Worm Breeder's Gazette 11(2): 22
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
With the eventual goal of setting up a general enhancer trap assay similar to that being used in Drosophila, we have been identifying segments of the genome which have tissue specific enhancer function. In a previous newsletter we reported the identification five random genomic segments which could enhance a myo-2-lacZ fusion outside of pharyngeal muscle. In each case the induced expression was in other muscle cells, leading to the suggestion that response of the myo-2 promoter to different enhancers was limited to muscle. We have used several of these random segments plus some of the identified myosin enhancer segments with different promoters to determine patterns of expression. In general these experiments have been done by staining the F1 after injection. [See Figure 1] Although it is difficult to explain this all with one simple model, several conclusions can be drawn. First the muscle promoters seem indeed to have some underlying muscle specificity, since they respond specifically in muscle in the vicinity of enhancers which can act in other tissues. Second, the internal enhancer present in unc-54 is capable of specifying proper tissue specificity even in the absence of a muscle specific promoter, since that enhancer can specifically enhance glp-1- -gal fusions (and unc-6- -gal fusions) specifically in body wall muscle. The P element and HSP70 promoter elements fail to respond to the unc-54 internal enhancer, and it is worth noting that these are both standard 'TATA' box containing promoters. An evident conclusion is that different promoters and enhancers can interact to generate a variety of specificities. In thinking about this it is worth keeping in mind that the 'enhancer' segments from random genomic DNA could each contain several different enhancer segments, and that the promoter segments too could include elements fortuitously responsive to different signals. The failure of the P element promoter to respond to the unc-54 enhancer is actually a blessing in that these constructs can be co- transformed with the unc-54 enhancer containing antisense 'twitcher plasmids' without spurious induction in body wall muscle. Most but not all of the twitching lines derived with an antisense plasmid containing the unc-54 enhancer mixed with glp-1 or myosin derived enhancer test constructs give strong body wall muscle expression of - gal.The constructs that show limited numbers of larval gut cells staining have reproducible patterns, indicating that some feature distinguishes posterior, anterior and middle gut cells in young larvae. Current view of myosin regulation We've previously described sequences both upstream of and inside the unc-54 gene which are independently capable of specifying body wall muscle specific expression. We have now narrowed down these sequences Internal: An 82 bp minimal region sufficient for body wall muscIe specific enhancer function has been defined. Within this region are several short direct and inverted repeats. At the 5' end is a palindrome TCAATTGA. A deletion of a single A residue (TCATTGA) completely abolishes activity. The palindrome is not sufficient for enhancer activity, with sequences at least 24bp downstream also required for enhancer function. Upstream: Sequences between -169 and +12 are sufficient to promote body wall muscle specific expression, while sequences from -138 to +12 are insufficient. The region upstream of -138 can be inverted with retention of activity. This region is functionally interchangable ( although weaker) than the internal enhancer, and probably represents a second tissue specific albeit weak enhancer. At the 5' boundry of the upstream element is a different palindrome from that above: 'CTACGCGTAG'. A clustered point mutation eliminating this palindrome eliminates specific expression. Consistent with the suggestion that the two palindromes might interact with different factors, the relative activities of the two elements in different body muscle subclasses appears to be different, with the upstream region having relatively poorer activity in vulval muscles. Promoter sequences downstream of -138, although not sufficient for any expression, seem to control the start point of the RNA based on 5' mapping with and without upstream and internal enhancer sequences. Mapping of the myo-2 promoter has revealed an upstream element which stimulates expression but does not act as a tissue specific enhancer, and a segment near the start site (minimal size now 254 bp) which acts both as a weak pharyngeal specific promoter and a pharyngeal enhancer ( when assayed on the myo-3 gene).