Interplay between FLI-1 and the LDB1 complex in murine erythroleukemia cells and during megakaryopoiesis.

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.

p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

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.

Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors.

This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.

Instructive role of M-CSF on commitment of bipotent myeloid cells involves ERK-dependent positive and negative signaling.

M-CSF and G-CSF are instructive cytokines that specifically induce differentiation of bipotent myeloid progenitors into macrophages and granulocytes, respectively. Through morphology and colony assay studies, flow cytometry analysis of specific markers, and expression of myeloid transcription factors, we show here that the Eger/Fms cell line is composed of cells whose differentiation fate is instructed by M-CSF and G-CSF, thus representing a good in vitro model of myeloid bipotent progenitors. Consistent with the essential role of ERK1/2 during macrophage differentiation and defects of macrophagic differentiation in native ERK1(-/-) progenitors, ERK signaling is strongly activated in Eger/Fms cells upon M-CSF-induced macrophagic differentiation but only to a very small extent during G-CSF-induced granulocytic differentiation. Previous in vivo studies indicated a key role of Fli-1 in myeloid differentiation and demonstrated its weak expression during macrophagic differentiation with a strong expression during granulocytic differentiation. Here, we demonstrated that this effect could be mediated by a differential regulation of protein kinase Cδ (PKCd) on Fli-1 expression in response to M-CSF and G-CSF. With the use of knockdown of PKCd by small interfering RNA, we demonstrated that M-CSF activates PKCd, which in turn, inhibits Fli-1 expression and granulocytic differentiation. Finally, we studied the connection between ERK and PKCd and showed that in the presence of the MEK inhibitor U0126, PKCd expression is decreased, and Fli-1 expression is increased in response to M-CSF. Altogether, we demonstrated that in bipotent myeloid cells, M-CSF promotes macrophagic over granulocytic differentiation by inducing ERK activation but also PKCd expression, which in turn, down-regulates Fli-1 expression and prevents granulocytic differentiation.

Spi-1, Fli-1 and Fli-3 (miR-17-92) oncogenes contribute to a single oncogenic network controlling cell proliferation in friend erythroleukemia.

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.

Spi-1/PU.1 activates transcription through clustered DNA occupancy in erythroleukemia.

Acute leukemias are characterized by deregulation of transcriptional networks that control the lineage specificity of gene expression. The aberrant overexpression of the Spi-1/PU.1 transcription factor leads to erythroleukemia. To determine how Spi-1 mechanistically influences the transcriptional program, we combined a ChIP-seq analysis with transcriptional profiling in cells from an erythroleukemic mouse model. We show that Spi-1 displays a selective DNA-binding that does not often cause transcriptional modulation. We report that Spi-1 controls transcriptional activation and repression partially through distinct Spi-1 recruitment to chromatin. We revealed several parameters impacting on Spi-1-mediated transcriptional activation. Gene activation is facilitated by Spi-1 occupancy close to transcriptional starting site of genes devoid of CGIs. Moreover, in those regions Spi-1 acts by binding to multiple motifs tightly clustered and with similar orientation. Finally, in contrast to the myeloid and lymphoid B cells in which Spi-1 exerts a physiological activity, in the erythroleukemic cells, lineage-specific cooperating factors do not play a prevalent role in Spi-1-mediated transcriptional activation. Thus, our work describes a new mechanism of gene activation through clustered site occupancy of Spi-1 particularly relevant in regard to the strong expression of Spi-1 in the erythroleukemic cells.

Inducible Fli-1 gene deletion in adult mice modifies several myeloid lineage commitment decisions and accelerates proliferation arrest and terminal erythrocytic differentiation.

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.

Spi-1 and Fli-1 directly activate common target genes involved in ribosome biogenesis in Friend erythroleukemic cells.

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.

Distinct modes of gene regulation by a cell-specific transcriptional activator.

