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.

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.

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.

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.

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.