Nuclear Lamin for Gene Silencing

Abstract

In the Drosophila ovary, germline stem cells (GSCs) divide to produce one cell that differentiates (a cystoblast) and one stem cell. The stem cell population is maintained by the release of Decapentaplegic (Dpp, a member of the bone morphogenetic protein family) from the stromal somatic cap cells (niche cells), which silences the gene bag-of-marbles ( bam ), which is required for cystoblast differentiation. Jiang et al . identified otefin ( ote ), which encodes a nuclear lamin protein of the LEM family (containing an LEM domain), in a screen for female sterile flies. The germaria (the site of the stem cell population and cystoblast formation) of the mutant flies contained very few germ cells. When GSCs were marked with green fluorescent protein (GFP), the authors found that the number of GSCs declined to nearly absent in Ote-deficient GSCs, whereas wild-type GSCs persisted, which is consistent with excessive differentiation and lack of self-renewal of the GSC population in the ote mutants. Ote was required in the GSCs because reintroduction of ote into the somatic cells only failed to rescue the mutant ovary phenotype of the Ote-deficient animals. Overexpression of Ote led to an increased number of GSC-like cells, which suggests that excess Ote may suppress or delay differentiation. Bam was detected by expression of a fusion protein, and in Ote-deficient GSCs Bam-GFP was abundant, whereas in wild-type GSCs Bam-GFP was undetectable. Bam expression is inhibited by Dpp signaling. The authors showed that a genetic interaction occurred between signaling components of the Dpp pathway and Ote, and that when an inhibitor of Dpp signaling was overexpressed, forced overexpression of Ote led to a loss of GSCs. Thus, Dpp signaling appeared to be required for Ote to stimulate stem cell self-renewal. Ote required nuclear membrane localization and the LEM domain to function in the GSCs because introduction of Ote lacking the transmembrane domain or the LEM domain or targeted to the plasma membrane with a different transmembrane domain failed to rescue the mutant ovary phenotype. Endogenous Ote and Smad4 (called Medea in flies) coimmunoprecipitated when a tagged form of Medea was transfected into S2 cells; this interaction required the LEM domain of Ote. Ote and Medea were also found bound to the Bam silencer region in chromatin immunoprecipitation experiments with S2 cells, and the binding of Ote was lost if endogenous Medea was knocked down with siRNA. Thus, signal-dependent gene silencing in the GSCs appears to involve an interaction between the chromatin, the nuclear envelope through Ote, and the transcription factor Medea. X. Jiang, L. Xia, D. Chen, Y. Yang, H. Huang, L. Yang, Q. Zhao, L. Shen, J. Wang, D. Chen, Otefin, a nuclear membrane protein, determines the fate of germline stem cells in Drosophila via interaction with Smad complexes. Dev. Cell 14 , 494-506 (2008). [PubMed]