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Stuart H Orkin

Publications and source records attributed to Stuart H Orkin.

At least 19 recordsLinked to original sources
PubMed2003

Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression.

Reactive oxygen species are involved in many cellular metabolic and signalling processes and are thought to have a role in disease, particularly in carcinogenesis and ageing. We have generated mice with targeted inactivation of Prdx1, a member of the peroxiredoxin family of antioxidant enzymes. Here we show that mice lacking Prdx1 are viable and fertile but have a shortened lifespan owing to the development beginning at about 9 months of severe haemolytic anaemia and several malignant cancers, both of which are also observed at increased frequency in heterozygotes. The haemolytic anaemia is characterized by an increase in erythrocyte reactive oxygen species, leading to protein oxidation, haemoglobin instability, Heinz body formation and decreased erythrocyte lifespan. The malignancies include lymphomas, sarcomas and carcinomas, and are frequently associated with loss of Prdx1 expression in heterozygotes, which suggests that this protein functions as a tumour suppressor. Prdx1-deficient fibroblasts show decreased proliferation and increased sensitivity to oxidative DNA damage, whereas Prdx1-null mice have abnormalities in numbers, phenotype and function of natural killer cells. Our results implicate Prdx1 as an important defence against oxidants in ageing mice.

Aging↗
PubMed2003

GATA-1 as a regulator of mast cell differentiation revealed by the phenotype of the GATA-1low mouse mutant.

Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neo Delta HS or GATA-1(low) mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue(+) mast cells and apoptotic metachromatic(-) mast cell precursors in connective tissues and peritoneal lavage and numerous (60-70% of all the progenitors) "unique" trilineage cells committed to erythroid, megakaryocytic, and mast pathways in the bone marrow and spleen. These abnormalities, which were mirrored by impaired mast differentiation in vitro, were reversed by retroviral-mediated expression of GATA-1 cDNA. These data indicate an essential role for GATA-1 in mast cell differentiation.

3T3 Cells↗
PubMed2003

Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene.

The production of blood cells is sustained throughout the lifetime of an individual by haematopoietic stem cells (HSCs). Specification of HSCs from mesoderm during embryonic development requires the stem cell leukaemia SCL/tal-1 gene product. Forced expression of SCL/tal-1 strongly induces blood formation in embryos, indicating that this gene has a dominant role in commitment to haematopoiesis. In the adult haematopoietic system, expression of SCL/tal-1 is enriched in HSCs and multipotent progenitors, and in erythroid and megakaryocytic lineages, consistent with roles for this factor in adult haematopoiesis. Here we assess by conditional gene targeting whether SCL/tal-1 is required continuously for the identity and function of HSCs. We find that SCL/tal-1 is dispensable for HSC engraftment, self-renewal and differentiation into myeloid and lymphoid lineages; however, the proper differentiation of erythroid and megakaryocytic precursors is dependent on SCL/tal-1. Thus, SCL/tal-1 is essential for the genesis of HSCs, but its continued expression is not essential for HSC functions. These findings contrast with lineage choice mechanisms, in which the identity of haematopoietic lineages requires continuous transcription factor expression.

Animals↗
PubMed2003

Intrinsic requirement for zinc finger transcription factor Gfi-1 in neutrophil differentiation.

We report essential roles of zinc finger transcription factor Gfi-1 in myeloid development. Gene-targeted Gfi-1(-/-) mice lack normal neutrophils and are highly susceptible to abscess formation by gram-positive bacteria. Arrested, morphologically atypical, Gr1(+)Mac1(+) myeloid cells expand with age in the bone marrow. RNAs encoding primary but not secondary or tertiary neutrophil (granulocyte) granule proteins are expressed. The atypical Gr1(+)Mac1(+) cell population shares characteristics of both the neutrophil and macrophage lineages and exhibits phagocytosis and respiratory burst activity. Reexpression of Gfi-1 in sorted Gfi-1(-/-) progenitors ex vivo rescues neutrophil differentiation in response to G-CSF. Thus, Gfi-1 not only promotes differentiation of neutrophils but also antagonizes traits of the alternate monocyte/macrophage program.

Animals↗
PubMed2003

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival.

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.

Animals↗
PubMed2002

The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1.

The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. Here we show that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, we define the role of SCL as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, we demonstrate by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. We therefore conclude that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, our observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells.

3T3 Cells↗
PubMed2002

Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.

