DEVELOPMENT2695RESEARCH ARTICLEINTRODUCTIONThe ordered recruitment of factors required for proper geneexpression is crucial for animal development. For example,sequence-specific transcription factors recruit a variety of proteincomplexes that remodel chromatin to regulate the transcription oftarget genes either by using the energy of ATP hydrolysis to movenucleosomes or by chemically modifying histones (Narlikar et al.,2002). These two mechanisms for chromatin remodeling have beencharacterized extensively in vitro (Roth et al., 2001; Smith andPeterson, 2005), but efforts to understand how each functions in thedevelopment of metazoa have just begun.Studies of vulval development in the nematode Caenorhabditiselegans could help establish the roles of chromatin remodelingduring development. The vulva of the C. elegans hermaphrodite isformed by the 22 descendants of three ectodermal blast cells (P5.p,P6.p and P7.p) located along the ventral surface of the animal(Sulston and Horvitz, 1977). P(5-7).p, three of a set of six equipotentcells P(3-8).p called the vulval equivalence group, are induced togenerate vulval cells by an epidermal growth factor (EGF)-likesignal from the gonad (Sulston and White, 1980; Hill and Sternberg,1992). The inductive signal is received and transduced by aconserved receptor tyrosine kinase (RTK)/Ras pathway (Kornfeld,1997). Unlike the other cells of the vulval equivalence group (P3.p,P4.p and P8.p), which divide once and fuse with the nearbyhypodermal syncytium (hyp7), P(5-7).p divide three times togenerate the cells that form the adult vulva (Sulston and Horvitz,1977). Mutations that reduce or eliminate the function of the let-23RTK/let-60 Ras pathway can result in a vulvaless (Vul) animal inwhich no cells of the vulval equivalence group express vulval fates;by contrast, mutations that increase the function of this pathway cancause the ectopic adoption of vulval cell fates by P3.p, P4.p andP8.p, and result in a multivulva (Muv) animal (Beitel et al., 1990;Han et al., 1990; Katz et al., 1996).Loss-of-function mutations in the synthetic multivulva (synMuv)genes also can cause a Muv phenotype (Horvitz and Sulston, 1980;Ferguson and Horvitz, 1989). These genes have been grouped intothree classes: A, B and C (Ferguson and Horvitz, 1989; Ceol andHorvitz, 2004). Loss-of-function mutations within any one class donot cause a Muv phenotype, whereas mutations in any two geneswithin two different classes cause a Muv phenotype (Ferguson andHorvitz, 1989; Ceol and Horvitz, 2004). The class A synMuv genesencode novel, nuclear components (Clark et al., 1994; Huang et al.,1994; Davison et al., 2005). Many class B synMuv genes encodehomologs of transcriptional repressors and factors that remodelchromatin, including LIN-35 Rb (Lu and Horvitz, 1998), the EFL-1/DPL-1 E2F heterodimeric transcription factor (Ceol and Horvitz,2001), the HDA-1 HDAC1, LET-418 Mi2, LIN-53 RbAp48 NuRDcomplex (Lu and Horvitz, 1998; Tong et al., 1998; Xue et al., 1998;von Zelewsky et al., 2000; Unhavaithaya et al., 2002) and HPL-2Heterochromatin Protein 1 (HP1) (Couteau et al., 2002). TheDrosophila melanogaster homologs of some class B synMuvproteins form a complex, identified by two different groups andcalled Myb-MuvB or dREAM, that is likely to repress thetranscription of genes through chromatin remodeling (Korenjak etal., 2004; Lewis et al., 2004). Class C synMuv genes encodehomologs of a putative Tip60/NuA4 histone acetyltransferasecomplex (Ceol and Horvitz, 2004). Because of these homologies,the synMuv genes, which negatively regulate the vulval cell fate,probably act by repressing the transcription of genes that promotethe expression of vulval cell fates.In this study, we describe the identification of a C. elegansortholog of Drosophila ISWI, called isw-1, as a suppressor of thesynMuv phenotype. ISWI is an ATP-dependent chromatin-remodeling enzyme identified by homology to S. cerevisiaeC. elegans ISWI and NURF301 antagonize an Rb-like pathwayin the determination of multiple cell fatesErik C. Andersen, Xiaowei Lu* and H. Robert Horvitz†The class A, B and C synthetic multivulva (synMuv) genes act redundantly to negatively regulate the expression of vulval cell fates inCaenorhabditis elegans. The class B and C synMuv proteins include homologs of proteins that modulate chromatin and influencetranscription in other organisms similar to members of the Myb-MuvB/dREAM, NuRD and Tip60/NuA4 complexes. To determinehow these chromatin-remodeling activities negatively regulate the vulval cell-fate decision, we isolated a suppressor of the synMuvphenotype and found that the suppressor gene encodes the C. elegans homolog of Drosophila melanogaster ISWI. The C. elegansISW-1 protein likely acts as part of a Nucleosome Remodeling Factor (NURF) complex with NURF-1, a nematode ortholog ofNURF301, to promote the synMuv phenotype. isw-1 and nurf-1 mutations suppress both the synMuv phenotype and the multivulvaphenotype caused by overactivation of the Ras pathway. Our data suggest that a NURF-like complex promotes the expression ofvulval cell fates by antagonizing the transcriptional and chromatin-remodeling activities of complexes similar to Myb-MuvB/dREAM,NuRD and Tip60/NuA4. Because the phenotypes caused by a null mutation in the tumor-suppressor and class B synMuv gene lin-35Rb and a gain-of-function mutation in let-60 Ras are suppressed by reduction of isw-1 function, NURF complex proteins might beeffective targets for cancer therapy.KEY WORDS: ISWI, NURF, Rb, Ras, C. elegansDevelopment 133, 2695-2704 (2006) doi:10.1242/dev.02444Howard Hughes Medical Institute, Department of Biology, Room 68-425, MIT,77 Massachusetts Avenue, Cambridge, MA 02139, USA.*Present address: Department of Cell Biology, University of Virginia School ofMedicine, Charlottesville, VA 22908, USA.†Author for correspondence (e-mail: [email protected])Accepted 15 May 2006DEVELOPMENT2696Snf2/Swi2 (Elfring et al., 1994). We show that ISW-1 probably actsas a component of a Nucleosome Remodeling Factor (NURF)-likecomplex with the Drosophila NURF301 ortholog NURF-1 toantagonize the synMuv genes. Our observations reveal theantagonistic functions of a NURF-like chromatin remodelingcomplex and complexes similar to Myb-MuvB/dREAM, NuRD andTIP60/NuA4 in the determination of multiple cell fates, includingthe antagonistic regulation of at least one putative target of synMuvtranscriptional repression.MATERIALS AND METHODSStrains and geneticsC. elegans