Ecdysterone induces the transcription of four heat-shock genes in Drosophila S3 cells and imaginal discs

DEVELOPMENTAL

BIOLOGY

93,

Ecdysterone

&%-507

(1982)

Induces the Transcription of Four Heat-Shock in Drosophila S3 Cells and lmaginal Discs

Genes

ROBERT C.IRELAND,* EDWARD BERGER,**~ KARL SIROTKIN,~; MARY ALICE YUND,$ DAVID OSTERBUR,$ AND JAMES FRISTROM$ *Biology

Department, Knoxville,

Dartmouth Tennessee

College, Hanover, New Hampshire 37996; and $ Genetics Department, Received

February

037,55,5; tDepartment of Microbiology, University of Tennessee, 7Jniversity of California, Berkeley, California 94704

26, 1982; accepted

in revised

form

June

14, 1982

Ecdysteronestimulates a 2- to 15-fold increase in the synthesis of four small heat-shock proteins (hsp) in Drosophila line S3 cells. This is accompanied by a rapid and coincident increase in the abundance of small hsp transcripts. A parallel in vivo situation is now described in imaginal discs isolated from pupariating larvae. In neither sytem are the high-molecular-weight heat-shock proteins, or their RNAs, induced by hormone. Nor does induction occur in variant cell lines which have acquired resistance to ecdysterone. Other genes within or contiguous to the small hsp cluster are not induced by either ecdysterone or high temperature. Induction by ecdysterone appears to be a primary hormone response. INTRODUCTION

Embryonic Drosophila cells in continuous culture exhibit a variety of responses when exposed to physiological doses of the steroid moulting hormone, ecdysterone. Of these the cessation of cell division occurs in all unselected lines (Courgeon, 1972a,b; Rosset, 1978; Wyss, 1976; Berger et al., 1978; Stevens et al., 1980), whereas other responses, including cell elongation, cell aggregation, or the induction of specific proteins or enzymes occur in only one or a few lines (Courgeon, 1972a,b; Cherbas et al., 1977, 1980a,b; Best-Belpomme et al., 1978; Berger et al., 1978, 1980; Berger and Wyss, 1980; Savakis et al., 1980). In the S3 line (Wyss, 1980) a temporal program of hormone responses has been described which includes the loss of mitotic activity, cell elongation, (Berger et al., 1978), and a remarkable pattern of acetylcholinesterase inactivation and reinduction (Berger and Wyss, 1980). In addition, a marked stimulation was found in the rate of synthesis of several polypeptides (Berger et al., 1980). A recent study (Ireland and Berger, 1982) has shown that four of the polypeptides whose synthesis is stimulated by ecdysterone are the four small heat-shock proteins (hsps) designated hsp 22, hsp 23, hsp 26, and hsp 27, encoded by four closely linked genes in the 67Bl region of the Drosophila polytene chromosome (Corces et al., 1980; Wadsworth et al., 1980 Craig and McCarthy, 1980; Voellmy et al., 1981). Several lines of evidence support the contention that small hsp induction by ecdysterone in S3 cells is not a stress response, but rather represents a bonafide and specific hormone-mediated event (Ireland and Berger, ’ To whom all correspondence

should be addressed.

1982). First, the induction does not occur in other independently derived hormone responsive cell lines, nor does it occur in S3 cells exposed to very low doses of hormone, to the solvent alone, or to high concentrations of mammalian steroids. Second, the high-molecularweight heat-shock protein genes, hsp 68 and hsp 70, are not activated, a distinction unique to this situation (Ashburner and Bonner, 1979). Finally, cell lines that have acquired resistance to the growth inhibition effect of ecdysterone, by mutation or by cell fusion, simultaneously lose small hsp inducibility by hormone, but retain the normal heat-shock response. In this report evidence is presented that the stimulation of small hsp synthesis by ecdysterone is accomplished by the rapid accumulation of small hsp transcripts. This accumulation does not require protein synthesis, but does depend upon the continuous presence of hormone, and the presence of ecdysterone receptor activity in the cells. In addition, we show that the expression of small hsp genes occurs as a normal developmental event in the imaginal discs of Drosophila entering puparium formation and that this expression is further enhanced by exogenous ecdysterone. Finally, we demonstrate that while the expression of small hsp genes is being stimulated by hormone, the transcription of genes in the midst, and flanking the hsp cluster, remains absent. We conclude that the domain of transcriptional regulation, in this case at least, appears to be the single gene. MATERIALSANDMETHODS Cell lines and imaginal

