(CT)N.(GA)N REPEATS AND HEAT-SHOCK ELEMENTS HAVE DISTINCT ROLES IN CHROMATIN STRUCTURE AND TRANSCRIPTIONAL ACTIVATION OF THE DROSOPHILA-HSP26 GENE

Previous analysis of the hsp26 gene of Drosophila melanogaster has sho wn that in addition to the TATA box and the proximal and distal heat s hock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT). repeats at -135 to -85 is r equired for full heat shock inducibility (R. L. Glaser, G. H. Thomas, E. S. Siegfried, S. C. R. Elgin, and J. T. Lis, J. Mol. Biol. 211:751- 761, 1990). This (CT)n element appears to contribute to formation of t he wild-type chromatin structure of hsp26, an organized nucleosome arr ay that leaves the HSEs in nucleosome-free, DNase 1-hypersensitive (DH ) sites (Q. Lu, L. L. Wallrath, B. D. Allan, R. L. Glaser, J. T. Lis, and S. C. R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of th e sequences upstream of hsp26 has revealed an additional (CT). element at -347 to -341, adjacent to the distal HSE. We have analyzed the con tribution of this distal (CT). element (-347 to -341), the proximal (C T)n element (-135 to -85), and the two HSEs both to the formation of t he chromatin structure and to heat shock inducibility. hsp26 construct s containing site-directed mutations, deletions, substitutions, or rea rrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin struc ture of the transgenes was analyzed (prior to gene activation) by DNas e I or restriction enzyme treatment of isolated nuclei, and heat-induc ible expression was monitored by measuring beta-galactosidase activity . The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin s tructure and heat-inducible expression of the transgenes. These (CT), repeats are associated with a nonhistone protein(s) in vivo and are bo und by a purified Drosophila protein, the GAGA factor, in vitro. In co ntrast, the HSEs are required for heat-inducible expression but play o nly a minor role in establishing the chromatin structure of the transg enes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, a n abundant protein factor required for normal expression of many Droso phila genes, and heat shock factor, a specific transcription factor ac tivated upon heat shock, play distinct roles in gene regulation: the G AGA factor establishes and/or maintains the DH sites prior to heat sho ck induction, while the activated heat shock factor recognizes and bin ds HSEs located within the DH sites to trigger transcription.