Joint Functions of METT-10 and Dynein in the Caenorhabditis elegans Germ Line

During normal development as well as in diseased states such as cancer, extracellular niches often provide cues to proximal cells and activate intracellular pathways. Activation of such signaling pathways in turn instructs cellular proliferation and differentiation. In the C.elegans gonad, GLP-1/Notch signaling instructs germ line stem cells to self renew through mitotic cell division. As germ cells progressively move out of the niche, they differentiate by entering meiosis and eventually form gametes. Using this model system, I uncovered a cooperative role for the METT-10 putative methyltransferase and the dynein motor complex in regulating the balance between germ cell proliferation and differentiation. I demonstrate that Dynein Light Chain-1: DLC-1), and its partner, Dynein Heavy Chain-1, inhibit the proliferative cell fate, in part through regulation of METT-10 levels and nuclear accumulation. I further show that the methyltransferase domain of METT-10 is required for normal inhibition of germ cell proliferation, and that DLC-1 and METT-10 act antagonistically to activation of the GLP-1 Notch pathway, which plays conserved roles in stem cell fate specification. Moreover, I find that regulation of germ cell proliferative fate is only one of multiple joint functions of METT-10 and the dynein motor complex, and propose that they function together in multiple cellular contexts, including mitotic cell division. The finding that METT-10 and dynein inhibit germ cell proliferative fate, despite promoting mitotic cell division of those cells that do proliferate, highlights a genetic difference between the specification of proliferative fate and its execution

A Role for Dynein in the Inhibition of Germ Cell Proliferative Fate▿

During normal development as well as in diseased states such as cancer, extracellular “niches” often provide cues to proximal cells and activate intracellular pathways. Activation of such signaling pathways in turn instructs cellular proliferation and differentiation. In the Caenorhabditis elegans gonad, GLP-1/Notch signaling instructs germ line stem cells to self-renew through mitotic cell division. As germ cells progressively move out of the niche, they differentiate by entering meiosis and eventually form gametes. In this model system, we uncovered an unexpected role for the dynein motor complex in promoting normal differentiation of proliferating germ cells. We demonstrate that dynein light chain 1 (DLC-1) and its partner, dynein heavy chain 1, inhibit the proliferative cell fate, in part through regulation of METT-10, a conserved putative methyltransferase. We show that DLC-1 physically interacts with METT-10 and promotes both its overall levels and nuclear accumulation. Our results add a new dimension to the processes controlled by the dynein motor complex, demonstrating that dynein can act as an antiproliferative factor

METT-10, A Putative Methyltransferase, Inhibits Germ Cell Proliferative Fate in Caenorhabditis elegans

Germ-line stem cells are unique because they either self-renew through mitosis or, at a certain frequency, switch to meiosis and produce gametes. The switch from proliferation to meiosis is tightly regulated, and aberrations in switching result in either too little or too much proliferation. To understand the genetic basis of this regulation, we characterized loss-of-function mutations and a novel tumorous allele of Caenorhabditis elegans mett-10, which encodes a conserved putative methyltransferase. We show that METT-10 is a nuclear protein that acts in the germ line to inhibit the specification of germ-cell proliferative fate. METT-10 also promotes vulva, somatic gonad, and embryo development and ensures meiotic development of those germ cells that do differentiate. In addition, phenotypic analysis of a mett-10 null allele reveals that METT-10 enables mitotic cell cycle progression. The finding that METT-10 functions to inhibit germ-cell proliferative fate, despite promoting mitotic cell cycle progression of those germ cells that do proliferate, separates the specification of proliferative fate from its execution

Nipped-A, the Tra1/TRRAP Subunit of the Drosophila SAGA and Tip60 Complexes, Has Multiple Roles in Notch Signaling during Wing Development

The Notch receptor controls development by activating transcription of specific target genes in response to extracellular signals. The factors that control assembly of the Notch activator complex on target genes and its ability to activate transcription are not fully known. Here we show, through genetic and molecular analysis, that the Drosophila Nipped-A protein is required for activity of Notch and its coactivator protein, mastermind, during wing development. Nipped-A and mastermind also colocalize extensively on salivary gland polytene chromosomes, and reducing Nipped-A activity decreases mastermind binding. Nipped-A is the fly homologue of the yeast Tra1 and human TRRAP proteins and is a key component of both the SAGA and Tip60 (NuA4) chromatin-modifying complexes. We find that, like Nipped-A, the Ada2b component of SAGA and the domino subunit of Tip60 are also required for mastermind function during wing development. Based on these results, we propose that Nipped-A, through the action of the SAGA and Tip60 complexes, facilitates assembly of the Notch activator complex and target gene transcription

Evolution of the Drosophila Feminizing Switch Gene Sex-lethal

In Drosophila melanogaster, the gene Sex-lethal (Sxl) controls all aspects of female development. Since melanogaster males lacking Sxl appear wild type, Sxl would seem to be functionally female specific. Nevertheless, in insects as diverse as honeybees and houseflies, Sxl seems not to determine sex or to be functionally female specific. Here we describe three lines of work that address the questions of how, when, and even whether the ancestor of melanogaster Sxl ever shed its non-female-specific functions. First, to test the hypothesis that the birth of Sxl's closest paralog allowed Sxl to lose essential ancestral non-female-specific functions, we determined the CG3056 null phenotype. That phenotype failed to support this hypothesis. Second, to define when Sxl might have lost ancestral non-female-specific functions, we isolated and characterized Sxl mutations in D. virilis, a species distant from melanogaster and notable for the large amount of Sxl protein expression in males. We found no change in Sxl regulation or functioning in the 40+ MY since these two species diverged. Finally, we discovered conserved non-sex-specific Sxl mRNAs containing a previously unknown, potentially translation-initiating exon, and we identified a conserved open reading frame starting in Sxl male-specific exon 3. We conclude that Drosophila Sxl may appear functionally female specific not because it lost non-female-specific functions, but because those functions are nonessential in the laboratory. The potential evolutionary relevance of these nonessential functions is discussed