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Perturbed epigenomic programs play key roles in tumorigenesis, and chromatin modulators are candidate therapeutic targets in various human cancer types. To define singular and shared dependencies on DNA and histone modifiers and transcription factors in poorly differentiated adult and pediatric cancers, we conducted a targeted shRNA screen across 59 cell lines of 6 cancer types. Here, we describe the TRPS1 transcription factor as a strong breast cancer-specific hit, owing largely to lineage-restricted expression. Knockdown of TRPS1 resulted in perturbed mitosis, apoptosis, and reduced tumor growth. Integrated analysis of TRPS1 transcriptional targets, chromatin binding, and protein interactions revealed that TRPS1 is associated with the NuRD repressor complex. These findings uncover a transcriptional network that is essential for breast cancer cell survival and propagation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Enterocytes assemble dietary lipids into chylomicron particles that are taken up by intestinal lacteal vessels and peripheral tissues. Although chylomicrons are known to assemble in part within membrane secretory pathways, the modifications required for efficient vascular uptake are unknown. Here we report that the transcription factor pleomorphic adenoma gene-like 2 (PlagL2) is essential for this aspect of dietary lipid metabolism. PlagL2(-/-) mice die from postnatal wasting owing to failure of fat absorption. Lipids modified in the absence of PlagL2 exit from enterocytes but fail to enter interstitial lacteal vessels. Dysregulation of enterocyte genes closely linked to intracellular membrane transport identified candidate regulators of critical steps in chylomicron assembly. PlagL2 thus regulates important aspects of dietary lipid absorption, and the PlagL2(-/-) animal model has implications for the amelioration of obesity and the metabolic syndrome.

Megakaryocyte (MK) differentiation is well described in morphologic terms but its molecular counterparts and the basis for platelet release are incompletely understood. We profiled mRNA expression in populations of primary mouse MKs representing successive differentiation stages. Genes associated with DNA replication are highly expressed in young MKs, in parallel with endomitosis. Intermediate stages are characterized by disproportionate expression of genes associated with the cytoskeleton, cell migration, and G-protein signaling, whereas terminally mature MKs accumulate hemostatic factors, including many membrane proteins. We used these expression profiles to extract a reliable panel of molecular markers for MKs of early, intermediate, or advanced differentiation and establish the value of this marker panel using mouse models of defective thrombopoiesis resulting from absence of GATA1, NF-E2, or tubulin beta1. Computational analysis of the promoters of late-expressed MK genes identified new candidate targets for NF-E2, a critical transcriptional regulator of platelet release. One such gene encodes the kinase adaptor protein LIMS1/PINCH1, which is highly expressed in MKs and platelets and significantly reduced in NF-E2-deficient cells. Transactivation studies and chromatin immunoprecipitation implicate Lims1 as a direct target of NF-E2 regulation. Attribution of stage-specific genes, in combination with various applications, thus constitutes a powerful way to study MK differentiation and platelet biogenesis.

PPARgamma is a dominant regulator of fat cell differentiation. However, this nuclear receptor also plays an important role in the differentiation of intestinal and other epithelial cell types. The mechanism by which PPARgamma can influence the differentiation of such diverse cell lineages is unknown. We show here that PPARgamma interacts with Hic-5, a coactivator protein expressed in gut epithelial cells. Hic-5 and PPARgamma colocalize to the villus epithelium of the small intestine, and their expression during embryonic gut development correlates with the transition from endoderm to a specialized epithelium; expression of both these factors is reduced in tumors. Forced expression of Hic-5 in colon cancer cells enhances the PPARgamma-mediated induction of several gut epithelial differentiation/maturation markers such as L-FABP, kruppel-like factor 4 (KLF4), and keratin 20. siRNA directed against Hic-5 specifically reduces PPARgamma-mediated induction of gut epithelial genes in colon cells and in an ex vivo model of embryonic gut differentiation. Finally, forced expression of Hic-5 during 3T3-L1 preadipocyte differentiation inhibits adipogenesis while inducing inappropriate expression of several mRNAs characteristic of gut epithelium in these mesenchymal cells. These results indicate that Hic5 is an important component in determining an epithelial differentiation program induced by PPARgamma.

