Caenorhabditis elegans unc-37/groucho Interacts Genetically With Components of the Transcriptional Mediator Complex

Corresponding author: Scott W. Emmons, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461., emmons{at}aecom.yu.edu (E-mail)

Communicating editor: P. ANDERSON

	ABSTRACT

TOP ABSTRACT Positive requirement for unc-37... Positive requirement for unc-37... unc-37 interacts synergistically... Synthetic lethal interactions... LITERATURE CITED

Groucho functions as a general corepressor by modulating chromatin structure and has a widespread role in many developmental processes. Here we show that Groucho may also interact with the basal transcriptional machinery. Mutations in Caenorhabditis elegans groucho interact with mutations in components of the transcriptional Mediator complex, resulting in synthetic lethality and loss of male sensory neurons.

TRANSCRIPTION initiation in eukaryotes requires the recruitment and assembly of a multifactor complex on the promoter, including chromatin remodeling factors, gene-specific DNA-binding proteins, general transcription factors (TAFs), the Mediator complex, and core RNA polymerase (BJORKLUND et al. 1999 ). The Mediator complex is one component of the holoenzyme that is thought to contain targets of regulatory factors (CARLSON 1997 ). How the transcription initiation apparatus integrates multiple inputs to specify gene expression during cell fate determination in multicellular development is a major question in developmental biology.

The postembryonic development of Caenorhabditis elegans male rays provides an opportunity to study the problem of how regulation of a transcriptional cascade leads to the differentiation of defined cell types at predetermined sites in the body. There are nine pairs of rays in the adult male tail (Fig 1A and Fig B), which collectively develop from three bilateral pairs of embryonic seam cells, V5, V6, and T. Male-specific postembryonic proliferation of V6 initiates with expression of the caudal homolog pal-1, which acts cell autonomously to turn on the expression of Hox gene mab-5 (HUNTER et al. 1999 ). MAB-5 in turn directly or indirectly activates the expression of another Hox gene egl-5 and bHLH protein lin-32 (EMMONS 1999 ; FERREIRA et al. 1999 ).

View larger version (76K): In this window In a new window Download PPT slide   Figure 1. An unc-37(0) male has a Pal phenotype. (A) Nomarski photomicrograph of a wild-type adult male, lateral view. Each side has nine rays. Ray 1 is derived from seam cell V5, rays 2–6 are derived from seam cell V6, rays 7–9 are derived from seam cell T. (B) Expression of a pkd-2::gfp reporter gene in wild-type male rays. pkd-2 encodes the homolog of human polycystic kidney disease gene (BARR and STERNBERG 1999 ) and is expressed in all nine rays as well as ray axons (L. JIA and S. W. EMMONS, unpublished data). The reporter is expressed in 10% of ray 6 in the integrated line used in this work [EM773: bxIs14(pkd-2::gfp, pha-1) was derived from PS32- 28A by integration of the transgene]. (C) V6 produces alae instead of rays in an unc-37 null (unc-37[wd17dm wd22]) male. Alae are absent both anterior and posterior of V6, suggesting that rays from V5 and T are still produced, though they are not visible due to abnormal tail morphogenesis. (D) Expression of a pkd-2::gfp reporter gene in an unc-37 null (unc-37[wd17dm wd22]) male. The reporter shows that V6 rays are missing whereas V5 and T rays are still generated.

In wild type, pal-1 expression in V6 requires the function of a cis-regulatory element lying within an intron (ZHANG and EMMONS 2000 ). We showed previously that pal-1 can also be activated by an alternate pathway that under normal circumstances is repressed by two proteins, SOP-1 and SOP-3, both putative components of the transcriptional Mediator complex. pal-1 expression in V6 is blocked if the cis-regulatory element is mutated [in the mutant pal-1(e2091)]. Expression can be restored via the alternate pathway if the activity of either SOP-1 or SOP-3 is reduced or eliminated (ZHANG and EMMONS 2000 , ZHANG and EMMONS 2001 ).