Murine hematopoietic stem cells (HSCs) originate from mesoderm in a process that requires the transcription factor SCL/Tal1. To define steps in the commitment to blood cell fate, we compared wild-type and SCL(-/-) embryonic stem cell differentiation in vitro and identified CD41 (GpIIb) as the earliest surface marker missing from SCL(-/-) embryoid bodies (EBs). Culture of fluorescence-activated cell sorter (FACS) purified cells from EBs showed that definitive hematopoietic progenitors were highly enriched in the CD41(+) fraction, whereas endothelial cells developed from CD41(-) cells. In the mouse embryo, expression of CD41 was detected in yolk sac blood islands and in fetal liver. In yolk sac and EBs, the panhematopoietic marker CD45 appeared in a subpopulation of CD41(+) cells. However, multilineage hematopoietic colonies developed not only from CD45(+)CD41(+) cells but also from CD45(-)CD41(+) cells, suggesting that CD41 rather than CD45 marks the definitive culture colony-forming unit (CFU-C) at the embryonic stage. In contrast, fetal liver CFU-C was CD45(+), and only a subfraction expressed CD41, demonstrating down-regulation of CD41 by the fetal liver stage. In yolk sac and EBs, CD41 was coexpressed with embryonic HSC markers c-kit and CD34. Sorting for CD41 and c-kit expression resulted in enrichment of definitive hematopoietic progenitors. Furthermore, the CD41(+) c-kit(+) population was missing from runx1/AML1(-/-) EBs that lack definitive hematopoiesis. These results suggest that the expression of CD41, a candidate target gene of SCL/Tal1, and c-kit define the divergence of definitive hematopoiesis from endothelial cells during development. Although CD41 is commonly referred to as megakaryocyte-platelet integrin in adult hematopoiesis, these results implicate a wider role for CD41 during murine ontogeny.

Animals↗
PubMed2002

X-linked thrombocytopenia with thalassemia from a mutation in the amino finger of GATA-1 affecting DNA binding rather than FOG-1 interaction.

Transcription factor GATA-1 is essential for the development of erythroid cells and megakaryocytes. Each of its 2 zinc fingers is critical for normal function. The C-terminal finger is necessary for DNA binding. The N finger mediates interaction with FOG-1, a cofactor for GATA-1, and also modulates DNA-binding affinity, notably at complex or palindromic GATA sites. Residues of the N finger-mediating interaction with FOG-1 lie on the surface of the N finger facing away from DNA. Strong sequence conservation of residues facing DNA suggests that this other surface may also have an important role. We report here that a syndrome of X-linked thrombocytopenia with thalassemia in humans is caused by a missense mutation (Arg216Gln) in the GATA-1 N finger. To investigate the functional consequences of this substitution, we used site-directed mutagenesis to alter the corresponding residue in GATA-1. Compared with wild-type GATA-1, Arg216Gln GATA-1 shows comparable affinity to single GATA sites but decreased affinity to palindromic sites. Arg216Gln GATA-1 interacts with FOG-1 similarly with wild-type GATA-1. Arg216Gln GATA-1 supports erythroid maturation of GATA-1 erythroid cells, albeit at reduced efficiency compared with wild-type GATA-1. Together, these findings suggest that residues of the N finger of GATA-1-facing DNA contribute to GATA-1 function apart from interaction with the cofactor FOG-1. This is also the first example of beta-thalassemia in humans caused by a mutation in an erythroid transcription factor.

Amino Acid Substitution↗
PubMed2002

Dysregulation of protein modification by ISG15 results in brain cell injury.

UBP43 (USP18) is a protease that removes the ubiquitin-like modifier ISG15 from conjugated proteins. Here we present the first report of dysregulation of protein ISG15 modification by the generation of UBP43 knockout mice. In the absence of UBP43, brain tissue showed an elevated level of ISG15 conjugates, and cellular necrosis was evident in the ependyma. Such disruption of the blood-brain barrier resulted in severe neurologic disorders. These results demonstrate that UBP43 plays a critical role in maintaining the homeostatic balance of ISG15-conjugated protein, and that regulation of cellular levels of ISG15 protein modification is essential for brain cell function.

Animals↗
PubMed2002

GATA-factor dependence of the multitype zinc-finger protein FOG-1 for its essential role in megakaryopoiesis.

The function of GATA transcription factors in diverse developmental contexts depends in part on physical interaction with cofactors of the Friend of GATA (FOG) family. However, previous studies indicate that FOG-1 may play a GATA-1-independent role in early megakaryopoiesis, suggesting that FOG proteins might act in a GATA factor-independent manner. Here, we have generated mouse knock-in (KI) mutants harboring a critical valine-to-glycine substitution in the amino-terminal zinc fingers of GATA-1 and GATA-2 to ablate FOG interaction. In contrast to male GATA-1(KI) (GATA-1 is located on the X-chromosome) or GATA-2(KI/KI) mice, compound GATA-1(KI) GATA-2(KI/KI) mutant mice display complete megakaryopoietic failure, a phenocopy of FOG-1(-/-) mice. We conclude that FOG-1 requires an interaction with either GATA-1 or -2 as part of its essential role in early megakaryopoiesis. On the basis of these and previous reports, we infer that GATA factor dependence is a critical aspect of FOG protein function.

Amino Acid Sequence↗
PubMed2002

Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo.