Unlabelled: Mice deficient in GATA-1 or NF-E2 have a 200-300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6-fold when cultured with megakaryocytes. A megakaryocyte-osteoblast interaction plays a role in the increased bone formation in these mice., Introduction: GATA-1 and NF-E2 are transcription factors required for the differentiation of megakaryocytes. Mice deficient in these factors have phenotypes characterized by markedly increased numbers of immature megakaryocytes, a concomitant drastic reduction of platelets, and a striking increased bone mass. The similar bone phenotype in both animal models led us to explore the interaction between osteoblasts and megakaryocytes., Materials and Methods: Histomorphometry, microCT, and serum and urine biochemistries were used to assess the bone phenotype in these mice. Wildtype and mutant osteoblasts were examined for differences in proliferation, alkaline phosphatase activity, and osteocalcin secretion. In vitro osteoclast numbers and resorption were measured. Because mutant osteoblasts and osteoclasts were similar to control cells, and because of the similar bone phenotype, we explored the interaction between cells of the osteoblast lineage and megakaryocytes., Results: A marked 2- to 3-fold increase in trabecular bone volume and bone formation indices were observed in these mice. A 20- to 150-fold increase in trabecular bone volume was measured for the entire femoral medullary canal. The increased bone mass phenotype in these animals was not caused by osteoclast defects, because osteoclast number and function were not compromised in vitro or in vivo. In contrast, in vivo osteoblast number and bone formation parameters were significantly elevated. When wildtype or mutant osteoblasts were cultured with megakaryocytes from GATA-1- or NF-E2-deficient mice, osteoblast proliferation increased over 3- to 6-fold by a mechanism that required cell-to-cell contact., Conclusions: These observations show an interaction between megakaryocytes and osteoblasts, which results in osteoblast proliferation and increased bone mass, and may represent heretofore unrecognized anabolic pathways in bone.

Key molecular lesions in colorectal and other cancers cause beta-catenin-dependent transactivation of T cell factor (Tcf)-dependent genes. Disruption of this signal represents an opportunity for rational cancer therapy. To identify compounds that inhibit association between Tcf4 and beta-catenin, we screened libraries of natural compounds in a high-throughput assay for immunoenzymatic detection of the protein-protein interaction. Selected compounds disrupt Tcf/beta-catenin complexes in several independent in vitro assays and potently antagonize cellular effects of beta-catenin-dependent activities, including reporter gene activation, c-myc or cyclin D1 expression, cell proliferation, and duplication of the Xenopus embryonic dorsal axis. These compounds thus meet predicted criteria for disrupting Tcf/beta-catenin complexes and define a general standard to establish mechanism-based activity of small molecule inhibitors of this pathogenic protein-protein interaction.

Megakaryocytes release platelets by reorganizing the cytoplasm into proplatelet extensions. Fundamental to this process is the need to coordinate transport of products and organelles in the appropriate abundance to nascent platelets. The importance of the Rab family of small GTPases (guanosine 5'-triphosphatases) in platelet biogenesis is revealed in gunmetal (gm/gm) mice, which show deficient Rab isoprenylation and macrothrombocytopenia with few granules and abnormal megakaryocyte morphology. Although some Rab proteins are implicated in vesicle and organelle transport along microtubules or actin, the role of any Rab protein in platelet biogenesis is unknown. The limited number of Rab proteins with defective membrane association in gm/gm megakaryocytes prominently includes Rab27a and Rab27b. Normal expression of Rab27b is especially increased with terminal megakaryocyte differentiation and dependent on nuclear factor-erythroid 2 (NF-E2), a transcription factor required for thrombopoiesis. Chromatin immunoprecipitation demonstrates recruitment of NF-E2 to the putative Rab27B promoter. Inhibition of endogenous Rab27 function in primary megakaryocytes causes severe quantitative and qualitative defects in proplatelet formation that mimic findings in gm/gm cells. Rab27b localizes to alpha and dense granules in megakaryocytes. These results establish a role for Rab27 in platelet synthesis and suggest that Rab27b in particular may coordinate proplatelet formation with granule transport, possibly by recruiting specific effector pathways.

