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One important environmental/health challenge is to determine, in a feasible way, the potential carcinogenic risk associated with environmental agents/exposures. Since a significant proportion of tumors have an environmental origin, detecting the potential carcinogenic risk of environmental agents is mandatory, as regulated by national and international agencies. The challenge mainly implies finding a way of how to overcome the inefficiencies of long-term trials with rodents when thousands of agents/exposures need to be tested. To such an end, the use of in vitro cell transformation assays (CTAs) was proposed, but the existing prevalidated CTAs do not cover the complexity associated with carcinogenesis processes and present serious limitations. To overcome such limitations, we propose to use a battery of assays covering most of the hallmarks of the carcinogenesis process. For the first time, we grouped such assays as early, intermediate, or advanced biomarkers which allow for the identification of the cells in the initiation, promotion or aggressive stages of tumorigenesis. Our proposal, as a novelty, points out that using a battery containing assays from all three groups can identify if a certain agent/exposure can pose a carcinogenic risk; furthermore, it can gather mechanistic insights into the mode of the action of a specific carcinogen. This structured battery could be very useful for any type of in vitro study, containing human cell lines aiming to detect the potential carcinogenic risks of environmental agents/exposures. In fact, here, we include examples in which these approaches were successfully applied. Finally, we provide a series of advantages that, we believe, contribute to the suitability of our proposed approach for the evaluation of exposure-induced carcinogenic effects and for the development of an alternative strategy for conducting an exposure risk assessment.

Prostate cancer is a major public health concern and one of the most prevalent forms of cancer worldwide. The definition of altered signaling pathways implicated in this complex disease is thus essential. In this context, abnormal expression of the receptor of Macrophage Colony-Stimulating Factor-1 (M-CSF or CSF-1) has been described in prostate cancer cells. Yet, outcomes of this expression remain unknown. Using mouse and human prostate cancer cell lines, this study has investigated the functionality of the wild-type CSF-1 receptor in prostate tumor cells and identified molecular mechanisms underlying its ligand-induced activation. Here, we showed that upon CSF-1 binding, the receptor autophosphorylates and activates multiple signaling pathways in prostate tumor cells. Biological experiments demonstrated that the CSF-1R/CSF-1 axis conferred significant advantages in cell growth and cell invasion in vitro. Mouse xenograft experiments showed that CSF-1R expression promoted the aggressiveness of prostate tumor cells. In particular, we demonstrated that the ligand-activated CSF-1R increased the expression of spp1 transcript encoding for osteopontin, a key player in cancer development and metastasis. Therefore, this study highlights that the CSF-1 receptor is fully functional in a prostate cancer cell and may be a potential therapeutic target for the treatment of prostate cancer.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q− syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle–arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Summary Transcription factors are key players in a broad range of cellular processes such as cell-fate decision. Understanding how they act to control these processes is of critical importance for therapy purposes. FLI-1 controls several hematopoietic lineage differentiation including megakaryopoiesis and erythropoiesis. Its aberrant expression is often observed in cancer and is associated with poor prognosis. We showed that FLI-1 interacts with the LDB1 complex, which also plays critical roles in erythropoiesis and megakaryopoiesis. In this study, we aimed to unravel how FLI-1 and the LDB1 complex act together in murine erythroleukemia cells and in megakaryocyte. Combining omics techniques, we show that FLI-1 enables the recruitment of the LDB1 complex to regulatory sequences of megakaryocytic genes and to enhancers. We show as well for the first time that FLI-1 is able to modulate the 3D chromatin organization by promoting chromatin looping between enhancers and promoters most likely through the LDB1 complex., Graphical abstract, Highlights • FLI-1 is important for the recruitment of the LDB1 complex • FLI-1 is important for chromatin looping • FLI-1 and the LDB1 complex co-regulate megakaryopoiesis, Molecular Biology; Chromosome Organization

