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Lineage-specific combinatorial action of enhancers regulates mouse erythroid Gata1 expression.
Precise spatiotemporal control of Gata1 expression is required in both early hematopoietic progenitors to determine erythroid/megakaryocyte versus granulocyte/monocyte lineage output and in the subsequent differentiation of erythroid cells and megakaryocytes. An enhancer element upstream of the mouse Gata1 IE (1st exon erythroid) promoter, mHS-3.5, can direct both erythroid and megakaryocytic expression. However, loss of this element ablates only megakaryocytes, implying that an additional element has erythroid specificity. Here, we identify a double DNaseI hypersensitive site, mHS-25/6, as having erythroid but not megakaryocytic activity in primary cells. It binds an activating transcription factor complex in erythroid cells where it also makes physical contact with the Gata1 promoter. Deletion of mHS-25/6 or mHS-3.5 in embryonic stem cells has only a modest effect on in vitro erythroid differentiation, whereas loss of both elements ablates both primitive and definitive erythropoiesis with an almost complete loss of Gata1 expression. Surprisingly, Gata2 expression was also concomitantly low, suggesting a more complex interaction between these 2 factors than currently envisaged. Thus, whereas mHS-3.5 alone is sufficient for megakaryocytic development, mHS-3.5 and mHS-25/6 collectively regulate erythroid Gata1 expression, demonstrating lineage-specific differences in Gata1 cis-element use important for development of these 2 cell types.
Uncoupling VEGFA functions in arteriogenesis and hematopoietic stem cell specification.
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.
SCL-mediated regulation of the cell-cycle regulator p21 is critical for murine megakaryopoiesis.
Megakaryopoiesis is a complex process that involves major cellular and nuclear changes and relies on controlled coordination of cellular proliferation and differentiation. These mechanisms are orchestrated in part by transcriptional regulators. The key hematopoietic transcription factor stem cell leukemia (SCL)/TAL1 is required in early hematopoietic progenitors for specification of the megakaryocytic lineage. These early functions have, so far, prevented full investigation of its role in megakaryocyte development in loss-of-function studies. Here, we report that SCL critically controls terminal megakaryocyte maturation. In vivo deletion of Scl specifically in the megakaryocytic lineage affects all key attributes of megakaryocyte progenitors (MkPs), namely, proliferation, ploidization, cytoplasmic maturation, and platelet release. Genome-wide expression analysis reveals increased expression of the cell-cycle regulator p21 in Scl-deleted MkPs. Importantly, p21 knockdown-mediated rescue of Scl-mutant MkPs shows full restoration of cell-cycle progression and partial rescue of the nuclear and cytoplasmic maturation defects. Therefore, SCL-mediated transcriptional control of p21 is essential for terminal maturation of MkPs. Our study provides a mechanistic link between a major hematopoietic transcriptional regulator, cell-cycle progression, and megakaryocytic differentiation.
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.
Differences in the chromatin structure and cis-element organization of the human and mouse GATA1 loci: implications for cis-element identification.
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.
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 SCL's 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.
Characterization of a megakaryocyte-specific enhancer of the key hemopoietic transcription factor GATA1.
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 megakaryocyte erythroid transcription factor GATA1. Hemopoietic-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, the 3' 179 bp of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show mHS-3.5 binds key hemopoietic transcription factors in vivo and is required to maintain histone acetylation at the mGata1 locus in primary megakaryocytes. Analysis of GATA1-LacZ reporter gene expression in transgenic mice shows that a 25-bp element within the 3'-179 bp in mHS-3.5 is critical for megakaryocyte expression. In vitro three DNA binding activities A, B, and C bind to the core of the 25-bp element, and these binding sites are conserved through evolution. Activity A is the zinc finger transcription factor ZBP89 that also binds to other cis elements in the mGata1 locus. Activity B is of particular interest as it is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows sequence corresponding to the binding site for activity B is a previously unrecognized motif, present in the cis elements of the Fli1 gene, another important megakaryocyte-specific transcription factor. In summary, we have identified a motif and a DNA binding activity likely to be important in directing a megakaryocyte gene expression program that is distinct from that in red cells.
Structural Basis for LMO2-Driven Recruitment of the SCL: E47bHLH Heterodimer to Hematopoietic-Specific Transcriptional Targets
Cell fate is governed by combinatorial actions of transcriptional regulators assembling into multiprotein complexes. However, the molecular details of how these complexes form are poorly understood. One such complex, which contains the basic-helix-loop-helix heterodimer SCL:E47 and bridging proteins LMO2:LDB1, critically regulates hematopoiesis and induces Tcell leukemia. Here, we report the crystal structure of (SCL:E47)bHLH:LMO2:LDB1LID bound to DNA, providing a molecular account of the network of interactions assembling this complex. This reveals an unexpected role for LMO2. Upon binding to SCL, LMO2 induces new hydrogen bonds in SCL:E47, thereby strengthening heterodimer formation. This imposes a rotation movement onto E47 that weakens the heterodimer:DNA interaction, shifting the main DNA-binding activity onto additional protein partners. Along with biochemical analyses, this illustrates, at an atomic level, how hematopoietic-specific SCL sequesters ubiquitous E47 and associated cofactors and supports SCL'sreported DNA-binding-independent functions. Importantly, this work will drive the design of small molecules inhibiting leukemogenic processes. © 2013 The Authors.
Structural basis for LMO2-driven recruitment of the SCL:E47bHLH heterodimer to hematopoietic-specific transcriptional targets.
Cell fate is governed by combinatorial actions of transcriptional regulators assembling into multiprotein complexes. However, the molecular details of how these complexes form are poorly understood. One such complex, which contains the basic-helix-loop-helix heterodimer SCL:E47 and bridging proteins LMO2:LDB1, critically regulates hematopoiesis and induces T cell leukemia. Here, we report the crystal structure of (SCL:E47)bHLH:LMO2:LDB1LID bound to DNA, providing a molecular account of the network of interactions assembling this complex. This reveals an unexpected role for LMO2. Upon binding to SCL, LMO2 induces new hydrogen bonds in SCL:E47, thereby strengthening heterodimer formation. This imposes a rotation movement onto E47 that weakens the heterodimer:DNA interaction, shifting the main DNA-binding activity onto additional protein partners. Along with biochemical analyses, this illustrates, at an atomic level, how hematopoietic-specific SCL sequesters ubiquitous E47 and associated cofactors and supports SCL's reported DNA-binding-independent functions. Importantly, this work will drive the design of small molecules inhibiting leukemogenic processes.