Catherine Porcher

The T Cell Leukemia Oncoprotein SCL/tal-1 Is Essential for Development of All Hematopoietic Lineages

The T cell leukemia oncoprotein SCL/tal-1, a basic-helix-loop-helix transcription factor, is required for production of embryonic red blood cells in the mouse yolk sac. To define roles in other lineages, we studied the hematopoietic potential of homozygous mutant SCL/tal-1 -/- embryonic stem cells upon in vitro differentiation and in vivo in chimeric mice. Here we show that in the absence of SCL/tal-1, hematopoiesis, Including the generation of red cells, myeloid cells, megakaryocytes, mast cells, and both T and B lymphoid cells, is undetectable. These findings suggest that SCL/tal-1 functions very early in hematopoietic development, either in specification of ventral mesoderm to a blood cell fate, or in formation or maintenance of immature progenitors.

Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia

The relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.

Genome-wide identification of TAL1's functional targets: Insights into its mechanisms of action in primary erythroid cells

Coordination of cellular processes through the establishment of tissue-specific gene expression programs is essential for lineage maturation. The basic helix-loop-helix hemopoietic transcriptional regulator TAL1 (formerly SCL) is required for terminal differentiation of red blood cells. To gain insight into TAL1 function and mechanisms of action in erythropoiesis, we performed ChIP-sequencing and gene expression analyses from primary fetal liver erythroid cells. We show that TAL1 coordinates expression of genes in most known red cell-specific processes. The majority of TAL1s genomic targets require direct DNA-binding activity. However, one-fifth of TAL1s target sequences, mainly among those showing high affinity for TAL1, can recruit the factor independently of its DNA binding activity. An unbiased DNA motif search of sequences bound by TAL1 identified CAGNTG as TAL1-preferred E-box motif in erythroid cells. Novel motifs were also characterized that may help distinguish activated from repressed genes and suggest a new mechanism by which TAL1 may be recruited to DNA. Finally, analysis of recruitment of GATA1, a protein partner of TAL1, to sequences occupied by TAL1 suggests that TAL1s binding is necessary prior or simultaneous to that of GATA1. This work provides the framework to study regulatory networks leading to erythroid terminal maturation and to model mechanisms of action of tissue-specific transcription factors.

ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis

ABSTRACT 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.

GATA1-Mediated Megakaryocyte Differentiation and Growth Control Can Be Uncoupled and Mapped to Different Domains in GATA1

ABSTRACT The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.

Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation.

The transcription factor GATA1 coordinates timely activation and repression of megakaryocyte gene expression. Loss of GATA1 function results in excessive megakaryocyte proliferation and disordered terminal platelet maturation, leading to thrombocytopenia and leukemia in patients. The mechanisms by which GATA1 does this are unclear. We have used in vivo biotinylated GATA1 to isolate megakaryocyte GATA1-partner proteins. Here, several independent approaches show that GATA1 interacts with several proteins in the megakaryocyte cell line L8057 and in primary megakaryocytes. They include FOG1, the NURD complex, the pentameric complex containing SCL/TAL-1, the zinc-finger regulators GFI1B and ZFP143, and the corepressor ETO2. Knockdown of ETO2 expression promotes megakaryocyte differentiation and enhances expression of select genes expressed in terminal megakaryocyte maturation, eg, platelet factor 4 (Pf4). ETO2-dependent direct repression of the Pf4 proximal promoter is mediated by GATA-binding sites and an E-Box motif. Consistent with this, endogenous ETO2, GATA1, and the SCL pentameric complex all specifically bind the promoter in vivo. Finally, as ETO2 expression is restricted to immature megakaryocytes, these data suggest that ETO2 directly represses inappropriate early expression of a subset of terminally expressed megakaryocyte genes by binding to GATA1 and SCL.

Structure of the Leukemia Oncogene Lmo2: Implications for the Assembly of a Hematopoietic Transcription Factor Complex.

The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis.

Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding-independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCLs action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

Uncoupling VEGFA Functions in Arteriogenesis and Hematopoietic Stem Cell Specification

Summary VEGFA signaling is critical for endothelial and hematopoietic stem cell (HSC) specification. However, blood defects resulting from perturbation of the VEGFA pathway are always accompanied by impaired vascular/arterial development. Because HSCs derive from arterial cells, it is unclear whether VEGFA directly contributes to HSC specification. This is an important question for our understanding of how HSCs are formed and for developing their production in vitro. Through knockdown of the regulator ETO2 in embryogenesis, we report a specific decrease in expression of medium/long Vegfa isoforms in somites. This leads to absence of Notch1 expression and failure of HSC specification in the dorsal aorta (DA), independently of vessel formation and arterial specification. Vegfa hypomorphs and isoform-specific (medium/long) morphants not only recapitulate this phenotype but also demonstrate that VEGFA short isoform is sufficient for DA development. Therefore, sequential, isoform-specific VEGFA signaling successively induces the endothelial, arterial, and HSC programs in the DA.

Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1

ABSTRACT The helix-loop-helix (HLH) domain is employed by many transcription factors that control cell fate choice in multiple developmental settings. Previously, we demonstrated that the HLH domain of the class II basic HLH (bHLH) protein SCL/Tal-1 is critical for hematopoietic specification. We have now identified residues in this domain that are essential for restoring hematopoietic development to SCL−/− embryonic stem cells and sufficient to convert a muscle-specific HLH domain to one able to rescue hematopoiesis. Most of these critical residues are distributed in the loop of SCL, with one in helix 2. This is in contrast to the case for MyoD, the prototype of class II bHLH proteins, where the loop seems to serve mainly as a linker between the two helices. Among the identified residues, some promote heterodimerization with the bHLH partners of SCL (E12/E47), while others, unimportant for this property, are still crucial for the biological function of SCL. Importantly, the residue in helix 2 specifically promotes interaction with a known partner of SCL, the LIM-only protein LMO2, a finding that strengthens genetic evidence that these proteins interact. Our data highlight the functional complexity of bHLH proteins, provide mechanistic insight into SCL function, and strongly support the existence of an active SCL/LMO2-containing multiprotein complex in early hematopoietic cells.