Assessment of autophagy activity has historically been limited to investigations of fixed tissue or bulk cell populations. To address questions of heterogeneity and relate measurements to functional properties of viable cells isolated from primary tissue, we created a lentiviral (RFP-GFP-MAP1LC3B) vector that allows the autophagosome and autolysosome content of transduced cells to be monitored at the single-cell level. Use of this strategy to analyze purified subsets of normal human mammary cells showed that both the luminal progenitor-containing (LP) subset and the basal cells (BCs) display highly variable but overall similar autophagic flux activity despite differences suggested by measurements of the proteins responsible (i.e., LC3B, ATG7 and BECLIN1) in bulk lysates. Autophagosome content was also highly variable in the clonogenic cells within both the LPs and BCs, but the proliferative response of the BCs was more sensitive to autophagy inhibition. In addition, use of this vector showed cells with the lowest autophagosome content elicited the fastest tumor growth in 2 different models of human mammary tumorigenesis. These results illustrate the utility of this vector to define differences in the autophagy properties of individual cells in primary tissue and couple these with their responses to proliferative and oncogenic stimuli.

International audience; Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and its porosity enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all cell lines tested. The soft cell confiner is thus a valuable tool to decipher the effects of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer.

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The BCR-ABL specific tyrosine kinase inhibitors (TKI) changed the outcome of chronic myeloid leukemia (CML), turning a life-threatening disease into a chronic illness. However, TKI are not yet curative, because most patients retain leukemic stem cells (LSC) and their progenitors in bone marrow and relapse following treatment cessation. At diagnosis, deregulation of the bone morphogenetic protein (BMP) pathway is involved in LSC and progenitor expansion. Here, we report that BMP pathway alterations persist in TKI-resistant patients. In comparison with patients in complete cytogenetic remission, TKI-resistant LSC and progenitors display high levels of BMPR1b expression and alterations of its cellular localization. In vitro treatment of immature chronic phase CML cells with TKI alone, or in combination with interferon-α, results in the preferential survival of BMPR1b+ cells. We demonstrated persistent and increasing BMP4 production by patients' mesenchymal cells with resistance. Patient follow-up revealed an increase of BMPR1b expression and in BMP4 expression in LSC from TKI-resistant patients in comparison with diagnosis, while remaining unchanged in sensitive patients. Both leukemic and nonleukemic cells exhibit higher BMP4 levels in the bone marrow of TKI-resistant patients. Exposure to BMP2/BMP4 does not alter BCR-ABL transcript expression but is accompanied by the overexpression of TWIST-1, a transcription factor highly expressed in resistant LSC. By modulating BMP4 or BMPR1b expression, we show that these elements are involved in TKI resistance. In summary, we reveal that persistence of BMP alterations and existence of an autocrine loop promote CML-primitive cells' TKI resistance.

Epithelia are widely found in human tissues where they play various functions, providing mechanical protection, regulating exchanges between a body cavity and the underlying tissue or secreting various molecules inside or outside the body. This plethora of epithelia is susceptible to a large number of inherited or sporadic diseases impacting their functions. To cite but a few, cystic fibrosis is an inherited autosomal recessive disorder due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene coding for a chloride ion channel (1). CFTR loss in lung, pancreas, liver kidney and intestine results in epithelia malfunctions, respiratory complications being the most life-threatening symptom of cystic fibrosis. Inherited mutations in the Breast Cancer susceptibility (BRCA) 1 or 2 tumor suppressor genes predispose to breast and ovarian cancers originating from epithelial cells transformation (2). The ability to grow normal and pathological primary human adult epithelial cells in culture and to subject them to genetic manipulations is then of paramount importance for the fundamental studies of these diseases and for the development of efficient treatments.