GATA transcription factors mediate cell differentiation in diverse tissues, and their dysfunction is associated with certain congenital human disorders. The six classical vertebrate GATA proteins, GATA-1 to GATA-6, are highly homologous, bear two tandem zinc fingers of the C(4) (GATA) type, and activate transcription. TRPS1, the only other vertebrate protein with the GATA motif, is a large, multitype zinc finger protein that harbors a single DNA-binding GATA domain and represses transcription. Monoallelic TRPS1 mutations cause two dominantly inherited human developmental disorders of the hair, face, and digits, tricho-rhino-phalangeal syndrome (TRPS) types I (MIM 190350) and III (MIM 190351); missense GATA domain mutations account for the more severe type III form. Here we report that heterozygous mice with deletions of the TRPS1 GATA domain (TRPS1(+/Deltagt)) display facial anomalies that overlap with findings for TRPS, whereas TRPS1(Deltagt/Deltagt) mice additionally reveal a complete absence of vibrissae. Unexpectedly, TRPS1(Deltagt/Deltagt) mice die of neonatal respiratory failure resulting from abnormalities of the thoracic spine and ribs. Heterozygotes also develop thoracic kyphoscoliosis with age and reveal structural deficits in cortical and trabecular bones. These findings directly implicate the GATA type zinc finger of TRPS1 in regulation of bone and hair development and suggest that skeletal abnormalities emphasized in descriptions of TRPS are only the extreme manifestations of a generalized bone dysplasia.

Known vertebrate GATA proteins contain two zinc fingers and are required in development, whereas invertebrates express a class of essential proteins containing one GATA-type zinc finger. We isolated the gene encoding TRPS1, a vertebrate protein with a single GATA-type zinc finger. TRPS1 is highly conserved between Xenopus and mammals, and the human gene is implicated in dominantly inherited tricho-rhino-phalangeal (TRP) syndromes. TRPS1 is a nuclear protein that binds GATA sequences but fails to transactivate a GATA-dependent reporter. Instead, TRPS1 potently and specifically represses transcriptional activation mediated by other GATA factors. Repression does not occur from competition for DNA binding and depends on a C-terminal region related to repressive domains found in Ikaros proteins. During mouse development, TRPS1 expression is prominent in sites showing pathology in TRP syndromes, which are thought to result from TRPS1 haploinsufficiency. We show instead that truncating mutations identified in patients encode dominant inhibitors of wild-type TRPS1 function, suggesting an alternative mechanism for the disease. TRPS1 is the first example of a GATA protein with intrinsic transcriptional repression activity and possibly a negative regulator of GATA-dependent processes in vertebrate development.

In previous studies, we identified a common site of retroviral integration designated Fli-2 in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia cell lines. Insertion of F-MuLV at the Fli-2 locus, which was associated with the loss of the second allele, resulted in the inactivation of the erythroid cell- and megakaryocyte-specific gene p45(NFE2). Frequent disruption of p45(NFE2) due to proviral insertion suggests a role for this transcription factor in the progression of Friend virus-induced erythroleukemias. To assess this possibility, erythroleukemia was induced by F-MuLV in p45(NFE2) mutant mice. Since p45(NFE2) homozygous mice mostly die at birth, erythroleukemia was induced in +/- and +/+ mice. We demonstrate that +/- mice succumb to the disease moderately but significantly faster than +/+ mice. In addition, the spleens of +/- mice were significantly larger than those of +/+ mice. Of the 37 tumors generated from the +/- and +/+ mice, 10 gave rise to cell lines, all of which were derived from +/- mice. Establishment in culture was associated with the loss of the remaining wild-type p45(NFE2) allele in 9 of 10 of these cell lines. The loss of a functional p45(NFE2) in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-type p45(NFE2) in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression of p45(NFE2) in these cells also slows tumor growth in vivo. These results indicate that p45(NFE2) functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.