498 0012.1606/82/100498-10$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

discs. The cell lines used are

designated MDR, S3, MDER,

and the MDER/S3

fusion

Small

Heat-Shock

hybrid, F6. Their origin and culture conditions have been described previously (Berger et al., 1980; Berger and Wyss, 1980). Imaginal discs were mass isolated according to the procedure of Eugene and Fristrom (1978), and all incubations were done in Robb’s medium. Ecdysterone (Calbiochem) was prepared as a 2 X lo-” M aqueous solution and diluted in medium. Hormone incubations were done at 25°C in Falcon flasks. RNA extraction. Total RNA was prepared from untreated and ecdysterone-treated S3 cells using the guanidine hydrochloride procedure of Scott et al. (1979). Plasmid DNA preparation. Bacterial cells containing recombinant plasmids were grown in L broth containing ampicillin (100 pg/ml) to OD6,,,, = 0.5-0.6, and treated with chloramphenicol (0.2 mg/ml) overnight. The cells were harvested and washed by low-speed centrifugation, treated with lysozyme, and lysed in a buffer containing 12.5% sucrose, 25 mM Tris, pH 8.0, 20 mM EDTA, and 5% Triton X-100. Chromosomal DNA and cellular debris were pelleted at 40,000 rpm for 30 min and plasmid DNAs were separated from contaminating chromosomal DNA by banding in CsCl. Following dialysis to remove CsCl, the DNA was recovered by ethanol precipitation. Nick translation of plasmid DNAs. Plasmid DNAs were radiolabeled by a modification of the nick translation procedure of Rigby et al. (1977). Labeling was performed in 30-~1 reactions containing 50 mM Tris, pH 7.5, 10 mM MgC12, 50 pg/ml BSA, 10 mM DTT, 30 mM each dTTP, dGTP, dATP, and 100 PC1 [a-““P]dCTP (1 mCi/0.0017 mmole). Between 0.75 and 1.0 pugof plasmid DNA was incubated at room temperature for 1 min with 2.5 pg DNase I (Worthington) and subsequently at 14°C for 2 hr with 1.6 pg DNA polymerase I (BRL). Labeled DNA was recovered by Sephadex G-75 chromatography. Dot blot analysis. Dot blot hybridization was carried out according to the procedure of Thomas (1980) using undenatured RNA. Each hybridization reaction contained 2 X lo6 cpm of probe in 5 ml. Following autoradiography, in some cases, the amount of hybridized “‘P-DNA was determined by excising the spots and counting. Over the range of RNA concentrations used we found the amounts of 32P cpm hybridized to be linear. Using tritium-labeled, undenatured RNA we found 73% retention after the prehybridization, hybridization, and washes were completed. Glyoxalated RNA was retained at a level of 87%. Protein labeling, sional polyacrylamide

cell fractionation, gel electrophoresis.

and

two-dimen-

For protein labeling, cells were incubated in methionine-free medium supplemented with [35S]methionine at 100 pCi/ml (New England Nuclear). Cells were fractionated by homogenization in a buffer containing 1 M sucrose, 3.3 mM

Protein

499

Induction

CaC12, and 0.1% Triton X-100, and nuclei were pelleted by low-speed centrifugation. The supernatant fraction was recentrifuged at SOOOg,5 min, to remove nuclei and cell debris. The nuclear pellet obtained from the first spin was washed twice in homogenization buffer and pelleted through a cushion of 2.5 M sucrose, 3.3 mM MgC12. Conditions for two-dimensional electrophoresis have been described previously (Berger et al., 1980b). [“HI Ponasterone A binding. Ecdysterone receptor activity was determined by a modification of the [“Hlponasterone A binding assay of Yund et al. (1978). About 10s saline washed cells were swollen in 20 vol of hypotonic TES buffer (10 mM Tris, 1.5 mM EDTA, 7 mM dithiothreitol, pH 7.4) and homogenized on ice in a small ground glass homogenizer. The extract was brought to 0.3 M KCl, with 1.2 M KC1 in TES, and rehomogenized (five strokes). After a 2-hr centrifugation at 48,OOOg, at 4”C, the supernatant was recovered and dialyzed against TE (10 mM Tris, 1.5 mM EDTA, pH 7.4) for 1 hr. The dialysate was made 7 mM DTT and 150-111 aliquots were incubated with 3.2 mM [“Hlponasterone A (PNA, 37,500 cpm) in the presence or absence of 100-fold unlabeled PNA. After 16 hr at 094°C the mixture was passed over a Bio-Rad PlO column to separate the free [“HIPNA from that bound to the macromolecular fraction. Aliquots were counted and protein concentration in the lysate was determined. RESULTS

Small

hsp Message

Accumulation

The dot blot hybridization procedure of Thomas (1980) was used to analyze the composition of total RNA extracted from control and hormone-treated ( lop6 M ecdysterone) S3 cells. The four probes used were clones designated JlS3, JlPR3, JlB, and T6BH2 (Fig. 1) each containing a different small hsp gene inserted into pBR322. They were originally isolated as subclones from the Jl and T6 clones, described in Craig and McCarthy (1980), and were generously provided by Craig. Figure 2 summarizes the results for hsp 22, hsp 26, and hsp 27. Dot blots for hsp 23 have been reported previously (Ireland and Berger, 1982). After 6 hr of hormone treatment the levels of small hsp message increased substantially over control. After 24 hr in hormone, the levels of hybridizable RNA appeared to be at, or slightly below, the 6-hr value. Using the B8 clone as a probe (Ingolia et al., 1980) which contains the unlinked hsp 70 gene, we could not detect levels of hybridization above our negative control, tRNA (Fig. 2D). The same experiment was done using total RNA extracted from three other cell lines. MDR is a hormone responsive subline of the Kc line (Wyss, 1980), and