Cis-element identification is a prerequisite to understand transcriptional regulation of gene loci. From analysis of a limited number of conserved gene loci, sequence comparison has proved a robust and efficient way to locate cis-elements. Human and mouse GATA1 genes encode a critical hematopoietic transcription factor conserved in expression and function. Proper control of GATA1 transcription is critical in regulating myeloid lineage specification and maturation. Here, we compared sequence and systematically mapped position of DNase I hypersensitive sites, acetylation status of histone H3/H4, and in vivo binding of transcription factors over approximately 120 kilobases flanking the human GATA1 gene and the corresponding region in mice. Despite lying in approximately 10 megabase (Mb) conserved syntenic segment, the chromatin structures of the 2 homologous loci are strikingly different. The 2 previously unidentified hematopoietic cis-elements, one in each species, are not conserved in position and sequence and have enhancer activity in erythroid cells. In vivo, they both bind the transcription factors GATA1, SCL, LMO2, and Ldb1. More broadly, there are both species- and regulatory element–specific patterns of transcription factor binding. These findings suggest that some cis-elements regulating human and mouse GATA1 genes differ. More generally, mouse human sequence comparison may fail to identify all cis-elements.

The critical myeloid transcription factor GATA1 can specify erythroid cells at expense of granulocytes/macrophages (GM) from the common myeloid progenitor (CMP). In red cells sustained GATA1 expression is required for terminal maturation. Conversely, GATA1 expression has to be extinguished to allow GM differentiation. Therefore, one component in dissecting myeloid specification will be to define how GATA1 expression is regulated. The level of GATA1 is mainly controlled transcriptionally. Here, we show that murine(m) GATA1 mRNA rises 6-fold as CMPs differentiate to MEPs (megakaryocyte-erythroid progenitor) and another 4-fold as MEPs differentiate to Ter119+ erythroid cells. As a step towards understanding the molecular basis of erythroid-specific GATA1 expression, we have been characterising GATA1 cis-elements. Previously, we and others showed that an upstream enhancer (mHS−3.5) is required to direct erythroid-specific GATA1 expression in cooperation with sequences near the mGata1 gene (IE promoter and intron element mHS+3.5) in transgenic mice. Though a mGata1 transgene regulated by mHS−3.5-IE-mHS+3.5 grossly rescues erythropoiesis in GATA1 knock out mice, germ line deletion of mHS−3.5 leaves red cell GATA1 expression unaffected. This suggests other cis-elements in the mGata1 locus can substitute for mHS−3.5 in red cells. Recently, we identified an erythroid-specific DNase I hypersensitive site (DHS), mHS-25, with enhancer activity in erythroid cell lines and where the chromatin associated with it is hyperacetylated at histone H3/H4. We now demonstrate by fine DHS mapping that mHS-25 is more complex, being composed of two adjacent DHSs ~500 bp apart (mHS-25 and mHS-26). These DHSs are present only in primary red cells and not other primary cells. Chromatin immunoprecipitation (ChiP) shows that H3 and H4 associated with both sites is hyperacetylated only in Ter119+ cells. We tested mHS25/6 function in mice transgenic for a mGata1-LacZ reporter construct regulated by mHS-25/6-IE-mHS+3.5. β-galactosidase expression was quantitated in myeloid lineages by FACS analysis using lineage-specific antibodies and the β-galactosidase substrate FDG. 6 out of 7 F0 transgenic embryos expressed β-galactosidase in 5–23% of fetal liver Ter119+ cells. FDG staining was not detected in CD61+Mac1- megakaryocytes or Mac-1+ macrophages.

Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

Mona/grb2 related adapter downstream of shc is a molecular adapter expressed in platelets, T lymphocytes and myelomonocytic cells. Using human hematopoietic cell lines, we have previously shown that lineage-specific Mona expression is achieved through the production of two transcripts (named 1A and 1B) differing by their 5′ untranslated region (5′UTR). Thus, platelets and megakaryocytic cell lines K562 and HEL (Human Erythro-Leukemia) specifically express 1B messenger RNA (mRNA). We report here characterization of the (−2031/+72) genomic region relative to the putative transcription start site of 1B mRNA. We show this region is sufficient to ensure specific reporter gene expression in megakaryocytic cell lines, and that most promoter activity is contained in the (−225/+72) fragment. Electro-mobility shift assay and mutational analyses indicated that GATA-1 and a yet unidentified E-26 family member transcription factor are required for 1B (−2031/+72) promoter activity. Thus, Mona 1B promoter exhibits typical features of megakaryocyte-specific promoters.

