK1 knockout pro-B cells restored their ability to differentiate in vitro into mature B cells.

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          Most cited references 60

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          MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb.

          MiR-150 is a microRNA (miRNA) specifically expressed in mature lymphocytes, but not their progenitors. A top predicted target of miR-150 is c-Myb, a transcription factor controlling multiple steps of lymphocyte development. Combining loss- and gain-of-function gene targeting approaches for miR-150 with conditional and partial ablation of c-Myb, we show that miR-150 indeed controls c-Myb expression in vivo in a dose-dependent manner over a narrow range of miRNA and c-Myb concentrations and that this dramatically affects lymphocyte development and response. Our results identify a key transcription factor as a critical target of a stage-specifically expressed miRNA in lymphocytes and suggest that this and perhaps other miRNAs have evolved to control the expression of just a few critical target proteins in particular cellular contexts.
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            Transgenic mice with hematopoietic and lymphoid specific expression of Cre.

            Bacteriophage P1 Cre/loxP based systems can be used to manipulate the genomes ofmice in vivo and in vitro, allowing the generation of tissue-specific conditional mutants. We have generated mouse lines expressing Cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hCD2 promoter and locus control region (LCR). The R26R-EYFP Cre reporter mouse line was used to determine the pattern of Cre expression in each line and enabled the assessment of Cre activity at a single-cell level. Analysis showed that the vav promoter elements were able to direct Cre-mediated recombination in all cells of the hematopoietic system. The hCD2 promoter and LCR on the other hand were able to drive Cre-mediated recombination only in T cells and B cells, but not in other hematopoietic cell types. Furthermore, in the appropriate tissues, deletion of the floxed target was complete in all cells, thereby excluding the possibility of variegated expression of the Cre transgene. Both of these Cre-transgenic lines will be useful in generating tissue-specific gene deletions within all the cells of hematopoietic or lymphoid tissues.
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              B cell development pathways.

              B cell development is a highly regulated process whereby functional peripheral subsets are produced from hematopoietic stem cells, in the fetal liver before birth and in the bone marrow afterward. Here we review progress in understanding some aspects of this process in the mouse bone marrow, focusing on delineation of the earliest stages of commitment, on pre-B cell receptor selection, and B cell tolerance during the immature-to-mature B cell transition. Then we note some of the distinctions in hematopoiesis and pre-B selection between fetal liver and adult bone marrow, drawing a connection from fetal development to B-1/CD5(+) B cells. Finally, focusing on CD5(+) cells, we consider the forces that influence the generation and maintenance of this distinctive peripheral B cell population, enriched for natural autoreactive specificities that are encoded by particular germline V(H)-V(L) combinations.
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                Author and article information

                Journal
                EMBO J
                EMBO J
                The EMBO Journal
                Nature Publishing Group
                0261-4189
                1460-2075
                03 April 2013
                05 March 2013
                05 March 2013
                : 32
                : 7
                : 1008-1022
                Affiliations
                [1 ]MRC Protein Phosphorylation Unit, Sir James Black Centre, College of Life Sciences, University of Dundee , Dundee, UK
                [2 ]Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee , Dundee, UK
                [3 ]Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow , Glasgow, UK
                [4 ]Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee , Dundee, UK
                Author notes
                [a ]Lymphocyte Signalling and Development, The Babraham Institute , Cambridge, CB22 3AQ, UK. Tel.:+44 1223 496467; E-mail: ramkumar.venigalla@ 123456babraham.ac.uk
                [b ]Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee , Dundee, UK. Tel.:+44 1382 384003; Fax:+44 1382 385783; E-mail: j.s.c.arthur@ 123456dundee.ac.uk
                [*]

                These authors contributed equally to this work.

                Article
                emboj201340
                10.1038/emboj.2013.40
                3616287
                23463102
                Copyright © 2013, European Molecular Biology Organization

                This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To view a copy of this licence visit http://creativecommons.org/licenses/by/3.0/.

                Categories
                Article

                Molecular biology

                b cell, pax5, pdk1, preb, vdj recombination

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