Experimental study of the function of the excreted/secreted Leishmania LmSIR2 protein by heterologous expression in eukaryotic cell line

BACKGROUND: In yeast and Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells. RESULTS: Our results demonstrate that the LmSIR2 protein was properly expressed by fibroblasts and that LmSIR2 is localized both in the cytoplasm and the nucleus of all the transformed cell clones. Unexpectedly, we found that cells expressing LmSIR2 presents reduced saturation cell density ranging from 40% to 60% and expressed an acidic (pH6.0) β-galactosidase activity, which is known to be a senescence biomarker. As a consequence, we observed that LmSIR2 positive fibroblasts were more permissive towards Leihmania infection. CONCLUSIONS: LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help Leishmania to survive for a long period in a cell with reduced capacity to multiply or respond to immunologic stimuli. The potential implications of our finding during the in vivo infection process are discussed

High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq

International audienceCell-type specific regulation of gene expression requires the activation of promoters by distal genomic elements defined as enhancers. The identification and the characterization of enhancers are challenging in mammals due to their genome complexity. Here we develop CapStarr-Seq, a novel high-throughput strategy to quantitatively assess enhancer activity in mammals. This approach couples capture of regions of interest to previously developed Starr-seq technique. Extensive assessment of CapStarr-seq demonstrates accurate quantification of enhancer activity. Furthermore, we find that enhancer strength is associated with binding complexity of tissue-specific transcription factors and super-enhancers, while additive enhancer activity isolates key genes involved in cell identity and function. The CapStarr-Seq thus provides a fast and cost-effective approach to assess the activity of potential enhancers for a given cell type and will be helpful in decrypting transcription regulation mechanisms

RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement

V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus

Chromatin signatures of active enhancers

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Les complexes MafB/MaffB et MafB/c-Fos (dualité dans la prolifération)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Projet européen \textitBlueprint: Vers une caractérisation épigénomique du système immunitaire

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Development of Macrophages with Altered Actin Organization in the Absence of MafB

In the hematopoietic system the bZip transcription factor MafB is selectively expressed at high levels in monocytes and macrophages and promotes macrophage differentiation in myeloid progenitors, whereas a dominant-negative allele can inhibit this process. To analyze the requirement of MafB for macrophage development, we generated MafB-deficient mice and, due to their neonatal lethal phenotype, analyzed macrophage differentiation in vitro, in the embryo, and in reconstituted mice. Surprisingly we observed in vitro differentiation of macrophages from E14.5 fetal liver (FL) cells and E18.5 splenocytes. Furthermore we found normal numbers of F4/80(+)/Mac-1(+) macrophages and monocytes in fetal liver, spleen, and blood as well as in bone marrow, spleen, and peritoneum of adult MafB(−/−) FL reconstituted mice. MafB(−/−) macrophages showed intact basic macrophage functions such as phagocytosis of latex beads or Listeria monocytogenes and nitric oxide production in response to lipopolysaccharide. By contrast, MafB(−/−) macrophages expressed increased levels of multiple genes involved in actin organization. Consistent with this, phalloidin staining revealed an altered morphology involving increased numbers of branched protrusions of MafB(−/−) macrophages in response to macrophage colony-stimulating factor. Together these data point to an unexpected redundancy of MafB function in macrophage differentiation and a previously unknown role in actin-dependent macrophage morphology

SUMO Modification Regulates MafB-Driven Macrophage Differentiation by Enabling Myb-Dependent Transcriptional Repression.

During the execution of differentiation programs, lineage-specific transcription factors are in competition with antagonistic factors that drive progenitor proliferation. Thus, the myeloid transcription factor MafB promotes macrophage differentiation of myeloid progenitors, but a constitutively active Myb transcription factor (v-Myb) can maintain proliferation and block differentiation. Little is known, however, about the regulatory mechanisms that control such competing activities. Here we report that the small ubiquitin-like protein SUMO-1 can modify MafB in vitro and in vivo on lysines 32 and 297. The absence of MafB SUMO modification increased MafB-driven transactivation and macrophage differentiation potential but inhibited cell cycle progression and myeloid progenitor growth. Furthermore, we observed that direct repression of MafB transactivation by v-Myb was strictly dependent on MafB SUMO modification. Consequently, a SUMOylation-deficient MafB K32R K297R (K32,297R) mutant could specify macrophage fate even after activation of inducible Myb alleles and resist their differentiation-inhibiting activity. Our findings suggest that SUMO modification of MafB affects the balance between myeloid progenitor expansion and terminal macrophage differentiation by controlling MafB transactivation capacity and susceptibility to Myb repression. SUMO modification of lineage-specific transcription factors may thus modulate transcription factor antagonism to control tissue homeostasis in the hematopoietic system