Unknown,Transcriptomics,Genomics,Proteomics

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Bcl11a controls Flt3 expression in early hematopoietic progenitors and is required for pDC development in vivo

ABSTRACT: Bcl11a is a transcription factor known to regulate lymphoid and erythroid development. Recent bioinformatic analysis of global gene expression patterns has suggested a role for Bcl11a in the development of dendritic cell (DC) lineages. We tested this hypothesis by analyzing the development of DC and other lineages in Bcl11a(-/-) mice. We show that Bcl11a is required for expression of IL-7 receptor (IL-7R) and Flt3 in early hematopoietic progenitor cells. The loss of IL-7R(+) common lymphoid progenitors accounts for previously described lymphoid defects in Bcl11a(-/-) mice. In addition, we found severely decreased numbers of plasmacytoid dendritic cells (pDCs) in Bcl11a(-/-) fetal livers and in the bone marrow of Bcl11a(-/-) fetal liver chimeras. Moreover, Bcl11a(-/-) cells show severely impaired in vitro development of Flt3L-derived pDCs and classical DCs (cDCs). In contrast, we found normal in vitro development of DCs from Bcl11a(-/-) fetal liver cells treated with GM-CSF. These results suggest that the persistent cDC development observed in Bcl11a(-/-) fetal liver chimeras reflects derivation from a Bcl11a- and Flt3-independent pathway in vivo. We compared global gene expression by microarray for donor-derived wild-type and Bcl11a(-/-) populations isolated from chimeric bone marrow to identify Bcl11a target genes that explain its role in hematopoietic progenitors. GMP and MPP populations were sorted from fetal liver chimeras and pooled by donor genotype. RNA was isolated using an RNAqueous-Micro Kit (Ambion) and submitted for amplification, labeling and hybridization. Expression values were analyzed after RMA quantile normalization using ArrayStar software (DNASTAR).

ORGANISM(S): Mus musculus

SUBMITTER: Xiaodi Wu

PROVIDER: E-GEOD-46270 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Innate immune responses are important in combating various microbes during the early phases of infection. Natural killer (NK) cells are innate lymphocytes that, unlike T and B lymphocytes, do not express antigen receptors but rapidly exhibit cytotoxic activities against virus infected cells and produce various cytokines1,2. We report here a new type of innate lymphocyte present in a novel lymphoid structure associated with adipose tissues in the peritoneal cavity. These cells do not express lineage (Lin) markers but express c-Kit, Sca-1, IL-7R and IL-33R. Similar lymphoid clusters were found in both human and mouse mesentery and we term this tissue â??FALCâ?? for fat-associated lymphoid cluster. FALC Lin-c-Kit+Sca-1+ cells are distinct from lymphoid progenitors3 and lymphoid tissue inducer (LTi) cells4. These cells proliferate in response to IL-2 and produce large amounts of Th2 cytokines such as IL-5, IL-6 and IL-13. IL-5 and IL-6 regulate B cell antibody production and self-renewal of B1 cells5-7. Indeed, FALC Lin-c-Kit+Sca-1+ cells support the self-renewal of B1 cells and enhance IgA production. IL-5 and IL-13 mediate allergic inflammation and protection against helminth infection8,9. Upon helminth infection and in response to IL-33, FALC Lin-c-Kit+Sca-1+ cells produce large amounts of IL-13, which leads to goblet cell hyperplasia, a critical step for helminth expulsion. In mice devoid of FALC Lin-c-Kit+Sca-1+ cells such goblet cell hyperplasia was not induced. Thus, FALC Lin-c-Kit+Sca-1+ cells are Th2-type innate lymphocytes and we propose that these cells be called â??natural helper cellsâ??. Experiment Overall Design: Natural helper cells (Lin- c-kit+ Sca-1+) were isolated from mesentery of C57BL/6 for RNA extraction and hybridization on Affymetrix microarrays. For the control of this experiment, we use double negative cells (DN2) from thymus of C57BL/6 or lymphoid tissue inducer cells (LTi). Thymic DN2 cells were prepared from adult thymocytes. Thymocytes were stained with magnetic beads conjugated with anti-CD4 and anti-CD8Î± mAbs and CD4-CD8- double negative (DN) cells were negatively sorted by AutoMACS. DN cells were further stained with anti-CD25 and anti-CD44 mAbs and CD25+CD44+ cells were sorted as DN2 cells on a FACSAria.Thy-1+CD4- LTi cells were prepared from fetal liver cells as described previously. c-Kit+Î±4Î²7+IL-7RÎ±+ fetal liver cells from day 13 embryos were cultured on TSt4 cells for 17 days.

Project description:Basak A, Munschauer M, Lareau CA, Montbleau KE, Ulirsch JC, Hartigan CR, Schenone M, Lian J, Wang Y, Huang Y, Wu X, Gehrke L, Rice CM, An X, Christou HA, Mohandas N, Carr SA, Orkin SH, Chen JJ, Lander ES, and Sankaran VG. Increased production of the beta-like gamma-globin genes that form fetal hemoglobin can ameliorate the severity of sickle cell disease and beta-thalassemia, the major hemoglobin disorders. BCL11A is a key repressor of the gamma-globin genes and is expressed in a developmental stage-specific manner to regulate the physiologic fetal-to-adult hemoglobin switch. Despite extensive studies, the upstream mechanisms underlying the developmental expression of BCL11A and hemoglobin switching in humans have remained mysterious. Here we show that BCL11A is regulated at the level of mRNA translation during human hematopoietic development. While BCL11A mRNA is comparably expressed at all developmental stages in erythroid cells, robust protein expression only occurs in adult erythroid cells. Importantly, at the earlier stages of development, the observed reduction in protein expression is attributable to decreased synthesis and not increased degradation of BCL11A. While BCL11A protein is not well synthesized at these earlier stages of development, its mRNA curiously continues to be associated with ribosomes. Through unbiased proteomic analyses in erythroid cells, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a reciprocal pattern to BCL11A, directly interacts with ribosomes. We show that the observed suppression of BCL11A protein translation is mediated by LIN28B through a direct interaction with BCL11A mRNA and independent of its role in let-7 microRNA biogenesis. Finally, we show that BCL11A is the major functional target in LIN28B-mediated fetal hemoglobin induction. Our results reveal a previously unappreciated regulatory mechanism underlying human hemoglobin switching and illuminate opportunities for developing improved treatments for sickle cell disease and beta-thalassemia.

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Bcl11a controls Flt3 expression in early hematopoietic progenitors and is required for pDC development in vivo

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