The NK-2 family homeobox gene,
ceh-22, is an important regulator of gene expression in C. elegans pharyngeal muscle, and it is the first gene known to be expressed as cells commit to a pharyngeal muscle fate. To identify mechanisms controlling early steps in pharyngeal muscle differentiation, we are examining how
ceh-22 gene expression is regulated. A
ceh-22::lacZ fusion containing a 1.9 kb promoter fragment is expressed identically to the endogenous
ceh-22 gene in a subset of pharyngeal muscles beginning at the bean stage of embryogenesis. Within this promoter fragment are two transcriptional enhancers, designated the distal and proximal enhancers, that display distinct temporal and spatial patterns of activity when assayed in transgenic lines. Distal enhancer activity initiates as early as the bean stage, and it is detected in both muscle and non-muscle cells in the pharynx, as well as in a subset of non-pharyngeal tissues. In contrast, proximal enhancer activity initiates later, beginning at the 1-1/2 fold stage, but it is detected exclusively in the pharyngeal muscles expressing the endogenous
ceh-22 gene. Here we describe our characterization of the proximal enhancer. The proximal enhancer contains multiple positively and negatively acting subelements. One region near the 5 end of the proximal enhancer contributes substantially to activity. Within this region we have identified two separate segments that can enhance reporter gene expression specifically in
ceh-22 expressing pharyngeal muscles, similarly to the full-length proximal enhancer. One of these segments, termed PE39, binds CEH-22 in vitro, suggesting it may be an autoregulatory site. Consistent with this hypothesis, we have shown the CEH-22 binding site is essential for PE39 enhancer activity in vivo. Moreover, the PE39 enhancer can be activated in body wall muscles by ectopic
ceh-22 expression, and its activity is significantly reduced in a
ceh-22 mutant. In addition to the CEH-22 site, PE39 appears to contain sites for two additional factors that cooperate with CEH-22. We are currently using the PE39 sequence to identify these factors.