The GATA factor elt-1 regulates C. elegans developmental timing by promoting expression of the let-7 family microRNAs.

Postembryonic development in Caenorhabditis elegans is a powerful model for the study of the temporal regulation of development and for the roles of microRNAs in controlling gene expression. Stable switch-like changes in gene expression occur during development as stage-specific microRNAs are expressed and subsequently down-regulate other stage-specific factors, driving developmental progression. Key genes in this regulatory network are phylogenetically conserved and include the post-transcriptional microRNA repressor LIN-28; the nuclear hormone receptor DAF-12; and the microRNAs LIN-4, LET-7, and the three LET-7 family miRNAs (miR-48, miR-84, and miR-241). DAF-12 is known to regulate transcription of miR-48, miR-84 and miR-241, but its contribution is insufficient to account for all of the transcriptional regulation implied by the mutant phenotypes. In this work, the GATA-family transcription factor ELT-1 is identified from a genetic enhancer screen as a regulator of developmental timing in parallel to DAF-12, and is shown to do so by promoting the expression of the LET-7, miR-48, miR-84, and miR-241 microRNAs. The role of ELT-1 in developmental timing is shown to be separate from its role in cell-fate maintenance during post-embryonic development. In addition, analysis of Chromatin Immnoprecipitation (ChIP) data from the modENCODE project and this work suggest that the contribution of ELT-1 to the control of let-7 family microRNA expression is likely through direct transcription regulation.

Multiple mechanisms are involved in regulating the expression of the developmental timing regulator lin-28 in Caenorhabditis elegans.

The timing of postembryonic developmental programs in Caenorhabditis elegans is regulated by a set of so-called heterochronic genes, including lin-28 that specifies second larval programs. lin-66 mutations described herein cause delays in vulval and seam cell differentiation, indicating a role for lin-66 in timing regulation. A mutation in daf-12/nuclear receptor or alg-1/argonaute dramatically enhances the retarded phenotypes of the lin-66 mutants, and these phenotypes are suppressed by a lin-28 null allele. We further show that the LIN-28 protein level is upregulated in the lin-66 mutants and that this regulation is mediated by the 3'UTR of lin-28. We have also identified a potential daf-12-response element within lin-28 3'UTR and show that two microRNA (miRNA) (lin-4 and let-7)-binding sites mediate redundant inhibitory activities that are likely lin-66-independent. Quantitative PCR data suggest that the lin-28 mRNA level is affected by lin-14 and miRNA regulation, but not by daf-12 and lin-66 regulation. These results suggest that lin-28 expression is regulated by multiple independent mechanisms including LIN-14-mediated upregulation of mRNA level, miRNAs-mediated RNA degradation, LIN-66-mediated translational inhibition and DAF-12-involved translation promotion.

The Caenorhabditis elegans ect-2 RhoGEF gene regulates cytokinesis and migration of epidermal P cells.

A reduction-of-function mutation in ect-2 was isolated as a suppressor of the Multivulva phenotype of a lin-31 mutation. Analysis using markers indicates that this mutation causes defects in both the cytokinesis and migration of epidermal P cells, phenotypes similar to those caused by expressing a rho-1 dominant-negative construct. ect-2 encodes the Caenorhabditis elegans orthologue of the mouse Ect2 and Drosophila Pebble that function as guanine nucleotide exchange factors (GEFs) for Rho GTPases. The ect-2Colon, two colonsGFP reporter is expressed in embryonic cells and P cells. RNA interference of ect-2 causes sterility and embryonic lethality, in addition to the P-cell defects. We have determined the lesions of two ect-2 alleles, and described their differences in phenotypes in specific tissues. We propose a model in which ECT-2GEF not only activates RHO-1 for P-cell cytokinesis, but also collaborates with UNC-73GEF and at least two Rac proteins to regulate P-cell migration.

The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans.

In metazoans, microRNAs (miRNAs) carry out various regulatory functions through association with multiprotein miRNA-induced silencing complexes (miRISCs) that contain Dicer and Argonaute proteins. How miRNAs regulate the expression of their mRNA targets remains a major research question. We have identified the C. elegans ain-1 gene through a genetic suppressor screen and shown that it functions with the heterochronic genetic pathway that regulates developmental timing. Biochemical analysis indicates that AIN-1 interacts with protein complexes containing an Argonaute protein, Dicer, and miRNAs. AIN-1 shares homology with the candidate human neurological disease protein GW182, shown to localize in cytoplasmic processing bodies that are sites of mRNA degradation and storage. A functional AIN-1::GFP also localizes at the likely worm processing bodies. When coexpressed from transgenes, AIN-1 targets ALG-1 to the foci. These results suggest a model where AIN-1 regulates a subset of miRISCs by localization to the processing bodies, facilitating degradation or translational inhibition of mRNA targets.

A Caenorhabditis elegans TGF-beta, DBL-1, controls the expression of LON-1, a PR-related protein, that regulates polyploidization and body length.

Using cDNA-based array analysis combined with double-stranded RNA interference (dsRNAi), we have identified yk298h6 as a target gene of Caenorhabditis elegans TGF-beta signaling. Worms overexpressing dbl-1, a TGF-beta ligand, are 16% longer than wild type. Array analysis shows yk298h6 to be one of several genes suppressed in such worms. Disruption of yk298h6 function by dsRNAi also resulted in long worms, suggesting that it is a negative regulator of body length. yk298h6 was then mapped to, and shown to be identical to, lon-1, a known gene that affects body length. lon-1 encodes a 312 amino acid protein with a motif sequence that is conserved from plants to humans. Expression studies confirm that LON-1 is repressed by DBL-1, suggesting that LON-1 is a novel downstream component of the C.elegans TGF-beta growth regulation pathway. Consistent with this, LON-1 is expressed mainly in the larval and adult hypodermis and has dose-dependent effects on body length associated with changes in hypodermal ploidy, but not hypodermal cell proliferation.