International audience; We previously found that the NF-κB transcription factor is activated during the recovery period after heat shock; moreover, we demonstrated that NF-κB is essential for cell survival after heat shock by activating autophagy, a mechanism that probably helps the cell to cope with hyperthermic stress through clearance of damaged proteins. In this study, we analyze the involvement of NF-κB in basal and heat-stress-induced protein quality control, by comparing the level of multiubiquitylated and/or aggregated proteins, and proteasome and autophagic activity in NF-κB-competent and NF-κB-incompetent cells. We show that NF-κB has only a minor role in basal protein quality control, where it modulates autophagosome maturation. By contrast, NF-κB is shown to be a key player in protein quality control after hyperthermia. Indeed, NF-κB-incompetent cells show highly increased levels of multiubiquitylated and/or aggregated proteins and aggresome clearance defects; a phenotype that disappears when NF-κB activity is restored to normal. We demonstrate that during heat shock recovery NF-κB activates selective removal of misfolded or aggregated proteins--a process also called 'aggrephagy'--by controlling the expression of BAG3 and HSPB8 and by modulating the level of the BAG3-HspB8 complex. Thus NF-κB-mediated increase in the level of the BAG3-HspB8 complex leads to upregulation of aggrephagy and clearance of irreversibly damaged proteins and might increase cell survival in conditions of hyperthermia.

International audience; Clonal erythroleukemia developing in susceptible mice infected by Friend virus complex are associated with highly recurrent proviral insertions at one of three loci called Spi-1, Fli-1 or Fli-3, leading to deregulated expression of oncogenic Spi-1 or Fli-1 transcription factors or miR-17-92 miRNA cluster, respectively. Deregulated expression of each of these three oncogenes has been independently shown to contribute to cell proliferation of erythroleukemic clones. Previous studies showed a close relationship between Spi-1 and Fli-1, which belong to the same ETS family, Spi-1 activating fli-1 gene, and both Spi-1 and Fli-1 activating multiple common target genes involved in ribosome biogenesis. In this study, we demonstrated that Spi-1 and Fli-1 are also involved in direct miR-17-92 transcriptional activation through their binding to a conserved ETS binding site in its promoter. Moreover, we demonstrated that physiological re-expression of exogenous miR-17 and miR-20a are able to partially rescue the proliferation loss induced by Fli-1 knock-down and identified HBP1 as a target of these miRNA in erythroleukemic cells. These results establish that three of the most recurrently activated oncogenes in Friend erythroleukemia are actually involved in a same oncogenic network controlling cell proliferation. The putative contribution of a similar ETS-miR-17-92 network module in other normal or pathological proliferative contexts is discussed.

This study investigated the role of the ETS transcription factor Fli-1 in adult myelopoiesis using new transgenic mice allowing inducible Fli-1 gene deletion. Fli-1 deletion in adult induced mild thrombocytopenia associated with a drastic decrease in large mature megakaryocytes number. Bone marrow bipotent megakaryocytic-erythrocytic progenitors (MEPs) increased by 50% without increase in erythrocytic and megakaryocytic common myeloid progenitor progeny, suggesting increased production from upstream stem cells. These MEPs were almost unable to generate pure colonies containing large mature megakaryocytes, but generated the same total number of colonies mainly identifiable as erythroid colonies containing a reduced number of more differentiated cells. Cytological and fluorescence-activated cell sorting analyses of MEP progeny in semisolid and liquid cultures confirmed the drastic decrease in large mature megakaryocytes but revealed a surprisingly modest (50%) reduction of CD41-positive cells indicating the persistence of a megakaryocytic commitment potential. Symmetrical increase and decrease of monocytic and granulocytic progenitors were also observed in the progeny of purified granulocytic-monocytic progenitors and common myeloid progenitors. In summary, this study indicates that Fli-1 controls several lineages commitment decisions at the stem cell, MEP, and granulocytic-monocytic progenitor levels, stimulates the proliferation of committed erythrocytic progenitors at the expense of their differentiation, and is a major regulator of late stages of megakaryocytic differentiation.