Unlike the normal pathway, the alternate pathway is stimulated by bar-1, which encodes a ß-catenin homolog (EISENMANN et al. 1998 ; ZHANG and EMMONS 2000 , ZHANG and EMMONS 2001 ). ß-Catenin is a signal transduction component of the Wnt pathway that acts together with a high mobility group DNA-binding protein of the TCF/LEF family (POP-1 in C. elegans) to promote gene activation (KORSWAGEN et al. 2000 ; POLAKIS 2000 ). Mutations in bar-1 have no effect on ray development in an otherwise wild-type genetic background. Stimulation of pal-1(e2091) gene activity by bar-1 in sop-1 and sop-3 mutants suggests that sop-1 and sop-3 act in part by blocking action of the Wnt pathway under inappropriate circumstances.

In Drosophila and vertebrates, negative gene regulation, including negative regulation by the Wnt pathway, often requires proteins of the Groucho/transducin-like Enhancer of split (Gro/TLE) family, which act as corepressors (CAVALLO et al. 1998 ; FISHER and CAUDY 1998 ; LEVANON et al. 1998 ; ROOSE et al. 1998 ; MANNERVIK et al. 1999 ; CHEN and COUREY 2000 ). A similar corepressor function has been demonstrated for the C. elegans groucho homolog, unc-37 (PFLUGRAD et al. 1997 ). We therefore examined the effects of unc-37 mutations on ray development, alone and in combination with other mutations, to determine whether it played a role in negative regulation, possibly by the Wnt pathway.

	Positive requirement for unc-37 in expression of pal-1 activity

TOP ABSTRACT Positive requirement for unc-37... Positive requirement for unc-37... unc-37 interacts synergistically... Synthetic lethal interactions... LITERATURE CITED

unc-37 has one or more essential zygotic functions during C. elegans development; a large fraction of the homozygous null progeny of an unc-37(0/+) mutant die as embryos or larvae (PFLUGRAD et al. 1997 ). We examined the tails of adult male escapers of this embryonic and larval lethality and found that they had a Pal phenotype (Table 1, line 2; Fig 1C and Fig D). In Pal phenotype males, rays are replaced by posterior alae as a result of a cell fate transformation in which the posterior seam cell V6 has taken the fate appropriate to anterior analogs and has generated cuticular ridges called alae instead of rays (WARING and KENYON 1990 ). pal-1 and the Hox gene mab-5 are both required for the rays vs. alae cell fate choice (KENYON 1986 ). In mab-5 mutants, the rays descended from two seam cells, V5 (ray 1) and V6 (rays 2–6), are affected and transformed to alae, whereas in the pal-1(e2091) mutant, only those rays descended from V6 are transformed to alae (Table 1, line 3). In unc-37(0) mutants, ray 1 was still generated, and rays 7–9, descended from T, were also unaffected (Fig 1C and Fig D). This suggested that the Pal phenotype in unc-37(0) escapers was the specific result of lack of pal-1 activity in V6 (HUNTER et al. 1999 ).

In this experiment, the alternate pathway is presumptively blocked by the wild-type activities of SOP-1 and SOP-3. Thus pal-1 activation via the normal pathway (requiring the intronic enhancer) requires the function of unc-37. Since neither pathway is active, we also conclude that unc-37 is not necessary for repression of the alternate pathway. Thus UNC-37 is not acting as a cofactor of the Wnt pathway in maintaining repression of the alternate pathway.

