Dependence of the Sperm/Oocyte Decision on the Nucleosome Remodeling Factor Complex Was Acquired during Recent Caenorhabditis briggsae Evolution

Xiangmei Chen 0 1 Yongquan Shen 1 Ronald E. Ellis 1 0 Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey 1 Department of Molecular Biology, Rowan University-SOM The major families of chromatin remodelers have been conserved throughout eukaryotic evolution. Because they play broad, pleiotropic roles in gene regulation, it was not known if their functions could change rapidly. Here, we show that major alterations in the use of chromatin remodelers are possible, because the nucleosome remodeling factor (NURF) complex has acquired a unique role in the sperm/oocyte decision of the nematode Caenorhabditis briggsae. First, lowering the activity of C. briggsae NURF-1 or ISW-1, the core components of the NURF complex, causes germ cells to become oocytes rather than sperm. This observation is based on the analysis of weak alleles and null mutations that were induced with TALENs and on RNA interference. Second, qRT-polymerase chain reaction data show that the C. briggsae NURF complex promotes the expression of Cbr-fog-1 and Cbr-fog-3, two genes that control the sperm/oocyte decision. This regulation occurs in the third larval stage and affects the expression of later spermatogenesis genes. Third, double mutants reveal that the NURF complex and the transcription factor TRA-1 act independently on Cbr-fog-1 and Cbrfog-3. TRA-1 binds both promoters, and computer analyses predict that these binding sites are buried in nucleosomes, so we suggest that the NURF complex alters chromatin structure to allow TRA-1 access to Cbr-fog-1 and Cbr-fog-3. Finally, lowering NURF activity by mutation or RNA interference does not affect this trait in other nematodes, including the sister species C. nigoni, so it must have evolved recently. We conclude that altered chromatin remodeling could play an important role in evolutionary change. - To control development, gene expression must be regulated in time and space. The organization of DNA into chromatin plays a key role in this process, by restricting the accessibility of promoters and enhancers (Clapier and Cairns 2009). This restriction also increases precision, because chromatinremodeling complexes can actively cooperate with transcription factors to control gene expression. However, the pleiotropic phenotypes of most chromatin remodelers have made it difficult to evaluate their roles in evolutionary change. Nematodes provide an ideal way to address this problem. Most of the known chromatin-remodeling complexes exist in nematodes and control various aspects of development (Cui and Han 2007). Moreover, detailed molecular models have been established for two developmental processes in Caenorhabditis elegans. The first involves the specification of sensory rays in the male tailthe trithorax group of chromatin regulators promotes the expression of two Hox genes in the seam cells V5 and V6, whereas the Polycomb group blocks their expression (Chamberlin and Thomas 2000; Ross and Zarkower 2003; Zhang et al. 2003). The other involves the induction of vulval development by an EGF signal from the anchor cell. In the nearby hypodermis, the Nucleosome Remodeling Deacetylase (NURD) and Retinoblastoma (Rb) complexes from the SynMuvB group block the expression of the EGF gene lin-3 (Cui et al. 2006), preventing the inappropriate activation of the Ras pathway. Other chromatin remodeling complexes help regulate the development of the vulva, but their targets remain unknown (Fay and Yochem 2007). For example, the Tip60/NuA4 histone acetyl transferase (HAT) complex blocks vulval development (Ceol and Horvitz 2004), whereas the nucleosome remodeling factor (NURF) complex promotes vulval cell fates (Andersen et al. 2006). Chromatin regulators also play an important role in the establishment of the nematode germline (Schaner et al. 2003), and three observations raise the possibility that some of them influence the sperm/oocyte decision. First, the C. elegans tra-4 gene encodes a PLZF-containing protein that works with histone chaperones and deacetylases to promote many female cell fates (Grote and Conradt 2006), although a role in oogenesis has not been detected. Second, natural variation in the C. elegans NATH-10 acetyltransferase controls the number of sperm produced by hermaphrodites (Duveau and Felix 2012), though how it does so remains unknown. Third, the Tip60 HAT complex regulates the sperm/oocyte decision in both C. elegans and C. briggsae (Guo et al. 2013), although it remains unclear whether Tip60 directly acetylates the transcription factor TRA-1 or works with TRA-1 to acetylate histones in the promoters of targets like fog-3. With these cases in mind, we began investigating the role of chromatin remodelers in the sperm/oocyte decision. This regulatory decision is ideal for comparative evolutionary studies (Haag 2005), because it played a critical role in the origin of self-fertile hermaphrodites (Baldi et al. 2009). Both C. briggsae and C. elegans evolved hermaphroditic reproduction independently (Cho et al. 2004; Kiontke et al. 2004, 2011). In each species, the XX animals have female bodies but make sperm during the L4 larval stage and oocytes as adults, an arrangement that allows self-fertilization. Several studies have shown that this evolutionary step involved independent modifications to the sex-determination pathway, which controls the sperm/oocyte decision. In C. elegans, FOG-2 and GLD-1 cause spermatogenesis in hermaphrodites, by blocking the translation of tra-2 messages (Clifford et al. 2000). By contrast, there is no fog-2 gene in C. briggsae (Nayak et al. 2005), gld-1 has a different function (Nayak et al. 2005; Beadell et al. 2011) and tra-2 is regulated by the novel protein SHE-1 (Guo et al. 2009). The FEM complex is also required for male cell fates in C. elegans (Doniach and Hodgkin 1984; Kimble et al. 1984; Hodgkin 1986) but is dispensable for hermaphrodite spermatogenesis in C. briggsae (Hill et al. 2006). All of these regulatory genes act through the transcription factor TRA-1 to control fog-1 and fog-3, which promote spermatogenesis in both sexes (Chen and Ellis 2000; Chen et al. 2001; Jin et al. 2001). Because the Tip60 HAT complex is involved in this decision, we began studying the roles of other chromatin remodelers. Here, we show that NURF-1A and ISW-1, the C. briggsae homologs of Drosophila NURF301 and ISWI, promote spermatogenesis in both sexes. These proteins are the principal components of the NURF complex (Tsukiyama and Wu 1995; Tsukiyama et al. 1995), which is a member of the imitation switch family of chromatin remodelers (Corona and Tamkun 2004). These complexes use the energy of ATP hydrolysis to slide nucleosomes along the DNA, which increases chromatin fluidity and alters the accessibility of target sites to transcription factors and other regulatory proteins. In Drosophila, NURF301 and ISWI are essential for the maintenance of germline stem cells (Cherry and Matunis 2010). In (...truncated)