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Stuart H Orkin

Publications and source records attributed to Stuart H Orkin.

65 records · Page 4Linked to original sources
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↗
PubMed2002

Distinct domains of the GATA-1 cofactor FOG-1 differentially influence erythroid versus megakaryocytic maturation.

FOG family zinc finger proteins play essential roles in development through physical interaction with GATA factors. FOG-1, like its interacting partner GATA-1, is required for normal differentiation of erythroid and megakaryocytic cells. Here, we have developed a functional assay for FOG-1 based on its ability to rescue erythroid and megakaryocytic maturation of a genetically engineered FOG-1(-/-) cell line. We demonstrate that interaction through only one of FOG-1's four GATA-binding zinc fingers is sufficient for rescue, providing evidence against a model in which FOG-1 acts to bridge multiple GATA-binding DNA elements. Importantly, we find that distinct regions of FOG-1 differentially influence erythroid versus megakaryocyte maturation. As such, we propose that FOG-1 may modulate the fate of a bipotential erythroid/megakaryocytic precursor cell.

Animals↗
PubMed2002

Interaction between FOG-1 and the corepressor C-terminal binding protein is dispensable for normal erythropoiesis in vivo.

The hematopoietic, zinc-finger protein FOG-1 is essential for the development of the erythroid and megakaryocytic lineages. FOG-1's function in hematopoiesis is dependent on its ability to interact with the transcription factor GATA-1. FOG-1 has also been observed to interact with the corepressor molecule C-terminal binding protein (CtBP) through a peptide motif shared by all FOG family members. In this study, we confirmed that FOG-1 and CtBP interact by coimmunoprecipitation. We further demonstrate that a FOG-1 mutant unable to interact with CtBP has increased erythropoietic (but not megakaryocytic) rescue (relative to the wild type) of a FOG-1(-/-) cell line. To analyze further the physiological role of the FOG-1-CtBP interaction, we generated knock-in mice that express a FOG-1 variant unable to bind CtBP. Surprisingly, these mice are normal and fertile. Furthermore, erythropoiesis at all stages of development is normal in these mice. Erythrocyte production is similar in mutant and wild-type mice even under conditions of erythropoietic stress stimulated by either exogenously added erythropoietin or phenylhydrazine-induced anemia. Thus, despite conservation of the FOG-CtBP interaction site, the in vivo function of FOG-1 in erythroid development is not affected by its inability to interact with the corepressor CtBP.

Alcohol Oxidoreductases↗
PubMed2002

Gonadal differentiation, sex determination and normal Sry expression in mice require direct interaction between transcription partners GATA4 and FOG2.

In mammals, Sry expression in the bipotential, undifferentiated gonad directs the support cell precursors to differentiate as Sertoli cells, thus initiating the testis differentiation pathway. In the absence of Sry, or if Sry is expressed at insufficient levels, the support cell precursors differentiate as granulosa cells, thus initiating the ovarian pathway. The molecular mechanisms upstream and downstream of Sry are not well understood. We demonstrate that the transcription factor GATA4 and its co-factor FOG2 are required for gonadal differentiation. Mouse fetuses homozygous for a null allele of Fog2 or homozygous for a targeted mutation in Gata4 (Gata4(ki)) that abrogates the interaction of GATA4 with FOG co-factors exhibit abnormalities in gonadogenesis. We found that Sry transcript levels were significantly reduced in XY Fog2(-/-) gonads at E11.5, which is the time when Sry expression normally reaches its peak. In addition, three genes crucial for normal Sertoli cell function (Sox9, Mis and Dhh) and three Leydig cell steroid biosynthetic enzymes (p450scc, 3betaHSD and p450c17) were not expressed in XY Fog2(-/-) and Gata(ki/ki) gonads, whereas Wnt4, a gene required for normal ovarian development, was expressed ectopically. By contrast, Wt1 and Sf1, which are expressed prior to Sry and necessary for gonad development in both sexes, were expressed normally in both types of mutant XY gonads. These results indicate that GATA4 and FOG2 and their physical interaction are required for normal gonadal development.

Animals↗