	Positive requirement for unc-37 in activation of pal-1 by an alternate pathway

TOP ABSTRACT Positive requirement for unc-37... Positive requirement for unc-37... unc-37 interacts synergistically... Synthetic lethal interactions... LITERATURE CITED

Not only is UNC-37 not necessary to block pal-1 activation via the alternate pathway, it is positively required for generation of pal-1 activity via this pathway, just as it is for generation of pal-1 activity via the normal pathway. To address this issue, we used the viable unc-37(e262) allele (a missense mutation), because null mutations in unc-37 cause severe lethality. We found that, as concluded above, not only was unc-37 not required to repress the alternate pathway, that is, unc-37(e262) was not a pal-1(e2091) suppressor (Table 1, line 5), but the frequency of animals with posterior alae increased rather than decreased when unc-37(e262) was introduced into pal-1(e2091); sop-1 or sop-3; pal-1 (e2091) backgrounds (Table 1, lines 6–9). Thus unc-37 is required for generation of rays via the alternate pathway as it is for the normal pathway.

	unc-37 interacts synergistically with components of the Mediator complex at one or more later steps to promote ray development

TOP ABSTRACT Positive requirement for unc-37... Positive requirement for unc-37... unc-37 interacts synergistically... Synthetic lethal interactions... LITERATURE CITED

Since unc-37(e262) alone had little effect on activation of the V6 ray developmental program in a pal-1(+) background [none of the animals are Pal (Table 1, line 4)], it was possible to use this mutation to test whether unc-37 interacted synergistically with Mediator components during later steps of the ray transcription factor cascade. The conclusion that unc-37 was required at some later step in ray generation was suggested by the observation that a small percentage of V6 rays was missing in unc-37(e262) males (Table 1, line 4). This percentage was greatly increased by mutations or RNAi of sop-1 (Table 1, lines 16 and 23), sop-3 (Table 1, line 17), or another known component of the Mediator complex, sur-2 (Table 1, line 18). SUR-2 interacts with transcription factors targeted by the Ras/MAPK pathway (SINGH and HAN 1995 ; BOYER et al. 1999 ). sur-2 does not function in the same pathway as sop-1 and sop-3 in regulation of pal-1, since a mutation in sur-2 cannot suppress the Pal phenotype of pal-1(e2091) (ZHANG and EMMONS 2001 ). Whereas singly, mutation or RNAi of each of these Mediator genes resulted in a small percentage of V6 ray loss similar to unc-37(e262) (Table 1, lines 10–15), indicating a weak positive requirement for ray generation, in combination with unc-37(e262) there was extensive ray loss (Table 1, lines 16–18).

All the mutations tested in combinations in these experiments are nonnull. Synergistic interaction suggested that these genes could act in a single pathway to promote ray development. Alternatively, they might act in parallel pathways with additive effects. Consistent with either interpretation, sop-1, sop-3, and sur-2 mutations also resulted in extensive synthetic V6 ray loss in combinations with each other (Table 1, lines 19–21). Absence of an effect on generation of rays 1 and 7–9 indicated that unc-37, sop-1, sop-3, and sur-2 were required specifically for one or more steps of the transcription factor cascade in the V6 cell lineage.

	Synthetic lethal interactions between unc-37 and components of the Mediator complex

TOP ABSTRACT Positive requirement for unc-37... Positive requirement for unc-37... unc-37 interacts synergistically... Synthetic lethal interactions... LITERATURE CITED

unc-37(e262) interacted with sop-1, sop-3, and sur-2 not only for generation of V6 rays, but also for viability. This indicated that the putative pathway or pathways involving these genes were required for one or more essential steps during embryogenesis or postembryonic development, as well as for ray development. Whereas single alleles had little effect on viability, in combination with unc-37(e262) there was a large lethal sector (Table 1, lines 16–18 and 22–24). Once again consistent with all these genes acting either in a single pathway or in parallel pathways with a common effect, double mutants among sop-1, sop-3, and sur-2 also showed synthetic lethality, as we demonstrated previously (ZHANG and EMMONS 2001 ; Table 1, lines 19 and 25–27).