International audience; Spi-1 and Fli-1 are ETS transcription factors recurrently deregulated in mouse erythroleukemia induced by Friend viruses. Since they share the same core DNA binding site, we investigated whether they may contribute to erythroleukemia by common mechanisms. Using inducible knockdown, we demonstrated that Fli-1 contributes to proliferation, survival, and differentiation arrest of erythroleukemic cells harboring an activated fli-1 locus. Similarly, we used inducible Fli-1 knockdown and either hexamethylenebisacetamide (HMBA)- or small interfering RNA-mediated Spi-1 knockdown to investigate their respective contributions in erythroleukemic cells harboring an activated spi-1 locus. In these cells, simple or double knockdown of both Spi-1 and Fli-1 additively contributed to induce proliferation arrest and differentiation. Transcriptome profiling revealed that virtually all transcripts affected by both Fli-1 knockdown and HMBA are affected in an additive manner. Among these additively downregulated transcripts, more than 20% encode proteins involved in ribosome biogenesis, and conserved ETS binding sites are present in their gene promoters. Through chromatin immunoprecipitation, we demonstrated the association of Spi-1 and Fli-1 on these promoters in Friend erythroleukemic cells. These data lead us to propose that the oncogenicity of Spi-1, Fli-1, and possibly other ETS transcription factors may involve their ability to stimulate ribosome biogenesis.

Previous observations suggested that functional antagonism between FLI-1 and EKLF might be involved in the commitment toward erythrocytic or megakaryocytic differentiation. We show here, using inducible shRNA expression, that EKLF knockdown in mouse erythroleukemia (MEL) cells decreases erythrocytic and increases megakaryocytic as well as Fli-1 gene expression. Chromatin immunoprecipitation analyses revealed that the increase in megakaryocytic gene expression is associated with a marked increase in RNA pol II and FLI-1 occupancy at their promoters, albeit FLI-1 protein levels are only minimally affected. Similarly, we show that human CD34+ progenitors infected with shRNA lentivirus allowing EKLF knockdown generate an increased number of differentiated megakaryocytic cells associated with increased levels of megakaryocytic and Fli-1 gene transcripts. Single-cell progeny analysis of a cell population enriched in bipotent progenitors revealed that EKLF knockdown increases the number of megakaryocytic at the expense of erythrocytic colonies. Taken together, these data indicate that EKLF restricts megakaryocytic differentiation to the benefit of erythrocytic differentiation and suggest that this might be at least partially mediated by the inhibition of FLI-1 recruitment to megakaryocytic and Fli-1 gene promoters.

The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.

Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well-studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the transcription factor GATA1, that is required for normal maturation of these two lineages. Megakaryocyte- and erythroid-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, we previously showed that the 3′ 179 base pairs (bp) of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show that mHS-3.5 binds key hemopoietic transcription factors in vivo (GATA1, SCL/TAL-1) and is required to maintain histone acetylation in the mGata1 locus in primary megakaryocytes. When deletional constructs containing mHS-3.5 were used to direct GATA1-LacZ reporter gene expression in transgenic mice, a 25 bp element within the 3′ 179bp in mHS-3.5, was critical for megakaryocyte expression. In vitro three uncharacterized DNA-binding activities A, B and C bind to the core of the 25 bp element, and these binding sites are conserved through evolution. Of these, only activity B is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals that activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows that sequence corresponding to the binding site for activity B is a previously unrecognised motif present in the cis-elements of other megakaryocyte-specific genes. In summary, we have identified a motif and a DNA-binding activity that are likely to be important in directing a megakaryocyte gene expression program distinct from that in red cells.

GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.

