[show abstract][hide abstract] ABSTRACT: While hematopoietic stem cell (HSC) self-renewal is well studied, it remains unknown whether distinct control mechanisms enable HSC divisions that generate progeny cells with specific lineage bias. Here, we report that the monocytic transcription factor MafB specifically restricts the ability of M-CSF to instruct myeloid commitment divisions in HSCs. MafB deficiency specifically enhanced sensitivity to M-CSF and caused activation of the myeloid master-regulator PU.1 in HSCs in vivo. Single-cell analysis revealed that reduced MafB levels enabled M-CSF to instruct divisions producing asymmetric daughter pairs with one PU.1(+) cell. As a consequence, MafB(-/-) HSCs showed a PU.1 and M-CSF receptor-dependent competitive repopulation advantage specifically in the myelomonocytic, but not T lymphoid or erythroid, compartment. Lineage-biased repopulation advantage was progressive, maintained long term, and serially transplantable. Together, this indicates that an integrated transcription factor/cytokine circuit can control the rate of specific HSC commitment divisions without compromising other lineages or self-renewal.
[show abstract][hide abstract] ABSTRACT: The function of the Ets-1 transcription factor is regulated by two regions that flank its DNA-binding domain. A previously established mechanism for auto-inhibition of monomeric Ets-1 on DNA response elements with a single ETS-binding site, however, has not been observed for the stromelysin-1 promoter containing two palindromic ETS-binding sites. We present the structure of Ets-1 on this promoter element, revealing a ternary complex in which protein homo-dimerization is mediated by the specific arrangement of the two ETS-binding sites. In this complex, the N-terminal-flanking region is required for ternary protein-DNA assembly. Ets-1 variants, in which residues from this region are mutated, loose the ability for DNA-mediated dimerization and stromelysin-1 promoter transactivation. Thus, our data unravel the molecular basis for relief of auto-inhibition and the ability of Ets-1 to function as a facultative dimeric transcription factor on this site. Our findings may also explain previous data of Ets-1 function in the context of heterologous transcription factors, thus providing a molecular model that could also be valid for Ets-1 regulation by hetero-oligomeric assembly.
The EMBO Journal 07/2008; 27(14):2006-17. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Molecular and Cellular Biology 09/2007; 27(15):5554-64. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Molecular and Cellular Biology 10/2006; 26(18):6808-18. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity on histone and various other cellular substrates. Functional analyses of such proteins have been carried out in a number of prokaryotes and eukaryotes organisms but until now, none have described an essential function for any SIR2 genes. Here using genetic approach, we report that a cytosolic SIR2 homolog in Leishmania is determinant to parasite survival. L. infantum promastigote tolerates deletion of one wild-type LiSIR2 allele (LiSIR2+/-) but achievement of null chromosomal mutants (LiSIR2-/-) requires episomal rescue. Accordingly, plasmid cure shows that these parasites maintain episome even in absence of drug pressure. Though single LiSIR2 gene disruption (LiSIR2+/-) does not affect the growth of parasite in the promastigote form, axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Taken together these data support a stage specific requirement and/or activity of the Leishmania cytosolic SIR2 protein and reveal an unrelated essential function for the life cycle of this unicellular pathogenic organism. The lack of an effective vaccine against leishmaniasis, and the need for alternative drug treatments, makes LiSIR2 protein a new attractive therapeutic target.
[show abstract][hide abstract] ABSTRACT: Silent information regulator 2 (SIR2) proteins are NAD-dependant deacetylases found in organisms ranging from bacteria to human. In eukaryotes, these proteins are involved in many biological processes including transcriptional repression, metabolism, ageing, or apoptosis. Here, we have shown that Sirtinol, a commercially available inhibitor of SIR2 deacetylases, significantly inhibits the in vitro proliferation of Leishmania infantum axenic amastigotes in a dose-dependent manner. This activity is stage specific since sirtinol did not affect the in vitro growth of parasite promastigotes. Growth arrest in amastigotes is associated with genomic DNA fragmentation, a process reminiscent of apoptosis. Interestingly parasites carrying extra copies of the LmSIR2 gene were less susceptible to the sirtinol mediated cell death. Altogether, these results constitute novel evidences that Leishmania SIR2 proteins play a role in the control of the parasite apoptotic phenomenon.
[show abstract][hide abstract] ABSTRACT: 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) beta-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.
Kinetoplastid Biology and Disease 02/2005; 4(1):1.