The cellular and molecular bases of platelet release by terminally differentiated megakaryocytes represent important questions in cell biology and hematopoiesis. Mice lacking the transcription factor NF-E2 show profound thrombocytopenia, and their megakaryocytes fail to produce proplatelets, the microtubule-based precursors of blood platelets. Using mRNA subtraction between normal and NF-E2-deficient megakaryocytes, cDNA was isolated encoding beta1 tubulin, the most divergent beta tubulin isoform. In NF-E2-deficient megakaryocytes, beta1 tubulin mRNA and protein are virtually absent. The expression of beta1 tubulin is exquisitely restricted to platelets and megakaryocytes, where it appears late in differentiation and localizes to microtubule shafts and coils within proplatelets. Restoring NF-E2 activity in a megakaryoblastic cell line or in NF-E2-deficient primary megakaryocytes rescues the expression of beta1 tubulin. Re-expressing beta1 tubulin in isolation does not, however, restore proplatelet formation in the defective megakaryocytes, indicating that other critical factors are required; indeed, other genes identified by mRNA subtraction also encode structural and regulatory components of the cytoskeleton. These findings provide critical mechanistic links between NF-E2, platelet formation, and selected microtubule proteins, and they also provide novel molecular insights into thrombopoiesis. (Blood. 2000;96:1366-1373)

Expression of the p45 subunit of transcription factor NF-E2 is restricted to selected blood cell lineages, including megakaryocytes and developing erythrocytes. Mice lacking p45 NF-E2 show profound thrombocytopenia, resulting from a late arrest in megakaryocyte differentiation, and a number of red blood cell defects, including anisocytosis and hypochromia. Here we report results of studies aimed to explore the pathophysiology of these abnormalities. Mice lacking NF-E2 produce very few platelet-like particles that display highly disorganized ultrastructure and respond poorly to platelet agonists, features consistent with the usually lethal hemorrhage in these animals. Thrombocytopenia was evident during fetal life and was not corrected by splenectomy in adults. Surprisingly, fetal NF-E2-deficient megakaryocyte progenitors showed reduced proliferation potential in vitro. Thus, NF-E2 is required for regulated megakaryocyte growth as well as for differentiation into platelets. All the erythroid abnormalities were reproduced in lethally irradiated wild-type recipients of hematopoietic cells derived from NF-E2-null fetuses. Whole blood from mice lacking p45 NF-E2 showed numerous small red blood cell fragments; however, survival of intact erythrocytes in vivo was indistinguishable from control mice. Considered together, these observations indicate a requirement for NF-E2 in generating normal erythrocytes. Despite impressive splenomegaly at baseline, mice lacking p45 NF-E2 survived splenectomy, which resulted in increased reticulocyte numbers. This reveals considerable erythroid reserve within extra-splenic sites of hematopoiesis and suggests a role for the spleen in clearing abnormal erythrocytes. Our findings address distinct aspects of the requirements for NF-E2 in blood cell homeostasis and establish its roles in proper differentiation of megakaryocytes and erythrocytes.

In the absence of the hematopoietic transcription factor GATA-1, mice develop thrombocytopenia and an increased number of megakaryocytes characterized by marked ultrastructural abnormalities. These observations establish a critical role for GATA-1 in megakaryopoiesis and raise the question as to how GATA-1 influences megakaryocyte maturation and platelet production. To begin to address this, we have performed a more detailed examination of the megakaryocytes and platelets produced in mice that lack GATA-1 in this lineage. Our analysis demonstrates that compared with their normal counterparts, GATA-1-deficient primary megakaryocytes exhibit significant hyperproliferation in liquid culture, suggesting that the megakaryocytosis seen in animals is nonreactive. Morphologically, these mutant megakaryocytes are small and show evidence of retarded nuclear and cytoplasmic development. A significant proportion of these cells do not undergo endomitosis and express markedly lower levels of mRNA of all megakaryocyte-associated genes tested, including GPIbalpha, GPIbbeta, platelet factor 4 (PF4), c-mpl, and p45 NF-E2. These results are consistent with regulation of a program of megakaryocytic differentiation by GATA-1. Bleeding times are significantly prolonged in mutant animals. GATA-1-deficient platelets show abnormal ultrastructure, reminiscent of the megakaryocytes from which they are derived, and exhibit modest but selective defects in platelet activation in response to thrombin or to the combination of adenosine diphosphate (ADP) and epinephrine. Our findings indicate that GATA-1 serves multiple functions in megakaryocyte development, influencing both cellular growth and maturation.

