Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5-17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.

NF-ATp is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In this study, we show that mice with a null mutation in the NF-ATp gene have splenomegaly with hyperproliferation of both B and T cells. They also display early defects in the transcription of multiple genes encoding cytokines and cell surface receptors, including CD40L and FasL. A striking defect in early IL-4 production was observed after ligation of the TCR complex by treatment with anti-CD3 in vivo. The transcription of other cytokines including IL-13, GM-CSF, and TNF alpha was also affected, though to a lesser degree. Interestingly, the cytokines IL-2 and IFN gamma were minimally affected. Despite this early defect in IL-4 transcription, Th2 development was actually enhanced at later timepoints as evidenced by increased IL-4 production and IgE levels in situations that favor the formation of Th2 cells both in vitro and in vivo. These data suggest that NF-ATp may be involved in cell growth, and that it is important for the balanced transcription of the IL-4 gene during the course of an immune response.

GA-binding protein (GABP) is a transcriptional regulator composed of two structurally dissimilar subunits. The alpha subunit contains a DNA-binding domain that is a member of the ETS family, whereas the beta subunit contains a series of ankyrin repeats. The crystal structure of a ternary complex containing a GABPalpha/beta ETS domain-ankyrin repeat heterodimer bound to DNA was determined at 2. 15 angstrom resolution. The structure shows how an ETS domain protein can recruit a partner protein using both the ETS domain and a carboxyl-terminal extension and provides a view of an extensive protein-protein interface formed by a set of ankyrin repeats. The structure also reveals how the GABPalpha ETS domain binds to its core GGA DNA-recognition motif.

The NF-AT family of transcription factors activates early immune response genes such as cytokines. In the adult, NF-ATc is expressed exclusively in the lymphoid system and is induced upon lymphocyte activation. NF-ATc null mutant mice die in utero of cardiac failure, precluding analysis of the role of NF-ATc in lymphocyte activation. By using RAG-2-deficient blastocyst complementation, we now demonstrate that young, highly chimeric mice lacking NF-ATc have impaired repopulation of both thymus and peripheral lymphoid organs. Furthermore, NF-ATc deficiency impaired T lymphocyte activation and secretion of IL-4. B lymphocytes displayed reduced proliferation and a selective loss of IL-4-driven immunoglobulin isotypes both in vivo and in vitro. Our data demonstrate that NF-ATc is essential for the optimal generation and function of mature T and B lineage cells, with an especially profound effect on IL-4-driven responses.

Nuclear factor of activated T cells (NFAT) is a family of four related transcription factors implicated in cytokine and early response gene expression in activated lymphocytes. Here we report that NFAT4, in contrast to NFATp and NFATc, is preferentially expressed in DP thymocytes. Mice lacking NFAT4 have impaired development of CD4 and CD8 SP thymocytes and peripheral T cells as well as hyperactivation of peripheral T cells. The thymic defect is characterized by increased apoptosis of DP thymocytes. The increased apoptosis and hyperactivation may reflect heightened sensitivity to TcR-mediated signaling. Further, mice lacking NFAT4 have impaired production of Bcl-2 mRNA and protein. NFAT4 thus plays an important role in the successful generation and survival of T cells.

This report outlines three observations relating to GABPI3 , a polypeptide constituent of the heterotetrameric transcription factor GABP. Evidence is presented showing that the mouse genome encodes two highly related GABPI3 polypeptides, designated GABPI31-1 and GABPI32-1. Genomic and eDNA copies of the newly defined Gabpb2 gene were cloned and characterized, providing the conceptually translated amino acid sequence of GABPI32-1. The genes encoding these two proteins, as well as GABPc~, were mapped to three unlinked chromosomal loci. Although physically unlinked, the patterns of expression of the three genes were strikingly concordant. Finally, the molecular basis of GABPI3 dimerization was resolved. Carboxy-terminal regions of the two GABPI3 polypeptides, which mediate dimerization, bear highly related primary amino acid sequences. Both sequences are free of oL-helix destabilizing residues and, when displayed on idealized c~-helical projections, reveal marked amphipathy. Two observations indicate that these regions adopt an a-helical conformation and intertwine as coiled-coils. First, the dimer-forming region of GABPI32-1 can functionally replace the leucine zipper of a bZIP transcription factor. Second, a synthetic peptide corresponding to this region shows distinctive helical properties when examined by circular dichroism spectroscopy. Finally, evidence is presented showing that GABPI31-1 and GABPI32-1 can heterodimerize through this carboxy-terminal domain, but neither protein can heterodimerize via the dimer-forming region of the bZIP protein C/EBPI3.

Primer extension and RACE (rapid amplification of cDNA ends) assays were used to identify and sequence the 5' terminus of mouse ob mRNA. This sequence was used to obtain a recombinant bacteriophage containing the first exon of the encoding gene. DNA

qmfl is a quail myogenic regulatory gene that is transcribed in skeletal myoblasts and differentiated muscle and shows sequence homology to MyoD1 and MyfS. We used the qmfl transcript as an in situ hybridization marker for determined myogenic cells to study myogenic lineages in developing embryos. We present evidence for the temporal and spatial regulation of qmfl mRNA expression and slow cardiac troponin C (TnC), fast skeletal troponin T (TnT), and a-cardiac actin contractile protein mRNA expression in the somite myotome and limb buds. Our results show that qmfl is a marker for myogenic lineages during both somite formation and limb development and that qmfl mRNAs, but not contractile protein mRNAs, localize in dorsal medial lip (DML) cells of the somite dermatome. We propose that the DML is a site of myogenic lineage determination.