DEVELOPMENTAL BIOLOGY

500

VOLUME 93, 1982

I Kb

7 hsp27

gene1

hsp23

hsp26

hsP22

gene4

gene5

w

JIPR3

T6ElH2

w JIS3

IC5

KO

P

hDm67 p

0 Eco

RI

Q Born HI

9 SOI I v

3.5 ?#.A

2.7Kby

P

6.8 Kb

P

Pst I

Q Hind III FIG. I. Partial restriction map of the small heat-shock gene cluster constructed from the maps in Corces et al. (1980), Craig and McCarthy (1980), and Sirotkin and Davidson (1982). Arrows indicate the direction and location of transcription, while dotted lines signify uncertainty of location. The clones and subclones used as probes in this study are designated JlS3, JlPR3, JlB, T6HB2, T4, the 2.6- and 3.5-kb EcoRI fragments of XDm67, and the 0.9. and 1.2-kb Hind11 fragments generated by the digestion of the 6.8-kb EcoRI fragment of XDm67.

shows only a small increase in the level of small hsp synthesis following hormone treatment (p5 and p7 in Berger et al. (1980)). As expected, dot blot studies uncovered only a small, parallel increase in the level of small hsp RNA (blots not shown). This accumulation was absent in the ecdysterone-resistant MDER and F6 lines characterized previously (Wyss, 1980; Berger et al., 1980; Berger and Wyss, 1980). In all three lines the levels of hybridization to RNA from untreated cells were very low and indistinguishable from those seen in untreated line S3 cells. Induction

is a Primary

Response

A primary hormone response at the genetic level may be defined as one in which the hormone, presumably interacting with its receptor, directly evokes an alteration in the transcriptional activity of a “target” gene. A secondary response, in contrast, is operationally de-

fined as one in which the transcriptional activity of a gene is regulated by the product of the primary or target gene. In the case of ecdysterone responsiveness in the salivary glands of Drosophila, this target gene product has been shown to be a protein (Ashburner et al., 1974; Ashburner, 1974). It was of interest, then, to determine whether the stimulation of small hsp transcription by ecdysterone required simultaneous protein synthesis. In this same experiment we also asked whether hsp transcription required the continuous presence of hormone. S3 cells were incubated with lop6 ecdysterone, in the presence or absence of 10m4A4 cycloheximide. At this inhibitor concentration protein synthesis, measured by [35S]methionine incorporation, is reduced by over 98% within 20 min. Cycloheximide when used then, was added 20 min prior to the addition of ecdysterone. Two hours after hormone addition the RNAs were extracted from harvested cells and applied to filters that were then hybridized to 32P-labeled hsp 27 and hsp 26 probes. After

IRELAND

1

Small Heat-Shock

ET AL.

2

I

501

Induction

Cells continuously exposed to hormone continued to accumulate hsp 26 and hsp 27 transcripts for at least 5 additional hr, and in agreement with the results in Fig. 2, we found a decline in the relative abundance of these RNAs by 24 hr. For reasons we do not yet understand this decline did not occur in the hormone wash-out experiment.

3

Small

i9

Protein

A

B

c

D

hsp Transcription

in Imaginal

Discs

hsp 22 and hsp 26 transcripts appear abruptly and abundantly in third instar larvae, and accumulate throughout puparium formation (Sirotkin and Davidson, 1982). They are essentially absent, however, in total RNA extracted from embryos, adult flies, and older pupae. There is a simultaneous accumulation of transcripts encoded by three adjacent genes designated gene 1, gene 4, and gene 5 in Fig. 1. Despite the proximity of these three genes to the small hsp cluster, there is

0

FIG. 2. Dot blot hybridization of control and hormone-treated S3 cell RNA with probes for (A) hsp 22, (B) hsp 26, (C) hsp 27, and (D) et al., 1980) which contains the hsp 70 gene. The the B8 clone (Ignolia tracks designated 1, 2, and 3 contain RNA from control, 6-hr ecdysterone (10m6 M) treated, and 24.hr ecdysterone-treated cells. The amount of RNA in the three spots of each track were, from top to bottom, 20, 10, and 2 pg. The autoradiographs were exposed for 1 day.

3

0

11 autoradiography, t.he dots were excised and the amount of hybridization was determined. The results, presented in Fig. 3, indicate that the stimulation of small hsp transcription by hormone is initiated and continued for at least 2 hr in the absence of protein synthesis. Longer treatments with cycloheximide were toxic to the cells. The level of these RNAs in cells treated with cycloheximide, but in the absence of hormone, was similar to that of control cells. In a continuation of this experiment, we removed an aliquot of S, cells that were being incubated with hormone and collected them by centrifugation. After three washes in hormone-free medium the cells were divided into two equal aliquots, which were further incubated in medium with or without ecdysterone. At three subsequent periods, a portion of cells from each aliquot was harvested and their RNAs were extracted for dot blot analysis. To be certain that the washing procedure had successfully removed exogenous hormone we verified that medium collected from the final wash would not stimulate Kc cell differentiation (Cherbas et al., 1977, lYSOa,b). The results, summarized in Fig. 3, indicate that shortly after hormone was removed from the medium the relative concentration of hsp message plateaued.

m ‘0 ;2 a v

1

I/ q/ OA I -1-A ______-- -----0,’ / /A \ I

0

I 4

I

1 8 Time

I

I

I

I

12 (hrs

16

I

A

I 20

I

1 24

)

FIG. 3. Hybridization of labeled hsp 27 (circles) and hsp 26 (triangles), DNA to RNA from & cells continuously exposed to lo-” A4 ecdysterone (solid lines) or exposed for 2 hr and then washed free of hormone (dotted lines). The 2-hr time point also includes a value of hsp 27 (B) and hsp 26 (0) hybridization to RNA from cells incubated in the presence of cyclohexamide.