In Drosophila and vertebrates, Groucho functions as a corepressor for Hairy family proteins (FISHER and CAUDY 1998 ; MANNERVIK et al. 1999 ). lin-22 encodes the C. elegans Hairy/E(spl) homolog. In contrast to unc-37, mutations in lin-22 suppress the Pal phenotype of pal-1(e2091) (WARING and KENYON 1990 ; WRISCHNIK and KENYON 1997 ). Thus, unc-37 is not a lin-22 corepressor in regulation of pal-1. Consistent with this conclusion, LIN-22 does not contain the WRPW (Trp, Arg, Prv, Trp) domain that mediates protein-protein interactions between Hairy family proteins and Groucho (WRISCHNIK and KENYON 1997 ). Unlike sop-1, sop-3, and sur-2, the V6 rays in lin-22 or pal-1; lin-22 mutants are not affected by introduction of unc-37(e262) mutation (Table 1, lines 28 and 29), suggesting that lin-22 acts downstream of sop-1 and sop-3 to block ray development or blocks an independent activation pathway. unc-37 might promote V6 ray development by repressing lin-22 expression or function in V6. In activating an independent pathway, lin-22 mutation might result in activation of a ray developmental program normally expressed only in V5. In support of this conjecture, in lin-22 mutants, seam cells V1–V4 and V6 express the same cell lineage and cell fates as V5 (HORVITZ et al. 1983 ).

UNC-37, like other proteins of the Gro/TLE family, has been suggested to act as a corepressor. It directly interacts with homeodomain-containing protein UNC-4 to specify the identity of VA-type motor neurons by preventing the expression of VB-type motor neuron-specific genes (WINNIER et al. 1999 ). Here we demonstrated that unc-37 is required for the activation of the transcription factor cascade leading to the generation of V6 rays and this requirement is sensitized by mutations in the components of the Mediator complex sop-1, sop-3, and sur-2. This observation would be explained if, in the ray pathway, unc-37 functions as a coactivator rather than as a corepressor. Alternatively, unc-37 might act to repress the expression of a pal-1 repressor. One candidate repressor gene, mentioned above, is lin-22. An alternate candidate repressor is POP-1, the C. elegans TCF/LEF homolog. POP-1 might repress pal-1 in V6, and UNC-37 might function to repress expression of POP-1. Later in the ray cell lineage, a higher level of POP-1 in the unc-37 background might result in a more stringent requirement for SOP-1 and SOP-3, acting as positive activators, thus accounting for the later synergistic interaction between mutations in these genes and unc-37.

Given the structural and functional similarity between Groucho and yeast transcription corepressor Tup1, it is likely that Groucho and Tup1 function through common mechanisms (FISHER and CAUDY 1998 ). Tup1 interacts directly with histones H3, H4, and histone deacetylase (EDMONDSON et al. 1996 ; WATSON et al. 2000 ). In addition, at least seven genes encoding Mediator proteins, including Srb8, -9, -10, -11, Sin4, Rgr1, and Rox3, are genetically required for Tup1 to repress gene expression (WAHI et al. 1998 ). Therefore, Tup1 represses transcription both by modifying chromatin structure and by interacting with the basal transcriptional machinery. Drosophila Groucho also directly interacts with histones H1, H3, and histone deacetylase Rpd3. Mutations in groucho and rpd3 result in synergistic effects on embryonic viability (CHEN et al. 1999 ). Therefore, Groucho may repress transcription by modulating local chromatin structure. Additional unknown mechanisms have been implicated for the repression mediated by Groucho (CHEN et al. 1999 ). The data presented here provide genetic evidence that UNC-37/Groucho also interacts with the basal transcriptional machinery to regulate gene activity.

	FOOTNOTES

¹ Present address: MGH Cancer Center, Rm. 7119, Bldg. 149, 13th St., Charlestown, MA 02129.

	ACKNOWLEDGMENTS

We thank D. Miller for unc-37(wd17dm wd22) and M. Barr and P. W. Sternberg for the pkd-2::gfp transgene. We thank L. Jia for bxIs14 and C. Smith and J. Dimele for technical assistance. This work was supported by a grant from the National Institutes of Health (R01 GM39353). S.W.E. is the Siegfried Ullmann Professor of Molecular Genetics.

Manuscript received August 9, 2001; Accepted for publication November 21, 2001.

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