Transcription factor GATA-1 reprograms immature myeloid cells to three different hematopoietic lineages-erythroid cells, megakaryocytes, and eosinophils. GATA-1 is essential for maturation of erythroid and megakaryocytic precursors, as revealed by gene targeting in mice. Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss of the eosinophil lineage. These findings suggest that GATA-1 is required for specification of this lineage during hematopoietic development. Mice lacking the ability to produce eosinophils should prove useful in ascertaining the role of eosinophils in a variety of inflammatory or allergic disorders.

Animals↗
PubMed2002

Transcriptional regulation of erythropoiesis: an affair involving multiple partners.

Previous work has demonstrated that lineage-specific transcription factors play essential roles in red blood cell development. More recent studies have shown that these factors participate in critical protein-protein interactions in addition to binding DNA. The zinc finger transcription factor GATA-1, a central mediator of erythroid gene expression, interacts with multiple proteins including FOG-1, EKLF, SP1, CBP/p300 and PU.1. The mechanisms by which these interactions influence GATA-1 function, as well as any possible relationships between these seemingly disparate complexes, remain incompletely understood. However, several new findings have provided further insight into the functional significance of some of these interactions. Studies involving point mutants of GATA-1 have shown that a direct physical interaction between GATA-1 and FOG-1 is essential for normal human erythroid and megakaryocyte maturation in vivo. In addition, evidence has emerged that physical interaction between GATA-1 and the myeloid/lymphoid specific factor PU.1, an oncogene implicated in murine erythroleukemia, acts to functionally cross-antagonize one another. This provides a possible mechanism by which dysregulated expression of hematopoietic transcription factors leads to lineage maturation arrest in leukemias.

Animals↗
PubMed2002

The zinc-finger proto-oncogene Gfi-1b is essential for development of the erythroid and megakaryocytic lineages.

Gfi-1 and Gfi-1b are novel proto-oncogenes identified by retroviral insertional mutagenesis. By gene targeting, we establish that Gfi-1b is required for the development of two related blood lineages, erythroid and megakaryocytic, in mice. Gfi-1b(-/-) embryonic stem cells fail to contribute to red cells of adult chimeras. Gfi-1b(-/-) embryos exhibit delayed maturation of primitive erythrocytes and subsequently die with failure to produce definitive enucleated erythrocytes. The fetal liver of mutant mice contains erythroid and megakaryocytic precursors arrested in their development. Myelopoiesis is normal. Therefore, Gfi-1b is an essential transcriptional regulator of erythroid and megakaryocyte development.

Animals↗
PubMed2002

The use of altered specificity mutants to probe specific protein-protein interactions involved in the activation of GATA-1 target genes.

With the increasing popularity of the yeast two-hybrid screen, a large number of protein-protein interactions have been identified. In many cases, single proteins have been found to associate with a large number of cofactors. For example, the hematopoietic transcription factor GATA-1 interacts with a multitude of other nuclear proteins, including Friend of GATA-1 (FOG-1), EKLF, CBP/p300, and Lmo2. Furthermore, p300, besides associating with GATA-1, interacts with at least seven other hematopoietic transcription factors. Despite the numerous pairwise and higher-order interactions identified, assessment of their contribution to transcriptional control has been lacking. Here we describe a strategy that can be applied to assess the functional relevance of any protein-protein association. This approach involves the creation of altered specificity mutants though the use of a combination of two yeast two-hybrid screens. Once altered specificity factors are obtained, a researcher can then proceed to functional assays that address the role of a specific protein-protein interaction.

Carrier Proteins↗
PubMed2002

Highly penetrant, rapid tumorigenesis through conditional inversion of the tumor suppressor gene Snf5.

Recent data suggest the SWI/SNF chromatin remodeling complex may also act as a tumor suppressor. Utilizing a reversibly inactivating conditional allele, we demonstrate that loss of Snf5/Ini1/Baf47/SmarcB1, a core subunit of SWI/SNF, results in highly penetrant cancer predisposition with 100% of mice developing mature CD8(+) T cell lymphoma or rare rhabdoid tumors with a median onset of only 11 weeks. Notably, while loss of Snf5 predisposes to aggressive cancers, it is also required for survival of virtually all nonmalignant cells in vivo. Reversible gene targeting demonstrates a critical and specific role for Snf5 in tumor suppression, provides a novel system in which to explore the genetic pathways involved in tumor suppression by Swi/Snf, and should be of wide use in evaluating other essential tumor suppressor genes.

Animals↗
PubMed2002

Hematopoiesis and stem cells: plasticity versus developmental heterogeneity.

Hematopoietic stem cells (HSCs) provide for blood formation throughout the life of the individual. Studies of HSCs form a conceptual framework for the analysis of stem cells of other organ systems. We review here the origin of HSCs during embryological development, the relationship between hematopoiesis and vascular development and the potential plasticity of HSCs and other tissue stem cells. Recent experiments in the mouse have been widely interpreted as evidence for unprecedented transdifferentiation of tissue stem cells. The use of enriched, but impure, cell populations allows for alternative interpretation. In considering these findings, we draw a distinction here between the plasticity of adult stem cells and the heterogeneity of stem cell types that pre-exist within tissues.

Animals↗