GATA-1 is a key transcription factor essential for the differentiation of the erythroid, megakaryocytic and eosinophilic lineages. GATA-1 functions in erythropoiesis involve lineage-specific gene activation and repression of early hematopoietic transcription programs. GATA-1 is known to interact with other transcription factors, such as FOG-1, TAL-1 and Sp1 and also with CBP/p300 and the SWI/SNF chromatin remodeling complex in vitro. Despite this information the molecular basis of its essential functions in erythropoiesis remains unclear. We show here that GATA-1 is mostly present in a high (> 670kDa) molecular weight complex that appears to be dynamic during erythroid differentiation. In order to characterize the GATA-1 complex(es) from erythroid cells, we employed an in vivo biotinylation tagging approach in mouse erythroleukemic (MEL) cells1. Briefly, this involved the fusion of a small (23aa) peptide tag to GATA-1 and its specific, efficient biotinylation by the bacterial BirA biotin ligase which is co-expressed with tagged GATA-1 in MEL cells. Nuclear extracts expressing biotinylated tagged GATA-1 were bound directly to streptavidin beads and co-purifying proteins were identified by mass spectrometry. In addition to the known GATA-1-interacting transcription factors FOG-1, TAL-1 and Ldb-1, we describe novel interactions with the essential hematopoietic transcription factor Gfi-1b and the chromatin remodeling complexes MeCP1 and ACF/WCRF. Significantly, GATA-1 interaction with the repressive MeCP1 complex requires FOG-1. We also show in erythroid cells that GATA-1, FOG-1 and MeCP1 are stably bound to repressed genes representing early hematopoietic (e.g. GATA-2) or alternative lineage-specific (e.g. eosinophilic) transcription programs, whereas the GATA-1/Gfi1b complex is bound to repressed genes involved in cell proliferation. In contrast, GATA-1 and TAL-1 are bound to the active erythroid-specific EKLF gene. Our findings on GATA-1 complexes provide novel insight as to the critical roles that GATA-1 plays in many aspects of erythropoiesis by revealing the GATA-1 partners in the execution of specific functions.

Expression of the myeloid transcription factor GATA1 is required for early stages of eosinophil differentiation. Defining mechanisms regulating eosinophil GATA1 expression will be important to understand development of this lineage. However, the cis-elements required for eosinophil GATA1 expression are not fully characterized. Previous work identified HS 1 as a major GATA1 enhancer, but its role in eosinophil GATA1 expression is unclear. Here, we show that mouse HS 1 deletion leaves eosinophil GATA1 mRNA expression and eosinophil differentiation unaffected. Chromatin isolated from eosinophils and encompassing HS 1 is weakly enriched for acetylated histones H3/H4. HS 1 deletion does not alter eosinophil GATA1 locus histone acetylation. In eosinophils, GATA1 and CCAAT/enhancer binding protein ϵ (C/EBPϵ) do not bind HS 1 but bind selectively a cis-element in the first GATA1 intron. Thus, HS 1 is not required for eosinophil GATA1 expression. Instead, this study suggests a previously unsuspected role for the GATA1 intron element for this function.

Mona, also called Gads, is a molecular adapter that plays a key role in T-cell and platelet signalling by linking the adaptors Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (Slp-76) and linker for activation of T cells (LAT) upon T-cell receptor and collagen receptor activation. Platelets express a specific form of Mona mRNA, called 1B, which is transcribed from a megakaryocyte-specific promoter. Mona 1B mRNA differ from 1A transcripts found in T cells and some myeloid cells only by the 5'UTR. We report here that 1B mRNA expressing cells do not express detectable amounts of Mona protein, in contrast to 1A expressing cells, and we show that 1B 5'UTR contains upstream open reading frames (uORFs). Mutating the corresponding uAUG restored efficient Mona translation, or that of an unrelated ORF. This suggested that Mona protein expression in 1B mRNA expressing cells is tightly controlled at the translational level. Accordingly, Mona protein was not detected in resting platelets. Strikingly, platelet activation by thrombin resulted in the rapid induction of Mona protein expression, suggesting that translation inhibition of 1B mRNA may be relieved in activated platelets.

