Upregulation of the let-7 microRNA with precocious development in lin-12/Notch hypermorphic Caenorhabditis elegans mutants.

The lin-12/Notch signaling pathway is conserved from worms to humans and is a master regulator of metazoan development. Here, we demonstrate that lin-12/Notch gain-of-function (gf) animals display precocious alae at the L4 larval stage with a significant increase in let-7 expression levels. Furthermore, lin-12(gf) animals display a precocious and higher level of let-7 gfp transgene expression in seam cells at L3 stage. Interestingly, lin-12(gf) mutant rescued the lethal phenotype of let-7 mutants similar to other known heterochronic mutants. We propose that lin-12/Notch signaling pathway functions in late developmental timing, upstream of or in parallel to the let-7 heterochronic pathway. Importantly, the human microRNA let-7a was also upregulated in various human cell lines in response to Notch 1 activation, suggesting an evolutionarily conserved cross-talk between let-7 and the canonical lin-12/Notch signaling pathway.

Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans.

BACKGROUND: The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions. RESULTS: Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome. CONCLUSION: Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved.

Caenorhabditis elegans OSR-1 regulates behavioral and physiological responses to hyperosmotic environments.

The molecular mechanisms that enable multicellular organisms to sense and modulate their responses to hyperosmotic environments are poorly understood. Here, we employ Caenorhabditis elegans to characterize the response of a multicellular organism to osmotic stress and establish a genetic screen to isolate mutants that are osmotic stress resistant (OSR). In this study, we describe the cloning of a novel gene, osr-1, and demonstrate that it regulates osmosensation, adaptation, and survival in hyperosmotic environments. Whereas wild-type animals exposed to hyperosmotic conditions rapidly lose body volume, motility, and viability, osr-1(rm1) mutant animals maintain normal body volume, motility, and viability even upon chronic exposures to high osmolarity environments. In addition, osr-1(rm1) animals are specifically resistant to osmotic stress and are distinct from previously characterized osmotic avoidance defective (OSM) and general stress resistance age-1(hx546) mutants. OSR-1 is expressed in the hypodermis and intestine, and expression of OSR-1 in hypodermal cells rescues the osr-1(rm1) phenotypes. Genetic epistasis analysis indicates that OSR-1 regulates survival under osmotic stress via CaMKII and a conserved p38 MAP kinase signaling cascade and regulates osmotic avoidance and resistance to acute dehydration likely by distinct mechanisms. We suggest that OSR-1 plays a central role in integrating stress detection and adaptation responses by invoking multiple signaling pathways to promote survival under hyperosmotic environments.

The notch regulator MAML1 interacts with p53 and functions as a coactivator.

Members of the evolutionarily conserved Mastermind (MAM) protein family, including the three related mammalian Mastermind-like (MAML) proteins MAML1-3, function as crucial coactivators of Notch-mediated transcriptional activation. Given the recent evidence of cross-talk between the p53 and Notch signal transduction pathways, we have investigated whether MAML1 may also be a transcriptional coactivator of p53. Indeed, we show here that MAML1 is able to interact with p53. We show that MAML1-p53 interaction involves the N-terminal region of MAML1 and the DNA-binding domain of p53, and we use a chromatin immunoprecipitation assay to show that MAML1 is part of the activator complex that binds to native p53-response elements within the promoter of the p53 target genes. Overexpression of wild-type MAML1 as well as a mutant, defective in Notch signaling, enhanced the p53-dependent gene induction in mammalian cells, whereas MAML1 knockdown reduced the p53-dependent gene expression. MAML1 increases the half-life of p53 protein and enhances its phosphorylation/acetylation upon DNA damage of cells. Finally, RNA interference-mediated knockdown of the single Caenorhabditis elegans MAML homolog, Lag-3, led to substantial abrogation of p53-mediated germ-cell apoptotic response to DNA damage and markedly reduced the expression of Ced-13 and Egl-1, downstream pro-apoptotic targets of the C. elegans p53 homolog Cep-1. Thus, we present evidence for a novel coactivator function of MAML1 for p53, independent of its function as a coactivator of Notch signaling pathway.