We are characterizing the
myo-2 enhancer to identify factors controlling gene expression in pharyngeal muscle. To assay enhancer activity, DNA fragments cloned upstream of a lacZ fusion to the
myo-3 or
glp-1 promoter are tested for the ability to induce -gal in pharyngeal muscle. The
myo-2 enhancer is pharyngeal muscle-specific and is located within a 0.4kb NsiI-HpaI fragment approximately 200bp upstream of the transcriptional start site. It can be divided into 3 fragments (A, B, and C) which are independently incapable of inducing pharyngeal expression from a
myo-3 ::lacZfusion. However, any combination of two or more of these fragments (including duplications of any fragment) functions as an enhancer. [See Figure 1] As a working model, we propose fragments A, B, and C each contain a discrete enhancer element, and two or more elements are required for transcriptional enhancement. These elements are not functionally equivalent, however. We observe distinct patterns of pharyngeal muscle cell types expressing -gal when the entire
myo-2 enhancer or the duplicated enhancer elements are assayed upstream of a
glp-1 ::lacZfusion. The intact
myo-2 enhancer induces expression in the major pharyngeal muscle cell groups (
m3 -
m7 ),but not in the minor cell groups (
m1 and
m2 ).Although the duplicated B element induces expression in a similar pattern of cells, -gal expression appears reduced in
m5 and absent in
m6 .In contrast, the duplicated C element gives expression in all the pharyngeal muscle cell types, including
m1 and
m2 .Finally, the duplicated A element is unable to enhance transcription from the
glp-1 ::lacZfusion. These results suggest the
myo-2 enhancer elements contain binding sites for different transcriptional activators (or repressors), and the factors binding the B and C elements may be present in different sets of pharyngeal muscle cells. Furthermore, the observation that a functional enhancer requires multiple enhancer elements suggests the combination of factors present in any pharyngeal muscle cell type might determine the activity of the
myo-2 enhancer. Consistent with this hypothesis, an enhancer containing the B and C elements is most active only in cells which express from both the duplicated B element and the duplicated C element (
m3 ,
m4 ,
m5 ,and
m7 ). Following protocols from Ginny Stroeher and Jim McGhee, we have begun examining worm nuclear extracts for proteins binding the
myo-2 enhancer. Multiple retarded complexes are seen using the B element or the C element as a probe in gel retardation assays. These binding activities are specific for either the B or the C element. We are currently defining these binding sites to determine their role in
myo-2 expression.