502

VOLUME 93, 1982

DEVELOPMENTAL BIOLOGY

1

2

3

1

4

A

6

C

D

2

3

4

FIG. 4. Dot blot hybridization of S, cell and imaginal disc RNA with probes for (A) hsp 22, (B) hsp 26, (C) hsp 27, and (D) hsp 70. The tracks designated 1, 2, 3, and 4 contain, respectively, RNA from control S, cells, freshly isolated imaginal discs, imaginal discs incubated in Robb’s medium containing 10m7M ecdysterone for 4 hr, and the 4-hr hormone-less sham incubation. The amounts of RNA the three spots of each track were, from top to bottom, 20, 10, and 2 pg. The autoradiographs were exposed for 1 day.

no evidence of their involvement in the heat-shock response. Since the XDm67 clone isolated by Sirotkin and Davidson (1982) does not contain the hsp 23 and hsp 27 sequences (Fig. l), their developmental expression was not studied. The discovery of Sirotkin and Davidson’s (1982) seemed important for two reasons. First, it provided indisputable evidence for the normal developmental regulation of at least two small heat-shock genes. The timing of their expression is significant, for it corresponds to that period when ecdysterone titers are rapidly rising within the animal in preparation for metamorphosis (Borst et al., 1974; Hodgetts et al., 1977; Richards, 1981). Second, some of the tissue in the cultures from which the line was derived had the properties of imaginal anlagen (Schneider, 1972). It was logical to speculate, therefore, that the induction of hsp transcription seen in whole animals might be occurring in imaginal discs. Mass isolated imaginal discs were recovered from late-third-instar larvae and incubated in the presence or absence of 10m7M ecdysterone. RNA extracted from these discs was then analyzed by dot blot hybridization using probes for hsp 22, hsp 26, and hsp 27 and the high-molecular-weight heat-shock gene hsp 70. RNA extracted from control and hormone-treated S3 cells was included on the filters for purposes of comparison. Qualitatively (Fig. 4), it is apparent that freshly collected discs already contain a substantial amount of hsp 26 and hsp 27 RNA compared to control S3 cells. These

levels are significantly elevated in the RNA of discs incubated for four hours in hormone, but apparently not in the RNA of discs incubated for an equivalent time in medium alone. The same result was seen when the hsp 23 probe was used (data not shown). There was very little change in the abundance of hsp 22 RNA (Fig. 6A). These results were confirmed and extended in a more detailed experiment whose results are summarized in Fig. 5. In all three cases shown the level of hsp message increased and later declined when discs were incubated in the presence of hormone. A lower level of accumulation was found in sham-treated discs incubated in parallel. We attribute this result to hormone present in the discs at the time of their isolation. Since the three probes were hybridized to RNA samples extracted from the same batch of discs, we believe that the differences in timing of induction for the three genes are real. In a final experiment freshly isolated imaginal discs were incubated for 1 hr in Robb’s medium with or without lop7 M ecdysterone at 25°C or under similar conditions at 37°C. The RNA isolated from these discs was then analyzed by dot blot hybridization with the 32Plabeled probes specific for each of the four small heatshock protein genes. The hybridized dots were finally excised and counted. The results, summarized in Table 1, indicate that the level of mRNA for hsp 22 and hsp 23 produced under heat-shock conditions is not significantly increased by simultaneous exposure to hormone. In contrast, the levels of hsp 26 and hsp 27 mRNA were

IRELAND ~‘r Al.

Small Heat-Shock

Protein

503

Induction

TABLE 1 OF SMALL hsp GENE PROBES TO RNA

DOT BI,OT HYRRIDIZATIOK

FROM IMAGINAI.

DISCS

‘“P counts/30 Treatment 25°C 37°C

1 Oo

4

8

I2 0

163 2070

4,568

223

100

15,519

1861

2839

381

6,529

450

261

2090

18,904

3385

3926

(25°C) (37OC)

substantially higher in discs exposed to both stimuli than in discs exposed to either stimulus alone. In fact the hybridization levels achieved are much greater than the sum of the two individual responses. 3

23K

27K

26K

10 ’ M ecdysterone

(hours)

FIG. 5. The accumulation of (a) hsp 22, (b) hsp 26, and (c) hsp 27 RNA in sham-incubated (dotted lines) and hormone (10 7 M ecdysterone)-treated (solid lines) imaginal discs. All values of hybridization were measured by excising and counting spots from dot blot analysis. Because the specific radioactivity of our probes differed in the two duplicate blotting experiments averaged in this figure, the data were normalized to the maximum level of hybridization in hormone-treated discs. In terms of cpm hybridizing to 30 rg of RNA spotted, the maximum for hsp 22, hsp 26, and hsp 27 were 407, 918, 845 cpm, respectively. The inverted triangles correspond to values for control S,) cell RNA spotted on the same filters.

12

22K

10 ’ M ecdysterone

;ki--F-+ I2 0 Time

fig RNA probes*

4

5

6

A

Note. All treatments were done in Robb’s medium for 1 hr. Samples were counted for 10 min. ” Counts are corrected for nonspecific binding to 30 pg tRNA which was 20-42 cpm in these experiments.

