Previous studies have suggested that the GATA4 transcription factor plays an important role in regulating mammalian cardiac development. In the studies described in this report we have used gene targeting to produce GATA4-deficient mice. Homozygous GATA4-deficient (GATA4-/-) mice died between 8.5 and 10.5 days post coitum (dpc). GATA4-/- embryos displayed severe defects in both rostral-to-caudal and lateral-to-ventral folding, which were reflected in a generalized disruption of the ventral body pattern. This resulted in the defective formation of an organized foregut and anterior intestinal pore, the failure to close both the amniotic cavity and yolk sac, and the uniform lack of a ventral pericardial cavity and heart tube. Analysis of cardiac development in the GATA4-/- mice demonstrated that these embryos developed splanchnic mesoderm, which differentiated into primitive cardiac myocytes that expressed contractile proteins. However, consistent with the observed defect in ventral morphogenesis, these GATA4-/- procardiomyocytes failed to migrate to the ventral midline to form a linear heart tube and instead formed aberrant cardiac structures in the anterior and dorsolateral regions of the embryo. The defect in ventral migration of the GATA4-/- procardiomyocytes was not cell intrinsic because GATA4-/- cardiac myocytes and endocardial cells populated the hearts of GATA4-/- -C57BL/6 chimeric mice. Taken together, these results demonstrated that GATA4 is not essential for the specification of the cardiac cell lineages. However, they define a critical role for GATA4 in regulating the rostral-to-caudal and lateral-to-ventral folding of the embryo that is needed for normal cardiac morphogenesis.

Members of the Ets family of winged helix-loop-helix transcription factors play important roles in the development and function of multiple mammalian cell lineages. Elf-1 is an Ets-related transcription factor that is expressed at high levels in T cells and is known to regulate the expression of several T cell genes, including the granulocyte-macrophage colony stimulating factor (GM-CSF) gene, the interleukin-2 receptor alpha subunit (IL-2Ralpha) gene, and the CD4 gene. In the studies described in this report, we have characterized the pattern of expression of Elf-1 in the developing mouse embryo and in adult mouse tissues as well as in multiple immortalized human and murine cell lines. Elf-1 is expressed at high levels throughout thymocyte development, with equivalent levels of Elf-1 expression seen in all subsets of maturing thymocytes and T cells. Somewhat surprisingly, however, Elf-1 is also expressed at high levels in epithelial cells lining the oral cavity, the lung, the CNS, and the gastrointestinal and urinary tracts as well as in the skin of the developing mouse embryo and at lower levels in the adult mouse testis and liver. Western blot analyses of a large number of immortalized cell lines demonstrated high-level Elf-1 expression in T and B lymphocyte and macrophage cell lines as well as in two prostate carcinoma cell lines. Low-level expression was observed in fibroblasts, embryonic stem cells, and myoblasts. Taken together, our data suggest that in addition to its role in regulating T cell development and function, Elf-1 may regulate gene expression in the B cell and myelomonocytic lineages, as well as in multiple epithelial cell types during murine embryonic development.

The transcriptional regulatory elements of many inducible T-cell genes contain adjacent or overlapping binding sites for the Ets and NF-B/NFAT families of transcription factors. Similar arrays of functionally important NF-B/NFAT and Ets binding sites are present in the transcriptional enhancers of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2), suggesting that this pattern of nuclear protein binding sites reflects an evolutionarily conserved mechanism for regulating inducible T-cell gene expression that has been co-opted during HIV evolution. Despite these findings, the molecular mechanisms by which Ets and NF-B/ NFAT proteins cooperatively regulate inducible T-cell gene expression remained unknown. In the studies described in this report, we demonstrated a physical interaction between multiple Ets and NF-B/NFAT proteins both in vitro and in activated normal human T cells. This interaction is mediated by the Ets domain of Ets proteins and the C-terminal region of the Rel homology domains of NF-B/NFAT proteins. In addition, the Ets-NF-B/NFAT interaction requires the presence of DNA binding sites for both proteins, as it is abolished by the DNA intercalating agents propidium iodide and ethidium bromide and enhanced by the presence of synthetic oligonucleotides containing binding sites for Ets and NF-B proteins. A dominant-negative mutant of NF-B p50 that binds DNA but fails to interact with Ets proteins inhibits the synergistic activation of the HIV-1 and HIV-2 enhancers by NF-B (p50 ؉ p65) and Ets-1, suggesting that physical interaction between Ets and NF-B proteins is required for the transcriptional activity of the HIV-1 and HIV-2 enhancers. Taken together, these findings suggest that evolutionarily conserved physical interactions between Ets and NF-B/NFAT proteins are important in regulating the inducible expression of T-cell genes and viruses. These interactions represent a potential target for the development of novel immunosuppressive and antiviral therapies.