Pax transcription factors play an important role in organ development in all animals. Most Pax proteins function in the development of more than one organ or cell type. However, it is not well characterized how they can mediate different effects in different cell types. To better understand target gene selection by Pax proteins, we have characterized the function of the C. elegans Pax gene
egl-38 .
egl-3 8 plays an important role in the development of several C. elegans organs, including the egg-laying system, the excretory system, the male spicules, and the hindgut. Genetic analysis showed that four non-null alleles of
egl-38 (
n578 ,
sy287 ,
sy294 , and
gu22 ) disrupt different functions in different tissues, suggesting that EGL-38 regulates different target genes in different tissues. Each of these mutant alleles corresponds to an amino acid change in the DNA-binding domain of the EGL-38. In a previous study, we characterized
lin-48 , a direct, tissue-specific target for EGL-38 in hindgut cells, and identified one regulatory element in the
lin-48 promoter that binds EGL-38 (Johnson et al, 2001). We have used this EGL-38 binding sequence (termed
lre2) to understand the different properties of the
egl-38 tissue preferential alleles. Using EMSA analysis, we have demonstrated that
n578 and
gu22 proteins retained the DNA-binding affinity to
lre2, while
sy287 protein showed much lower affinity and
sy294 showed no detectable binding. These in vitro results are consistent with in vivo analysis of
egl-38 alleles, which showed that among these mutants,
sy294 disrupted hindgut development and
lin-48::gfp transcription to the greatest extent, whereas
n578 had little effect. These in vitro data also indicate that the mutations affect
egl-38 activity in the hindgut by altering EGL-38 DNA-binding properties. Using the in vitro random DNA oligomer selection method, we identified the DNA sequences bound by the EGL-38 wild type ( wt ) and mutant proteins. The mutant
sy294 or
n578 protein was found to select a single sequence each, while, like wt ,
sy287 and
gu22 proteins selected multiple sequences. The consensus sequence selected by wt protein is similar to that selected by Pax2 protein (Xu et al, 1995). All of the mutant proteins select sequences that differ from those selected by wt in the nucleotides contacted by ß-hairpin and linker region of the Pax DNA binding domain. To test the in vivo activity associated with these selected sequences, we used modified
lin-48::gfp reporter genes. We replaced
lre2 with the consensus sequence selected by wt protein or the sequence selected by
sy294 or
n578 protein. In animals bearing the modified transgenes,
lin-48::gfp was expressed in the hindgut cells but at levels much lower than transgenes bearing the endogenous
lre2. This result indicates that DNA binding affinity is just one feature that determines the sequence of a Pax-response element. These results provide additional information to better understand the interaction between Pax family proteins and DNA. Johnson et al. (2001). Development 128, 2857-2865. Xu et al. (1995). Cell 8, 639-650.