The Domain

of Genetic

The RNA samples extracted from cells and discs were next analyzed for the presence of sequences complementary to gene 1, gene 4, and gene 5. The gene 1 probe was a 2.7-kb EcoRI fragment subcloned from XDM67 (Fig. 1). The probes for genes 4 and 5 were, respectively, subclones containing the 0.9- and 1.2-kb Hind111 fragments found in the 6%kb EcoRI fragment of XDM67 (Fig. 1). In addition we probed these RNAs with clones containing the 3.5-kb EcoRI fragment, to the right of gene 5 in the XDM67 clone, and with a pBR322 clone designated T4, that contains a IO-kb insert found to the left of the hsp 27 gene (Fig. 1). There was essentially no hybridization detected using the gene 4, gene 5 (Fig. 6), or 3.5-kb EcoRI probes. RNAs homologous to gene 1 were found in low abundance (Fig. 6) in both cells and discs, but these levels were not significantly changed by hormone treatment, or by heat shock (data not shown). The T4 probe produced a very strong hybridization signal in all cell lines (Fig. 6), but the level of hybridization was not changed after hormone or heat treatment. Only a weak signal was seen in disc RNA. Craig and McCarthy (1980) have shown that the left end of the T4 insert contains a dispersed, repeated DNA sequence that is abundantly transcribed in Drosophila cell lines. These RNAs produced no protein product in a cell-free translation assay. Cell Localization

FIG. 6. Dot blot analysis of (A) gene 1, (B) gene hybridization to RNA from: (1) control S3 cells; (2) and (3) 24 hr hormone-treated S3 cells; and from (4) and imaginal discs incubated (5) in the presence; or lo-’ M ecdysterone for 4 hr.

5, and (C) T4 6-hr hormone; imaginal discs; (6) absence of

Regulation

of hsps

During the heat-shock response over half of the newly synthesized hsps accumulate within the nucleus (Arrigo et al., 1980; Velazquez et al., 1980) apparently bound to chromatin and/or the nuclear scaffolding matrix (Levinger and Varshavsky, 1981). hsp 82 is the exception and is almost entirely cytoplasmic. We investigated the

DEVELOPMENTAL BIOLOGY

VOLUME 93, 1982

FIG. 7. Localization of [%]methionine-labeled heat-shock proteins in heat-shocked and ecdysterone-treated S, cells. Cells were incubated for 2 hr in the presence of [3”S]methionine at 25, 37, or at 25°C in the presence of 10 s&f ecdysterone. Proteins extracted from the total homogenate (a,d,g) nuclear fraction (b,e,h), or the cytoplasmic fraction (c,f,i) of the three respective treatments were analyzed by two-dimensional gel electrophoresis. Approximately 5 X lo” cpm were loaded onto each gel, and the autoradiographs were exposed for 6 days. Proteins circumscribed by squares and circles represent normal cell proteins primarily showing a cytoplasmic or nuclear localization, respectively. The large heat-shock proteins, hsp 68, 70, and 82, are indicated by triangles; the four small heat-shock proteins are designated by arrows.

intracellular distribution of hsps in S3 cells under both conditions of heat-shock and ecdysterone treatment and the results are summarized in Fig. 7. To facilitate localization in Fig. 7, several marker proteins showing either a nuclear (bound by squares) or a cytoplasmic (bound by circles) distribution are indicated. We find that during heat-shock hsp 82 is almost exclusively found in the cytoplasm, while hsp 27 is highly concentrated within the nucleus. The remaining hsps are found in both cell compartments, although the relative ratio of hsp 68 + 70 to hsp 22 + 23 + 26 is sig-

nificantly higher in the nucleus (Figs. 7d, e, f). In a parallel fractionation of ecdysterone-treated cells (Figure 7g, h, i) we found an interesting contrast. Whereas there is no evidence of hsp 68 and hsp 70, the four small hsps are found almost entirely in the cytoplasmic compartment. While hsp 82 is present in hormone-treated cells, again concentrated in the cytoplasm, it is also found in control cells (Figs. 7a, b, c). Its rate of synthesis was apparently unaffected by hormone. This observation has also been made by others (O’Connor and Lis, 1981).

Small Heat-Shock

IRELAND ET AL.

TABLE ECDYSTERONE RECEPTOR ACTIVITY [“HIPNA cpm bound

Cell line F6 MDR Imaginal

discs

MDER S,{ Expt 1 S:, Expt 2 Si No KC1 extraction

Ecdysterone

IN SEVERAL

Noncompetable cpm bound

Protein

2 Drosophila

CELL

LINES

AND IMACINAL

cpm

Milligrams of protein per assay

Specific

139 2153 2676

100 125 45

39 2028 2631

0.18 0.62 0.15

6 1459 856 108

9 9 0 58

0 1450 856 50

0.15 0.18 0.14 0.11

Receptor Activity

Drosophila cell line Kc has been shown to contain ecdysterone receptor activity (Maroy et al., 1978). Using a modification of the [“Hlponasterone A binding assay originally developed for imaginal discs (Yund et al., 1978) we measured the level or hormone receptor in extracts of two responsive lines, S3 and MDR, and in the two ecdysterone-insensitive lines, MDER and the MDER/S3 fusion hybrid, F6. The results of this analysis, summarized in Table 2, clearly indicated that cells insensitive to ecdysterone have little or no hormone binding activity. Responsive cells, in contrast, contain high levels, similar to those found in imaginal discs. It is interesting to note that a high-speed cell supernatant prepared in the absence of 0.3 M KC1 extraction contained only 5% of the receptor activity found in lysates in which the salt extraction was done. This implies that the majority of receptor activity found in these cells is nuclear. DISCUSSION