Mechanisms of platelet production and release by mammalian megakaryocytes are poorly understood. We used thrombocytopenic knockout mice to better understand these processes. Proplatelets are filamentous extensions of terminally differentiated megakaryocytes that are thought to represent one mechanism of platelet release; however, these structures have largely been recognized in cultured cells and there has been no correlation between thrombocytopoiesis in vivo and proplatelet formation. Mice lacking transcription factor NF-E2 have a late arrest in megakaryocyte maturation, resulting in profound thrombocytopenia. In contrast to normal megakaryocytes, which generate abundant proplatelets, cells from these mice never produce proplatelets, even after prolonged stimulation with c-Mpl ligand. Similarly, megakaryocytes from thrombocytopenic mice with lineage-selective loss of transcription factor GATA-1 produce proplatelets very rarely. These findings establish a significant correlation between thrombocytopoiesis and proplatelet formation and suggest that the latter represents a physiologic mechanism of platelet release. We further show that proplatelet formation by normal megakaryocytes and its absence in cells lacking NF-E2 are independent of interactions with adherent (stromal) cells. Similarly, thrombocytopenia in NF-E2(-/-) mice reflects intrinsic defects in the megakaryocyte lineage. These observations improve our understanding of platelet production and validate the study of proplatelets in probing the underlying mechanisms., (Copyright 1998 by The American Society of Hematology.)

NF-E2 binding sites, located in distant regulatory sequences, may be important for high level alpha- and beta-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2-related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2-deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of alpha- and beta-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2-deficient mice is not the result of compensation by Nrf-2.

The transcription factor GATA-1 is specifically expressed in hematopoietic lineages. Prior gene knockout experiments established an essential role for GATA-1 in red blood cell production, but could not provide direct evidence with respect to a requirement in megakaryopoiesis. We summarize here recent lineage-selective gene targeting in mice that establishes critical functions for GATA-1 in controlling megakaryocyte growth and maturation, and platelet production. GATA-1 megakaryocytes are delayed in their cellular maturation, exhibit marked hyperproliferation and generate fewer than normal, yet enlarged, platelets in vivo. Thus GATA-1 is a central regulator in both the erythroid and megakaryocytic lineages.

Transcription factor p45 NF-E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF-E2. To find target genes for p45 NF-E2 in megakaryopoiesis, we used an in vivo immunoselection assay: genomic fragments bound to p45 NF-E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti-p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). We demonstrate that the TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF-E2. First, its promoter contains a functional NF-E2 binding site; second, the intronic NF-E2 binding site is located within a chromatin-dependent enhancer element; third, p45-null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function.

The mechanisms that regulate circulating levels of thrombopoietin (Tpo) are incompletely understood. According to one favored model, the rate of Tpo synthesis is constant, whereas the serum concentration of free Tpo is modulated through binding to c-Mpl receptor expressed on blood platelets. Additionally, a role for c-Mpl expressed on megakaryocytes is suggested, particularly by the observation that serum Tpo levels are not elevated in human immune thrombocytopenic purpura. Whereas direct binding of Tpo to platelets has been demonstrated in vitro and in vivo, the role of megakaryocytes in modulating serum Tpo levels has not been addressed experimentally. The profoundly thrombocytopenic mice lacking transcription factor p45 NF-E2 do not show the predicted increase in serum Tpo concentration. To evaluate the fate of the ligand in these animals, we injected 125I-Tpo intravenously into mutant and control mice. In contrast to normal littermates, NF-E2 knockout mice show negligible association of radioactivity with blood cellular components, consistent with an absence of platelets. There is no corresponding increase in plasma-associated radioactivity to suggest persistence in the circulation. However, a greater fraction of the radioligand is bound to hematopoietic tissues. In the bone marrow this is detected virtually exclusively in association with megakaryocytes, whereas in the spleen it is associated with megakaryocytes and small, abnormal, platelet-like particles or megakaryocyte fragments that are found within or in close contact with macrophages. These findings implicate the combination of megakaryocytes and the latter particles as a sink for circulating Tpo in NF-E2 knockout mice, and provide an explanation for the lack of elevated serum Tpo levels in this unique animal model of thrombocytopenia.

