Transcription of Drosophila small hsp-tk hybrid genes is induced by heat shock and by ecdysterone in transfected Drosophila cells.

Abstract

Hybrid genes containing the 5' region of Drosophila heat shock protein (hsp) genes, ligated to the herpesvirus thymidine kinase (tk) gene, were transfected into Drosophila line S3 cells. Constructions containing sequences upstream from hsp7O, or from any of the small hsp genes, showed heat-inducible tk expression. Ecdysterone-inducible tk expression was seen only in transfections with small hsp-tk hybrid genes. Plasmids containing a synthetic "heat shock consensus sequence" or a deletion of the hsp23 upstream region were totally inactive in transfection studies.The small heat shock protein (s-hsp) genes of Drosophila, designated hsp22, hsp23, hsp26, and hsp27, are expressed in response to high temperature shock (1) and at two points in development. During pupation, all four of the genes are transcribed and translated in imaginal discs (2, 3). During oogenesis, both hsp26 and hsp27 are expressed in ovarian nurse cells (4). The expression of the four s-hsp genes can also be induced in vitro in both established tissue culture cell lines and in immature imaginal discs (2, 5) by treatment with physiological concentrations of the molting hormone, ecdysterone.Our finding of regulated gene expression by two independently acting stimuli raises several questions at the molecular level. Among these is whether recognition and binding of the ecdysterone-receptor complex (6, 7) and of the endogenous factor(s) implicated in heat shock induction (8, 9) occur at the same upstream regulatory site.We have recently developed a functional assay for analyzing s-hsp gene regulation in a homologous transient expression system (10). Initial studies, using a cloned gene that specifies an electrophoretic hsp22 gene variant, demonstrated that the transfected gene could rapidly acquire the normal pattern of heat shock regulation. We now describe experiments designed to examine the regulated expression of hybrid genes containing upstream promoter regions from each of the s-hsp genes ligated to the herpesvirus thymidine kinase (tk) gene. We find that these hybrid genes acquire both heat shock induction and ecdysterone induction. Preliminary results on the nature and location of these regulatory sites are discussed. METHODS AND MATERIALSCell Culture and Transfection. Drosophila line S3 cells were cultured in Schneider's medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) at 25°C. Ecdysterone-insensitive line F6 cells were grown in ZH1 medium (11). For transfection, 37 ,tg of CsCl-purified plasmid DNA (12) was added as a CaCl2 coprecipitate (13) to each 75-cm2 flask of confluent cells in 10 ml of medium. For heat shock, the flasks were floated in a 370C water bath for 1 hr after a 24-hr transfection. In hormone studies, 6 hr after transfection began, ecdysterone (Calbiochem) was added to 1 uM final concentration, and the flasks were cultured for an additional 24 hr at 250C.Slot Blot Analysis. Total RNA was recovered from cells by using the guanidine-HCl/CsCl method (14) with the addition of a DNase digestion step (13). Aliqu...

Ecdysterone regulatory elements function as both transcriptional activators and repressors.

“…First, ecdysterone induction is based on an increased level of new transcription. This is true for expression of both endogenous s-hsp genes (55) and a reporter gene put under the regulatory control of an s-hsp gene promoter (32,33). There is no evidence to suggest that ecdysterone alters the stability of endogenous s-hsp transcripts (55), or mRNAs transcribed from reporter genes, such as the chloramphenicol acetyl * Corresponding author.…”

“…transferase gene (CAT) or thymidine kinase gene (tk) (32,33). In addition, the response is quite specific.…”

“…In both larval imaginal disks (4, 21) and continuous cell lines (20,(31)(32)(33), s-hsp gene expression can also be induced by treatment with physiological doses of the moulting hormone, ecdysterone. Functional tests involving the use of P-element transformation, or tratisient transfection, have shown that the cis-acting DNA regulatory elements involved in heat-induced expression and in ecdysterone-induced expression are physically separate and nonoverlapping (1, 3, 6, 17-19, 22, 26, 29, 30, 36, 37, 40, 41).…”

Sequences involved in temperature and ecdysterone-induced transcription are located in separate regions of a Drosophila melanogaster heat shock gene.

“…All four of these developmentally regulated "small heat shock genes", along with a fifth developmentally regulated non-heat shock gene (R), are located within a 13-kilobase region on the left arm of chromosome 3 at 67B1 (10,51). Workers in other laboratories have recently published transcriptional regulation studies of these developmentally regulated heat shock genes in both heterologous monkey cells (1) and D. melanogaster cells (34).…”

The ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene react very differently to perturbations of DNA sequence.

“…These studies have generally substantiated the importance of the heat shock consensus sequence (24), which binds the heat shock transcription factor both in vivo (35) and in vitro (22). The heat-induced regulation of the Drosophila small heat shock genes (hsp22, -23, and -27) has been studied in Xenopus oocytes (20), monkey cells (2,26), Drosophila cells (17,21), and germ line-transformed flies (5,(12)(13)(14)23 (3). It has been shown that a positioned array of nucleosomes occupies the coding but not the intergenic regions of these genes when in a transcriptionally inactive state.…”