The architectural layout of a eukaryotic RNA polymerase II core promoter plays a role in general transcriptional activation. However, its role in tissue-specific expression is not known. For example, differing modes of its recognition by general transcription machinery can provide an additional layer of control within which a single tissue-restricted transcription factor may operate. Erythroid Kruppel-like factor (EKLF) is a hematopoietic-specific transcription factor that is critical for the activation of subset of erythroid genes. We find that EKLF interacts with TATA binding protein-associated factor 9 (TAF9), which leads to important consequences for expression of adult beta-globin. First, TAF9 functionally supports EKLF activity by enhancing its ability to activate the beta-globin gene. Second, TAF9 interacts with a conserved beta-globin downstream promoter element, and ablation of this interaction by beta-thalassemia-causing mutations decreases its promoter activity and disables superactivation. Third, depletion of EKLF prevents recruitment of TAF9 to the beta-globin promoter, whereas depletion of TAF9 drastically impairs beta-promoter activity. However, a TAF9-independent mode of EKLF transcriptional activation is exhibited by the alpha-hemoglobin-stabilizing protein (AHSP) gene, which does not contain a discernable downstream promoter element. In this case, TAF9 does not enhance EKLF activity and depletion of TAF9 has no effect on AHSP promoter activation. These studies demonstrate that EKLF directs different modes of tissue-specific transcriptional activation depending on the architecture of its target core promoter.

EKLF restricts megakaryocytic differentiation at the benefit of erythrocytic differentiation.

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.

Activator-mediated recruitment of the MLL2 methyltransferase complex to the beta-globin locus.

MLL-containing complexes methylate histone H3 at lysine 4 (H3K4) and have been implicated in the regulation of transcription. However, it is unclear how MLL complexes are targeted to specific gene loci. Here, we show that the MLL2 complex associates with the hematopoietic activator NF-E2 in erythroid cells and is important for H3K4 trimethylation and maximal levels of transcription at the beta-globin locus. Furthermore, recruitment of the MLL2 complex to the beta-globin locus is dependent upon NF-E2 and coincides spatio-temporally with NF-E2 binding during erythroid differentiation. Thus, a DNA-bound activator is important initially for guiding MLL2 to a particular genomic location. Interestingly, while the MLL2-associated subunit ASH2L is restricted to the beta-globin locus control region 38 kb upstream of the beta(maj)-globin gene, the MLL2 protein spreads across the beta-globin locus, suggesting a previously undefined mechanism by which an activator influences transcription and H3K4 trimethylation at a distance.

Disruption of the mechanism of long range activation within the human alpha-globin complex.

The human alpha-globin complex lies at the tip of the short arm of chromosome 16. It comprises three functional globin genes (5'-zeta2-alpha2-alpha1-3'), the expression of which is strictly dependent on a positive regulatory element located 40-kb upstream, HS-40. This DNase I-hypersensitive site is the only known regulatory element displaying strong erythroid-specific enhancer activity within the human alpha-globin complex. How this enhancer activity is shared among different erythroid genes present in the same cluster without affecting the ubiquitous genes present within and around the complex is poorly understood. To address this issue, we used hybrid murine erythroleukemia cells containing a single copy of human chromosome 16 and targeted the insertion of different sequences downstream of HS-40 by recombinase-mediated cassette exchange. We thus demonstrate that (i). HS-40-mediated erythroid-specific activation of the alpha-globin genes is impaired solely by the insertion of a promoter sequence and not a coding sequence, unless it is methylated, and that (ii). the degree of transcriptional repression observed seems to be related directly to the transcriptional rate of the inserted promoter. Taken together, these results emphasize the importance of promoter sequences as the main targets for the activation mechanism of the human alpha-globin genes by HS-40.

Spi-1/PU.1 but not Fli-1 inhibits erythroid-specific alternative splicing of 4.1R pre-mRNA in murine erythroleukemia cells.

