The gene coding for apolipoprotein AI (apoAl), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine.Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAl gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXRea over the previously characterized RA receptors RARae and RARI3. In this study we investigated the combined effects of ARP-1 and RXRa on apoAl gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXRa-mediated transactivation of the apoAl gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXRoa bind to site A with similar affinities. These assays also revealed that ARP-1 and RXRai bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXRa alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAl gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXRai has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXRa together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXRai together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAl gene responsiveness to RXRa and RA and suggest that the magnitude of this responsiveness is regulated by the intracellular ratio of ARP-1 to RXRa. These observations raise the possibility that transcriptional repression is a general mechanism for switching gene transcription between alternative transcription activation pathways.A large body of epidemiologic, genetic, pharmacologic, and biochemical evidence suggests that high-density lipoprotein (HDL) levels in plasma play an important role in regulation of cellular cholesterol homeostasis and atherosclerosis progression and regression. Although changes in plasma HDL levels have been associated with a diverse number of dietary, hormonal, and str...

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Notch signaling has an evolutionarily conserved function for cell fate determination and stem cell maintenance. Previously, we identified a novel component of the Notch signaling pathway in zebrafish, mind bomb, which encodes an E3 ubiquitin ligase essential for Notch signal activation. Further studies showed that Mind bomb À/À mouse embryos exhibited pan-Notch phenotypes in various tissues, suggesting that Mind bomb function is conserved in mammals. Therefore we sought to understand the various molecular partners of Mind bomb using yeast two-hybrid screening. In this search we identified Sorting nexin 5 (Snx5) as a novel interacting partner of Mind bomb. Furthermore we demonstrated that Snx5 colocalizes with Mind bomb in early endosomal compartments, suggesting that Snx5 is important for Mind bomb trafficking. In addition, we identified zebrafish orthologue of Snx5 and showed that snx5 is predominantly expressed in hematopoietic and endothelial precursor cells in zebrafish. We also found defects in hematopoiesis and blood vessel development in snx5 morpholino-injected embryos. Taken together, we show that Snx5, a novel interacting partner of Mind bomb, may have an essential role for cell fate determination in early development.

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While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount in situ hybridization and antisense morpholino oligonucleotide (MO)-based gene knockdown. We find both smyd3 and setd7 are highly expressed within developing zebrafish heart and knock-down of these genes led to severe defects in cardiac morphogenesis without altering the expressions pattern of heart markers, including cmlc2, vmhc, and amhc. Furthermore, double knock-down by coinjection of smyd3 and setd7 MOs caused the synergistic defects in heart development. As similar to knock-down effect, overexpression of these genes also caused the heart morphogenesis defect in zebrafish. These results indicate that histone modifying enzymes, SMYD3 and SETD7, appear to function synergistically during heart development and their proper functioning is essential for normal heart morphogenesis during development.

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