The regulation of small heat-shock protein synthesis in Drosophila is under dual control. However, the nature of the induction in response to the two stimuli, heat and hormone, is different in several ways. Under conditions of high temperature the stimulation of hsp synthesis is rapid and accompanied by a marked decrease in the production of most normal cell proteins. Stimulation by ecdysterone is slower and prolonged (Berger et al., 1980; Ireland and Berger, 1982) and the pattern of cellular protein synthesis for the most part remains unchanged (Ireland and Berger, 1982). In addition, high temperature simultaneously evokes the massive synthesis of two larger proteins, hsp 68 and hsp 70, which are absent in cells responding to ecdysterone. Finally, the small heatshock proteins appear to accumulate in different cell compartments in response to the two stimuli, a point we will return to later. One major objective of this study was to determine whether the induction of small hsp synthesis in S3 cells

505

Induction

DISCS Specific [3H]PNA bound/ mg protein in assay 216 3,271 16,865 0 8,239 5,986 455

was paralleled by an increase in small hsp mRNA, as is the case under heat-shock conditions (reviewed in Ashburner and Bonner, 1979). Using a series of genomic clones, and the dot blot hybridization technique, we found that by 2 hr after hormone addition there is already a signiscant increase in the level of hsp 26 and hsp 27 transcripts. Similar results have been reported earlier for hsp 23 (Ireland and Berger, 1982). These levels continue to rise for at least 7 hr, and then, in the case of hsp 26 and hsp 27, plateau and decline (Fig. 3). hsp 22 induction is low (Berger et al., 1980; Ireland and Berger, 1982) (Fig. 7i) as is the level of message accumulation. The induction of hsp 26 and hsp 27 is a primary response in the sense that it occurs in the absence of protein synthesis (Fig. 3). The progressive accumulation of these messages requires the continuous presence of hormone (Fig. 3) as a sustaining stimulus. This requirement has previously been demonstrated for acetylcholinesterase induction in Kc cells (Cherbas et al., 1980a,b), although the commitment to cease cell proliferation is apparently made after less than 1 hr of exposure to hormone (Cherbas et al., 1980a,b). Finally, the presence of ecdysterone receptor activity appears to be a necessary but not a sufficient condition for small hsp induction. In agreement with earlier studies, in which patterns of protein synthesis were examined (Berger et al., 1978, 1980; Berger and Wyss, 1980; Ireland and Berger, 1982), we find that small hsp induction by ecdysterone is very low in the Kc subline, MDR. This line is highly responsive to hormone by several other criteria (Berger et al., 1978; Cherbas et al., 1980a,b), and we have shown here (Table 2) that these cells contain a level of hormone-receptor activity very similar to that found in S3 cells. The basis of cell line-specific hormone responses is unclear. It may either reflect differences in the cell type from which the independently derived lines were established, or be the result of physiological differences that arose during continued culture. In the ecdysterone-resistant variant cell lines, MDER

506

DEVELOPMENTAL BIOLOGY

and F6, there is neither an increase in the rate of small hsp synthesis following exposure to hormone (Ireland and Berger, 1982) nor an accumulation of small hsp mRNAs. We have also been unable to detect a significant level of ecdysterone receptor activity (Table 2) and presume that this deficiency is the basis of hormone insensitivity. The absence of receptor activity in the fusion hybrid, F6, is particularly intriguing, for these cells contain a complete and presumably normal complement of S3 chromosomes (Wyss, 1980). One parsimonious interpretation of this result is that the dominant mutation leading to insensitivity in the MDER parent leads to the production of a defective subunit that “poisons” the binding activity of multimeric receptors. Alternatively, the mutation may somehow lead to the repression of receptor synthesis. Until an assay for the physical presence of receptor is available this question will remain unresolved. One legitimate objection to the cell line system, in general, has been that the hormone responses observed have no in uivo counterpart. This criticism is particularly devastating if the hormone response under investigation for the most part has been considered a stress response. It was important to learn, then, that hsp 22 and hsp 26 transcription is normally induced to high levels in Drosophila during the late-third-instar and prepupal stages (Sirotkin and Davidson, 1982), a time when ecdysterone titers are high in the animal (Borst et al., 1974; Hodgetts et al., 1977; reviewed in Richards, 1981). Their results, combined with the evidence that S3 cells might have originally arisen from imaginal disc tissue (Schneider, 1972) led us to the experiments summarized in Figs. 4 and 5. Our major finding is that imaginal discs isolated from late-third-instar larvae already contain a high level of hsp 23, 26, and 27 transcripts. This level is increased substantially when the discs are further incubated in the presence of exogenous hormone. Recently Cheney and Shearn (1981; Cheney, manuscript in preparation) found that hsp 23, 26, and 27 synthesis is initiated in wing imaginal discs during the third larval instar, and continues to rise dramatically as puparium formation occurs. Thus, the small hsp genes are transcribed in imaginal discs, and the transcripts are apparently translated as well. Interestingly, for two of the four small hsp genes, transcription levels were much higher than predicted when the two stimuli were presented simultaneously. The significance of this result is unknown. While the genes designated 1, 4, and 5 (Fig. 1) by Sirotkin and Davidson (1982) lie within or adjacent to the small hsp cluster, and are actively transcribed in some larval and prepupal tissue, their expression was not abundant in either S3 cells or imaginal discs. The absence of gene 1 induction is especially provocative, for it lies between two genes, hsp 26 and hsp 23, that