The transcription factor GATA-1, which is expressed in several hematopoietic lineages and multipotential progenitors, is required for the development of red blood cells and platelets. To identify control elements of the mouse GATA-1 gene, we analyzed DNase I hypersensitivity of the locus in erythroid chromatin and the expression of GATA-1/Escherichia coli beta-galactosidase (lacZ) transgenes in mice. Transgenes with 2.7 kb of promoter sequences are expressed infrequently and only within adult (definitive) erythroid cells. We show that inclusion of an upstream hypersensitive site (HS I) markedly enhances the frequency of expressing transgenic lines and activates expression in primitive erythroid cells. This pattern recapitulates the proper pattern of GATA-1 expression during development. By breeding a GATA-1/lacZ transgene into a GATA-1(-) background, we also have shown that the activation or maintenance of GATA-1 expression does not require the presence of GATA-1 itself, thereby excluding simple models of positive autoregulation. The transgene cassette reported here should be useful in directing expression of foreign sequences at the onset of hematopoiesis in the embryo and may assist in the identification of upstream regulators of the GATA-1 gene.

Transcription factor GATA-1 is essential for red blood cell maturation and, therefore, for survival of developing mouse embryos. GATA-1 is also expressed in megakaryocytes, mast cells, eosinophils, multipotential hematopoietic progenitors and Sertoli cells of the testis, where its functions have been elusive. Indeed, interpretation of gene function in conventional knockout mice is often limited by embryonic lethality or absence of mature cells of interest, creating the need for alternate methods to assess gene function in selected cell lineages. Emerging strategies for conditional gene inactivation through site-specific recombinases rely on the availability of mouse strains with high fidelity of transgene expression and efficient, tissue-restricted DNA excision. In an alternate approach, we modified sequences upstream of the GATA-1 locus in embryonic stem cells, including a DNase I-hypersensitive region. This resulted in generation of mice with selective loss of megakaryocyte GATA-1 expression, yet sufficient erythroid cell levels to avoid lethal anemia. The mutant mice have markedly reduced platelet numbers, associated with deregulated megakaryocyte proliferation and severely impaired cytoplasmic maturation. These findings reveal a critical role for GATA-1 in megakaryocyte growth regulation and platelet biogenesis, and illustrate how targeted mutation of cis-elements can generate lineage-specific knockout mice.

The hematopoietic-restricted transcription factor GATA-1 is required for both mammalian erythroid cell and megakaryocyte differentiation. To define the mechanisms governing its transcriptional regulation, we replaced upstream sequences including a DNase I hypersensitive (HS) region with a neomycin-resistance cassette by homologous recombination in mouse embryonic stem cells and generated mice either harboring this mutation (neoDeltaHS) or lacking the selection cassette (DeltaneoDeltaHS). Studies of the consequences of these targeted mutations provide novel insights into GATA-1 function in erythroid cells. First, the neoDeltaHS mutation leads to a marked impairment in the rate or efficiency of erythroid cell maturation due to a modest (4- to 5-fold) decrease in GATA-1 expression. Hence, erythroid differentiation is dose-dependent with respect to GATA-1. Second, since expression of GATA-1 from the DeltaneoDeltaHS allele in erythroid cells is largely restored, transcription interference imposed by the introduced cassette must account for the "knockdown" effect of the mutation. Finally, despite the potency of the upstream sequences in conferring high-level, developmentally appropriate expression of transgenes in mice, other cis-regulatory elements within the GATA-1 compensate for its absence in erythroid cells. Our work illustrates the usefulness of targeted mutations to create knockdown mutations that may uncover important quantitative contributions of gene function not revealed by conventional knockouts.