Monocytic adaptor (Mona) also known as Gads is a Grb2-related adaptor whose expression is restricted to hematopoietic cells. It plays an important role in intracellular signaling in T cells, monocytic cells, and platelets. Here we investigated the regulatory aspects of Mona expression in human hematopoietic cells. This was carried out by combining nucleotide sequence analyzes and experimental approaches. We confirmed that Mona expression is restricted to T-cell, myeloid and platelet lineages. In the various cells examined, we detected two major Mona transcripts (1.9 and 4 kb), likely resulting from the alternative use of two polyadenylation sites. Consequently, Mona transcripts of the same size have identical 3' untranslated region (UTR), irrespective of the cell type. In contrast, Mona transcripts contain either 5' UTR-1A or -1B exons, that were detected in a cell-lineage specific manner. Thus, T cells and several myeloid cell lines express 5' UTR-1A-containing transcripts, whereas platelets and cell lines exhibiting megakaryocytic potential express 5' UTR-1B-containing transcripts. Interestingly, 5' UTR-1A is generated from an exon located approximately 45 kb upstream of exon 1B. This suggested that lineage-restricted transcription of the Mona gene is controlled by specific promoters. Indeed, 2-kb genomic fragments upstream of each 5'-UTR showed lineage-restricted ability to drive expression of luc reporter gene.

Genetic insults of ribosomal protein (RP) genes including heterozygous mutations in Diamond Blackfan Anemia (DBA) or RPS14 haploinsufficiency in acquired 5q- syndrome selectively impair erythropoiesis suggesting that the integrity of ribosome biogenesis (RB) is fully required for the production of 10e11erythrocytes per day in humans. Defective RB consecutive to RPL5, RPL11 mutation or RPS14 deletion is thought to induce cell cycle arrest and a various degree of apoptosis in erythroblasts (EB). However, recent data showed that, loss of RPL5/RPL11neither induces apoptosis nor arrests cell cycle, but hampers the progression through the G1 phase, in primary fibroblasts. Furthermore, RB coordinates cell cycle to cell growth for the maintenance of constant cell size. In this work, we investigate the role of RB in erythroid cell differentiation and cell size regulation. We first analyzed the renewal of ribosome during in vitro differentiation of human EB derived from cytapheresis CD34+ cells and mouse erythroblasts derived from mouse fetal liver by a pulsed SILAC (Stable Isotopic Labeling by Amino acids in Culture cell) riboproteomic assay. Ribosome biogenesis dramatically decreases with the disappearance of proEB and basophilic EB and the onset of poly- and ortho-chromatophilic EB. Importantly, inhibition of RNA polI by CX-5461 in proEB forced them to enter the final maturation steps with an increase of glycophorin A (GPA) expression. To study the effect of RPS14 heterozygous deletion on RB, UT7/EPO cell line was infected by a lentivirus containing an inducible GFP-shRNA RPS14. After a 48-h treatment with doxycyclin, Rps14 protein expression was reduced by half and sorted GFP-positive cells had an altered ribosome profile devoid of 40S small subunit or 80S entire particle. Consistently, RB inhibition induced a cell size reduction. Second, we compared RB level in cells responsive to SCF+EPO or EPO alone. RB was optimal when EB responded to SCF+EPO and this was correlated with cell size being higher in SCF+EPO-responsive cells compared to EPO-responsive cells. Both cytokines additively activate the cell growth regulator, p70S6K1. Third, inhibition of p70S6K1 by rapamycin, or a specific chemical S6K1 inhibitor significantly reduced RB as shown by a 50% decrease of ribosome renewal in pulsed-SILAC. Inhibition of RB by rapamycin led to a size reduction and to GPA acquisition, which are the features of erythroid cell differentiation. Our data shows that the collapse of RB due to the loss of c-Kit and reduced activation of p70S6K1 is a key step for cell growth inhibition and induction of terminal differentiation in human or mouse erythroblasts. Disclosures No relevant conflicts of interest to declare.