VOLUME 93, 1982

are highly responsive to high temperature and hormone. This implies that the domain of transcriptional regulation, in this system at least, is not the entire small hsp region, but rather the individual locus. This view is supported by two other observations. First, there is no stoichiometry in either the levels of small hsp message accumulation or the rates of small hsp synthesis (Ireland and Berger, 1982). Second, the time at which maximum hsp message accumulation occurs in imaginal discs is different for the three genes studied here (Fig. 5). A noncoordinate pattern of regulation has been described previously for these genes during the heat-shock response (Lindquist, 1980). Finally, we turn to the cellular localization of small hsps. Levinger and Varshavsky (1981) have recently confirmed and extended earlier studies (Arrigo et al., 1980; Velazquez et al., 1980) demonstrating that the majority of heat-shock proteins concentrate within the nucleus of Drosophila cells during exposure to high temperature. The single exception is hsp 82 which is almost exclusively cytoplasmic. They went on to show that the nuclear hsps were not associated with nucleosomes, but rather with the peripherally located nuclear lamella which contains, as they put it, “components of an intranuclear fibrous scaffold or matrix.” They postulated a role for hsps in the preservation of spatial organization in transcriptionally active chromatin, and predicted that this localization would be universal. Our findings indicate that during the ecdysone response small hsps remain almost entirely within the cytoplasm. We can interpret this distinction in two ways. First, if hsp 68 and hsp 70 were needed to fix or stabilize the small hsps within the nuclear lamalla, then small hsps produced in their absence would either remain in the cytoplasm, concentrate in the nucleus but leak out during cell fractionation, or transiently cycle through the nucleus following their synthesis. Alternatively, the small hsps may normally become structural components of a cytoplasmic matrix that collapses during heat shock. Their nuclear localization during heat shock would then be a consequence of damage produced by high temperature. We are extremely grateful to Dr. E. Craig for furnishing many of the clones used in these experiments. This work was supported by NIH Grants GM 22866 (E.B.), GM 23349 (M.A.Y.1, and GM 19937 (J.F.). REFERENCES ARRIGO, A., FAKAN, S., and TISSIERES, A. (1980). Localization of the heat shock-induced proteins in Drosophila melanogmter tissue culture cells. Dec. Biol. 78, 86-103. ASHRURNER, M. (1974). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogmter. II. The effects of inhibitors of protein synthesis. Deu. Biol. 39, 141-157. ASHBURNER, M., and BONNER, J. (1979). The induction of gene activity in Drosophila by heat shock. Cell 17, 241-254.

IKELAND XT AL.

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Heat-Shock

ASHBURNER, M., CHIHAKA, C., MELTZER, R., and RICHARDS, G. (1974). Temporal control of puffing activity in polytene chromosomes.

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BE:RGER, E., FRANK, M., and ARELL, M. C. (1980). Ecdysone induced changes in protein synthesis in embryonic Drosophila cells in culture in “Invertebrate Systems in Vitro” (E. Kurstak, K. Maramorosch, A. Dubendorfer, eds.), pp. 195-208. Elsevier/North-Holland, Amsterdam. BEKGER, E., IRELAND, R., and WYSS, C. (1980). Pattern of peptide synthesis in Drosophila cell lines and their hybrids. Sam. Cell Genet. 6, 719-729. BERC.EK, E., RINGLF,R, R., ALAHIOTIS, S., and FRANK, M. (1978). Ecdysone induced changes in morphology and protein synthesis in Drosophila cells. UCU. Biol. 62, 498-511. BER(:ER, E., and WYSS, C. (1980). Acetylcholinesterase induction by /j-erdysone in Drosophila cell lines and their hybrids. Sam. Cell Geart. 6, 63-640. BE:s’I’-BI%POMME, M., COURC.F,ON, A. M., and RAMBACH, A. (1978). fi-Galactosidase is induced by hormone in Drosophila melanogaster cell cultures. Proc. Nat. Acad. Sci. 1JSA 75, 61026106. BORST, D. W., BOLLENBACHEK, W. E., O’CONNOR, J. D., KING, D. W., and FRISTROM, J. W. (1974). Ecdysone levels during metamelanogaster. Deu. Biol. 39, 308-316. morphosis of Drosophila CHENEY, C. M., and SHEARN, A. (1981). Synthesis of a Drosophila heat shock protein is developmentally regulated. J. Cell Biol. 91, 376a. CHF,HRAS, P., CHERHAS, L., and WILLIAMS, C. M. (1977). Induction cell of acetylcholinesterase activity by fl-ecdysone in a Drosophila line. Science 197, 275-277. CHERHAS, L., CHERBAS, P., SAVAKIS, C., DEMETRI, G., MANTEUFFELCYMROROWSKA, M., YON~X, C. D., and WILLIAMS, C. M. (1980a). Studies of ecdysteroid action on a Drosophila cell line In “Invertebrate Systems in Vitro” (E. Kurstak, K. Maramorosch, and A. Dhbendorfer, eds.), pp. 217-228. Elsevier/North-Holland, Amsterdam. CHERHAS, L., YONGF,, C. D., CHERBAS, P., and WILLIAMS, C. M. (1980b). The morphological response of Kc-H cells to ecdysteroids: Hormonal specificity. Wilhelm Roux’s Arch. 189, 1-15. CORPUS, V., HOLMGREN, R., FREIJND, R., MORIMOTO, R., and MESELSON, M. (1980). Four heat shock proteins of Drosophila melaregion in chromosome subnogaster coded within an 12.Kilobase division 67B. Proc. Nat. Acad. Sci. USA 77, 5390-5393. Coumxo~, A. M. (1972a). Action of insect hormones at the cellular level. Exp. Cell. Res. 74, 327-336. COURGEON, A. M. (1972b). Effect of (Y- and @-ecdysone on in vitro diploid cell multiplication in Drosophila melanogaster. Nature New Riol. 238, 250-251. CHAIC., E. A., and MCCARTHY, B. J. (1980). Four Drosophila heat shock genes at 67B: Characterization of recombinant plasmids. Nucl. Acid Res. 8, 4441-4457. EIJGENE, 0. M., and FRISTROM, J. W. (1978). The Mass Isolation of Imaginal discs. In “The Genetics and Biology of Drosophila” (M. Ashburner and T. R. F. Wright, eds.), Vol. 2a, pp. 121-126. Academic Press, New York. HODGETTS, R. B., SA(:E, B., and O’CONNOR, J. D. (1977). Ecdysone titers during postembryonic development of Drosophila melanogaster. Deu. Biol. 60, 310-317. INGOLIA, T., CRAIG, E., and MCCARTHY, B. (1980). Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions. Cell 21, 669-679.