The inclusion of exon 16 in mature protein 4.1R mRNA arises from a stage-specific splicing event that occurs during late erythroid development. We have shown that mouse erythroleukemia (MEL) cells reproduce this erythroid-specific splicing event upon induction of differentiation. We here found that this splicing event is regulated specifically in erythroleukemic cells that have the potential to differentiate and produce hemoglobin, regardless of the nature of the differentiation inducer. Knowing that dysregulated expression of spi-1/pu.1 and fli-1 oncogenes is involved in MEL cell differentiation arrest, we looked at their effect on exon 16 erythroid splicing. We found that exon 16 inclusion requires Spi-1/PU.1 shutdown in MEL cells, and that enforced expression of Spi-1/PU.1 inhibits exon selection, regardless of the presence or absence of a chemical inducer. By contrast, endogenous overexpression or enforced expression of Fli-1 has no effect on exon selection. We further showed that Spi-1/PU.1 acts similarly on the endogenous and on a transfected exon 16, suggesting a promoter-independent effect of Spi-1/PU.1 on splicing regulation. This study provides the first evidence that Spi-1/PU.1 displays the unique property, not shared with Fli-1, to inhibit erythroid-specific pre-mRNA splicing in erythroleukemia cell context.

Induction of globin mRNA expression by interleukin-3 in a stem cell factor-dependent SV-40 T-antigen-immortalized multipotent hematopoietic cell line.

Erythropoiesis requires the stepwise action on immature progenitors of several growth factors, including stem cell factor (SCF), interleukin 3 (IL-3), and erythropoietin (Epo). Epo is required to sustain proliferation and survival of committed progenitors and might further modulate the level of expression of several erythroid genes, including globin genes. Here we report a new SCF-dependent immortalized mouse progenitor cell line (GATA-1 ts SCF) that can also grow in either Epo or IL-3 as the sole growth factor. When grown in SCF, these cells show an "open" chromatin structure of the beta-globin LCR, but do not significantly express globin. However, Epo or IL-3 induce globin expression and are required for its maintainance. This effect of IL-3 is unexpected as IL-3 was previously reported either to be unable to induce hemoglobinization, or even to antagonize it. This suggests that GATA-1 ts SCF cells may have progressed to a stage in which globin genes are already poised for expression and only require signal(s) that can be elicited by either Epo or IL-3. Through the use of inhibitors, we suggest that p38 may be one of the molecules modulating induction and maintenance of globin expression.

Functional cross-antagonism between transcription factors FLI-1 and EKLF.

FLI-1 is an ETS family transcription factor which is overexpressed in Friend erythroleukemia and contributes to the blockage of differentiation of erythroleukemic cells. We show here that FLI-1 represses the transcriptional activity of the beta-globin gene promoter in MEL cells and interacts with two of its critical transactivators, GATA-1 and EKLF. Unexpectedly, FLI-1 enhances the stimulating activity of GATA-1 on a GATA-1-responsive promoter but represses that of EKLF on beta-globin and an EKLF-responsive artificial promoters. This repressive effect of FLI-1 requires the ETS DNA binding domain and its association with either the N- or C-terminal domain, which themselves interact with EKLF but not with GATA-1. Furthermore, the FLI-1 ETS domain alone behaves as an autonomous repression domain when linked to the Gal4 DNA binding domain. Taken together, these data indicate that FLI-1 represses EKLF-dependent transcription due to the repression activity of its ETS domain and its indirect recruitment to erythroid promoters by protein-protein interaction with EKLF. Reciprocally, we also show that EKLF itself represses the FLI-1-dependent megakaryocytic GPIX gene promoter, thus further suggesting that functional cross-antagonism between FLI-1 and EKLF might be involved in the control of the erythrocytic versus megakaryocytic differentiation of bipotential progenitors.

Caspase cleavage of the transcription factor FLI-1 during preB leukemic cell death.

Programmed cell death (apoptosis) is a complex phenomenon that is mediated in mammals mainly via the selective cleavage of intracellular proteins by the large family of cysteine aspartate protease caspases. Apoptosis is tightly regulated by the competitive effect of numerous proteins displaying either pro-apoptotic or anti-apoptotic activity. The ETS-family transcription factor FLI-1, frequently associated with malignant transformation, has been shown to display anti-apoptotic activity in several cell types including avian erythroblasts, mouse fibroblasts or lymphoid cells. We show here that apoptosis of murine preB leukemic cells is accompanied with the specific cleavage of FLI-1 by a caspase-like activity. We also demonstrate that the two isoforms of FLI-1 are indeed cleaved at three conserved sites by caspase 3 in vitro. The conservation of these cleavage sites among species suggests that the caspase cleavage of the anti-apoptotic transcription factor FLI-1 may represent a critical step to ensure irreversible cell death.