The C. elegans gene
egl-38 encodes a Pax transcription factor that contains a DNA-binding paired domain that interacts with a 20bp DNA fragment (1, 2).
egl-38 plays an important role in the development of the hindgut, the egg-laying system, and the spicules of the male tail. Genetic analysis showed that mutations in
egl-38 resulted in a discrete set of tissue-preferential defects in the development of C. elegans. Four non-null alleles of
egl-38:
n578,
sy287,
sy294, and
gu22, disrupted different functions in different tissues, suggesting that EGL-38 regulates different target genes in different tissues. Each of these four alleles results from an amino acid substitution in the DNA-binding paired domain of the EGL-38. To understand how
egl-38 regulates different genes in different tissues, we have characterized a gene,
lin-48, which encodes a C2H2 zinc-finger protein similar to Drosophila OVO. Genetic and expression data show that
lin-48 is a direct, tissue-specific target for EGL-38 in hindgut cells.
lin-48 requires EGL-38 for its expression in the hindgut. Deletion analysis identified two important regulatory elements (
lre1 and
lre2) in the
lin-48 promoter that are necessary for the expression of
lin-48. EGL-38 appeared to bind to only one of these two elements (
lre2) in vitro. Here, we report the in vitro analysis to test the DNA binding to
lre2 by the proteins encoded by the four non-null alleles of
egl-38. We over-expressed the DNA-binding domain of these four
egl-38 alleles in E. coli and tested their DNA-binding ability to
lre2 by electrophoretic mobility shift assay. The results showed that EGL-38 (
n578) and EGL-38 (
gu22) retained high DNA-binding affinity to this hindgut specific element while EGL-38 (
sy287) showed much less affinity, and EGL-38 (
sy294) had no detectable binding. These in vitro results are consistent with the in vivo analysis of
egl-38 alleles, which showed that among these mutants,
sy294 disrupted hindgut development to the greatest extent while
n578 is weakest. These data also provide direct in vitro evidence that alteration in DNA-binding affinity is one way to specifically affect
egl-38 activity. We speculate that DNA elements from target genes important in other tissues would likewise exhibit different affinities for the different mutant proteins.1. Czerny et al. (1993). Genes Dev. 7, 2048-2061; 2. Xu et al. (1999). Genes Dev. 13, 1263-1275