Protein

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IKELAND, R., and BERGEN, E. (1982). Synthesis of low molecular weight heat shock polypeptides stimulated by ecdysterone in a culcell line. Proc. Nat. Acad. Sci. lJSA 79, 855-859. tured Drosophila LEVINCEH, L., and VARSHAVSKY, A. (1981). Heat-shock proteins of Drosophila are associated with nuclease-resistant, high-salt-resistant nuclear structures. J. Cell Biol. 90, 793-796. LINDQUIST, S. (1980). Varying patterns of protein synthesis in Drofor regulation. Dev. Riol. 77, sophila during heat shock: Implications 463-479.

MAROY, P., DENNIS, R., BECKFXS, C., SA(:E, B. A., and O’CONNOR, J. D. (1978). Demonstration of an ecdysteroid receptor in a cultured cell line of Drosophila melanogayter. Proc. Nat. Acad. Sci. CJSA 75, 6035-6038. O’CONNOK,

D., and Lrs, J. (1981). Two closely linked transcription units within the 63B heat shock puff locus of D. melanogasterdisplay strikingly different regulation. Nucl. Acids Res. 9, 5075-5092. RICHARDS, G. (1981). The radioimmune assay of ecdysteroid titres in Drosophila melanogaster. Mol. Cell. Endocrinol. 2 1, 181-197. RICBY, P. W. J., DIECKMANN, M., RHODES, C., and BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J. Mol. Riol. 113, 237251. cell line. ROSSET, R. (1978). Effects of ecdysterone on a Drosophila Exp. Cell Res. 111, 31-36. SAVAKIS, C., DEMETRI, G., and CHERRAS, P. (1980). Ecdysteroid-incell line. Cell 22, 655-674. ducible polypeptides in a Drosophila SCHNEIDER, I. (1972). Cell lines derived from late embryonic stages melanogaster. J. Embryol. Exp. Morphol. 27, 353-365. of Drosophila SCOTT, M. P., STOHTI, R. V., PARDUE, M. L., and RICH, A. (1979). Cell-free protein synthesis in lysates of Drosophila melanogaster cells. Biochemistry 18, 1588-1594. SIROTKIN, D., and DAVIDSON, N. (1982). Developmentally regulated transcription from Drosophila melanogaster chromosomal site 67B. Deu. Riol. 89, 196-210. STEVENS, B., ALVAREZ, C. M., BOHMAN, R., and O’CONNOR, J. D. (1980). An Ecdysteroid-induced alteration on the cell cycle of cultured Drosophila cells. Cell 22, 675-682. THOMAS, P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Nat. Acad. Sci. IJSA 77, 5201-5205. VELAZQUEZ, J., DIDOMENICO, B., and LINDQUIST, S. (1980). IntracelCell 20, 679lular localization of heat shock proteins in Drosophila. 689.

VOELLMY, R., GOLDSCHMIDT-CLERMONT, M., SOUTHGATE, R., TrsSIERES, A., LEVIS, R., and GEHRING, W. (1981). A DNA segment from chromosomal site 67B in D. melanogayter contains four closely linked heat-shock genes. Cell 23, 261-270. WAI~SWORTH, S., CRAIG, E. A., and MCCARTHY, B. J. (1980). Genes for three Drosophila heat shock-induced proteins at a single locus. Proc. Nat. Acad. Sci. IJSA 77, 2134-2137. WYSS, C. (1976). Juvenile hormone analogue counteracts growth stimulation and inhibition by ecdysones in clonal Drosophila cell line. Experientia 32, 127221274. WYSS, C. (1980). Cell hybrid analysis of ecdysone sensitivity and resistance in Drosophila cell lines. In “Invertebrate Systems In Vitro” (E. Kurstak, K. Maramorosh, and A. Diibendorfer, eds.), pp. 279289. Elsevier/North-Holland, Amsterdam. YUND, M. A., KING, D. S., and FRISTROM, J. W. (1978). Ecdysteroid receptors in imaginal discs of Drosophila melanogaster. Proc. Nat. Acad.

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