To determine the in vivo functions of the transcription factor NF-E2, we have disrupted the gene encoding its hematopoietic-specific p45 subunit in embryonic stem cells and generated knockout mice. These animals have a surprisingly mild erythroid cell abnormality but experience lethal hemorrhage as a result of profound thrombocytopenia. Impaired platelet formation is secondary to a cytoplasmic maturation arrest within megakaryocytes, characterized by a marked reduction in granule numbers. Although the proliferative response to recombinant thrombopoietin is intact, this is not accompanied by correction of the differentiation defect. Absence of the smaller (p18) subunit of NF-E2 does not have similar consequences. These findings implicate target genes of the NF-E2 transcription factor in critical aspects of late megakaryocyte maturation and platelet formation.

Previous studies in transgenic mice and cultured cells have indicated that the major enhancer function for erythroid cell expression of the globin genes is provided by the heterodimeric basic-leucine zipper transcription factor NF-E2. Globin gene expression within cultured mouse erythroleukemia cells is highly dependent on NF-E2. To examine the requirement for this factor in vivo, we used homologous recombination in embryonic stem cells to generate mice lacking the hematopoietic-specific subunit, p45 NF-E2. The most dramatic aspect of the homozygous mutant mice was an absence of circulating platelets, which led to the death of most animals due to hemorrhage. In contrast, the effect of loss of NF-E2 on the erythroid lineage was surprisingly mild. Although neonates exhibited severe anemia and dysmorphic red-cell changes, probably compounded by concomitant bleeding, surviving adults exhibited only mild changes consistent with a small decrease in the hemoglobin content per cell. p45 NF-E2-null mice responded to anemia with compensatory reticulocytosis and splenomegaly. Globin chain synthesis was balanced, and switching from fetal to adult globins progressed normally. Although these findings are consistent with the substitution of NF-E2 function in vivo by one or more compensating proteins, gel shift assays using nuclear extracts from p45 NF-E2-null mice failed to reveal novel complexes formed on an NF-E2 binding site. Thus, regulation of globin gene transcription through NF-E2 binding sites in vivo is more complex than has been previously appreciated.

Despite the importance of blood platelets in health and disease, the mechanisms regulating their formation within megakaryocytes are unknown. We generated mice lacking the hematopoietic subunit (p45) of the heterodimeric erythroid transcription factor NF-E2. Unexpectedly, NF-E2-/- mice lack circulating platelets and die of hemorrhage; their megakaryocytes show no cytoplasmic platelet formation. Though platelets are absent, serum levels of the growth factor thrombopoietin/MGDF are not elevated above controls. Nonetheless, NF-E2-/- megakaryocytes proliferate in vivo in response to thrombopoietin administration. Thus, as an essential factor for megakaryocyte maturation and platelet production, NF-E2 must regulate critical target genes independent of the action of thrombopoietin. These findings provide insight into the genetic analysis of megakaryocyte maturation and thrombopoiesis.

Chromosomal translocations associated with malignancies often result in deregulated expression of genes encoding transcription factors. In human T-cell leukaemias such regulators belong to diverse protein families and may normally be expressed widely (for example, Ttg-1/rbtn1, Ttg-2/rbtn2), exclusively outside the haematopoietic system (for example, Hox11), or specifically in haematopoietic cells and other selected sites (for example, tal-1/SCL, lyl-1). Aberrant expression within T cells is though to interfere with programmes of normal maturation. The most frequently activated gene in acute T-cell leukaemias, tal-1 (also called SCL), encodes a candidate regulator of haematopoietic development, a basic-helix-loop-helix protein, related to critical myogenic and neurogenic factors. Here we show by targeted gene disruption in mice that tal-1 is essential for embryonic blood formation in vivo. With respect to embryonic erythropoiesis, tal-1 deficiency resembles loss of the erythroid transcription factor GATA-1 or the LIM protein rbtn2. Profound reduction in myeloid cells cultured in vivo from tal-1 null yolk sacs suggests a broader defect manifest at the myelo-erythroid or multipotential progenitor cell level.