Stem cells are regulated by several cues from their microenvironment and it is becoming increasingly clear that perturbations of these signals can participate to transformation. Previously, our lab showed that BMP2 (B2) was able to induce the transformation of breast epithelial stem cells and it was overproduced in luminal breast cancer microenvironment. It was also found that a potential source of this B2 over-expression was the exposure to oestrogen (E2)-mimetic environmental pollutants such as BPA, which is also involved in breast cancer development due to its effect on oestrogen receptor ERα66. Several studies have already reported the dangerous effects of BPA on human health, leading to its replacement by other bisphenols, such as BPS and BPF, showing a lower ERα66 affinity. However, some studies suggest that these BPA’s alternatives could also be endocrine disruptors via ERα36 activation, an ERα66 isoform, calling into question their innocuity and the audit criteria used to evaluate their effect on human health. All these data leading us to postulate the hypothesis of bisphenols involvement in stem cells properties dysregulation and transformation, through the cooperation between BMP2 signalling and ERα36. We had followed the effect of a chronical exposure to E2, BPA, BPS, BPF, alone or in combination with B2, in the ERα66-/ ERα46-/ ERα36+ immature human mammary epithelial cell line’s activity and transformation. We found that chronical exposure to BPS or BPF act in collaboration with B2 in favour to a stem cell compartment enrichment. One molecular mechanism could be probably due to B2 hypersensitivity induction, leading to potentiation of its canonical pathway. Moreover, we also identify for the first time, the existence of a physical interaction between ERα36 and two major actors of B2 pathway: BMPR1B and SMAD1. Furthermore, we highlight that ERα36/BMPR1B interaction is lose after chronical exposure to bisphenols or E2, with B2 as well as without, suggesting existence of a crosstalk between ERα36 and B2 signalling pathway. This work allowed to better understand relation existing between ERα36 and B2 pathway, two important pathways independently involved in breast cancers development. Linking these two signalling allows to better understand how hormonal signals, endogen or from our environment, participate altogether to modify physiological cell regulation. In addition to its fundamental significance, this work strongly emphasizes the necessity to go further on the study of the innocuity and regulation of these E2-mimetic environmental pollutants, but also on the diagnostic and therapies in breast cancers; Stem cells are regulated by several cues from their microenvironment and it is becoming increasingly clear that perturbations of these signals can participate to transformation. Previously, our lab showed that BMP2 (B2) was able to induce the transformation of breast epithelial stem cells and it was overproduced in luminal breast cancer microenvironment. It was also found that a potential source of this B2 over-expression was the exposure to oestrogen (E2)-mimetic environmental pollutants such as BPA, which is also involved in breast cancer development due to its effect on oestrogen receptor ERα66. Several studies have already reported the dangerous effects of BPA on human health, leading to its replacement by other bisphenols, such as BPS and BPF, showing a lower ERα66 affinity. However, some studies suggest that these BPA’s alternatives could also be endocrine disruptors via ERα36 activation, an ERα66 isoform, calling into question their innocuity and the audit criteria used to evaluate their effect on human health. All these data leading us to postulate the hypothesis of bisphenols involvement in stem cells properties dysregulation and transformation, through the cooperation between BMP2 signalling and ERα36. We had followed the effect of a chronical exposure to E2, BPA, BPS, BPF, alone or in combination with B2, in the ERα66-/ ERα46-/ ERα36+ immature human mammary epithelial cell line’s activity and transformation. We found that chronical exposure to BPS or BPF act in collaboration with B2 in favour to a stem cell compartment enrichment. One molecular mechanism could be probably due to B2 hypersensitivity induction, leading to potentiation of its canonical pathway. Moreover, we also identify for the first time, the existence of a physical interaction between ERα36 and two major actors of B2 pathway: BMPR1B and SMAD1. Furthermore, we highlight that ERα36/BMPR1B interaction is lose after chronical exposure to bisphenols or E2, with B2 as well as without, suggesting existence of a crosstalk between ERα36 and B2 signalling pathway. This work allowed to better understand relation existing between ERα36 and B2 pathway, two important pathways independently involved in breast cancers development. Linking these two signalling allows to better understand how hormonal signals, endogen or from our environment, participate altogether to modify physiological cell regulation. In addition to its fundamental significance, this work strongly emphasizes the necessity to go further on the study of the innocuity and regulation of these E2-mimetic environmental pollutants, but also on the diagnostic and therapies in breast cancers