To understand the molecular mechanisms underlying organ development, we have identified and characterized several genes important for organogenesis of the C. elegans hindgut (Chamberlin et al., 1999, Genetics 153, 731). One of these genes (
egl-38) encodes a Pax transcription factor, and is important for proper development of several structures, including the hindgut and the egg-laying system (Chamberlin et al., 1997, Devt. 124, 3919). Another gene identified in our screen (
lin-48) encodes an OVO-related zinc finger protein. Genetic and expression data are consistent with a model that
lin-48 is a hindgut-specific target for EGL-38. Specifically, we have found mutations in
lin-48 disrupt hindgut development but not egg-laying system development, and
lin-48::gfp transgenes are expressed in hindgut cells, but not cells of the egg-laying system. This
lin-48::gfp expression in the hindgut is significantly reduced in
egl-38 mutants, suggesting
lin-48 is genetically downstream of
egl-38. We have begun to characterize the relationship between EGL-38 and
lin-48 in detail. A set of different
egl-38 mutant alleles have allowed us to dissect different functions for EGL-38 in regulating
lin-48 expression. Animals bearing a strong (but non-null) mutation,
egl-38(
sy294), show reduced
lin-48::gfp expression in hindgut cells. In contrast, animals bearing a weaker mutation,
egl-38(
sy287), express
lin-48::gfp normally. We have identified a potential EGL-38 binding element in the
lin-48 promoter (termed Pax binding site 1 or PBS1). Transgenes in which this site is mutated are expressed normally in wild-type animals, but exhibit reduced expression in
egl-38(
sy287) mutants. We interpret these results to suggest there are (at least) two
egl-38-responsive elements in the
lin-48 promoter: PBS1, and a second one which is
egl-38(
sy287)-sensitive. In this model, severely compromised EGL-38 (as in
egl-38(
sy294) mutants) can act through neither site. In
egl-38(
sy287) mutants, EGL-38 can act only through PBS1 and not the second site(s). We are interested in the nature of the
egl-38(
sy287)-sensitive element in the
lin-48 promoter. Based on the following data, we propose a model in which this element binds a heterodimer of EGL-38 and an Ets transcription factor. The
egl-38(
sy287) mutation results in a serine substitution at a glycine residue conserved among all Pax proteins (G9). This mutation affects the the b-hairpin sub-domain of the paired (DNA binding) domain. In vitro, the b-hairpin mediates interaction between mammalian Pax and Ets transcription factors, and the resulting Pax:Ets complexes bind unique promoter elements (Fitzsimmons et al., 1996, G&D 10, 2198; Wheat et al., 1999, MCB 19, 2231). Preliminary results suggest the Ets protein LIN-1 may be an in vivo partner for EGL-38.
lin-1(
e1777) animals show reduced hindgut expression of
lin-48::gfp transgenes in which PBS1 is mutant. In other words,
lin-1 mutants exhibit
lin-48::gfp expression defects similar to those seen in
egl-38(
sy287) mutants. However, since
lin-48::gfp expression in
lin-1 mutants is not affected to the same extent as in
egl-38(
sy287) mutants, another Ets factor may also participate.