NOTES
Biochem. Cell Biol. 1992.70:339-342. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/21/15. For personal use only.
Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis NICKOVSENEK' AND JOHNJ. HEIKKILA Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1 Received December 4, 1991 N., and HEIKKILA, J. J. 1992. Compensatory effect of distal promoter sequences on the basal expression OVSENEK, of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis. Biochem. Cell Biol. 70: 339-342. The promoter sequences involved in the basal expression of a human 70-kilodalton heat shock protein (HSP70) gene during Xenopus embryogenesis were analyzed by microinjection of mutant promoters of a HSP70 - chloramphenicol acetyltransferase fusion gene into fertilized eggs and following their expression during early development. While deletion of the HSP70 gene promoter to - 100 base pairs (bp) did not affect basal transcription in postmidblastula stage embryos, linker-scanner mutations in the CCAAT and purine box elements blocked expression. However, extension of the 5' boundary to - 188 bp restored full wild-type expression to these mutants. These results suggest that multiple redundant cis-acting regulatory elements present in the human HSP7O gene promoter can function during Xenopus embryogenesis. Key words: heat shock protein gene, Xenopus embryos, microinjection, linker-scanner mutations, transcription.
OVSENEK, N., et HEIKKILA, J. J. 1992. Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis. Biochem. Cell Biol. 70 : 339-342. Les stquences du promoteur permettant l'expression basale du gtne humain d'une prottine de choc thermique de 70 kilodaltons (kDa) (PCT70) durant I'embryogentse chez le Xenopus ont t t t analyskes en micro-injectant, dans des oeufs fertilids, des promoteurs mutants du gtne de fusion PCT7O - chloramphtnicol acktyltransftrase, puis en suivant leur expression au dtbut du dheloppement. Alors que la dtlttion d'une portion du promoteur du gtne de la PCT70, jusqu'h - 100 paires de bases, n'affecte pas le taux basal de la transcription dans les embryons ayant dtpasst le milieu du stade blastula, des mutations de liaison-lecture (linker-scanner) des tlkments de la skquence CCAAT et de la stquence riche en purines bloquent la transcription. Cependant, I'extension de la frontitre 5' du gtne a - 188 paires de bases permet l'expression de ces gtnes mutts au meme t a w que le gtne non mutt. Ces rtsultats suggtrent que de multiples sequences cis-rtgulatrices redondantes, prtsentes dans le promoteur du gtne humain de la PCT70, peuvent avoir une fonction durant l'embryogentse chez le Xenopus. Mots elks : gene de prottine de choc thermique, embryons du Xenopus, micro-injection, mutations de liaison-lecture (linker-scanner), transcription. [Traduit par la rtdaction]
Introduction During Xenopus Iaevis embryogenesis, zygote genome transcription is not activated until the midblastula stage (Newport and Kirschner 1982a, 19828; Kimelman et al. 1987). This point in development, which has been termed MBT is also characterized by a desynchronization of subsequent cell divisions and the onset of cell motility. Prior to MBT, the only detectable RNA synthesis in Xenopus embryos is from mitochondria1 genes at a low rate (Chase and Dawid 1972). Activation of the zygotic genome results in an abrupt 200-fold increase in the rate of RNA synthesis (Kimelman et al. 1987). The mechanism by which Xenopus embryos remain transcriptionally dormant until MBT and then activate selected genes is not fully understood. It has been suggested that the lack of transcriptional activity during cleavage stages is a result of an overabundance of either cytoplasmic M-phase-promoting factor or initiation factor, ABBREVIATIONS: HSP70, 70-kilodalton heat shock protein; bp, base pair(s); MBT, midblastula transition; CAT, chloramphenicol acetyltransferase; NEO, neomycin resistance; TLC, thin-layer chromatography; nt, nucleotide(s). 'present address: Center for Developmental Biology, Department of Zoology, University of Texas, Austin, TX 78712, U.S.A. Printed in Canada / Imprime au Canada
which prevents a G2phase in the cell cycle by rapidly triggering the onset of mitosis after S phase. Among the set of zygotic genes that are selectively transcribed or inducible at MBT are a number of the HSP genes including HSP70. Recently, we found that a microinjected human HSP70 promoter - CAT chimeric gene was strongly expressed under nonstress conditions at MBT, indicating that it shared common regulatory features associated with endogenous genes expressed at MBT (Ovsenek et al. 1990). The availability of various promoter mutants permitted the examination of the cis-acting sequences required for expression at MBT. Deletion mutant analysis revealed that only 74 bp of the 5' promoter region was required for constitutive expression. Also, we examined the role of specific sequences in the basal promoter by injecting HSP70-CAT vectors containing linker-scanner mutations in the basal elements such as CCAAT, purine-rich element, GC element, ATF/AP 1, and TATA. Interestingly, the alteration of any one of these elements in the basal promoter prevented expression at MBT. In the human HSP70 promoter there are a number of potentially functional cis-acting elements between - 100 and - 188 including CCAAT, AP-2, GC box, and HSE, some of which have been previously shown to interact with trans-acting factors
BIOCHEM. CELL
GC
CCAAT
AP2
BIOL. VOL. 70, 1992
CCAAT Purine Box GC
HSE
ATF/AP 1
TATA
AP2
...................... ...................... ......................
Biochem. Cell Biol. 1992.70:339-342. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/21/15. For personal use only.
FIG. 1. Diagram of the human HSP7O promoter. Major established promoter elements are indicated. LSPNILSN SERIES CCAAT BOX
-75 GCT
033
TGA TTG
GCc
gaG
atc
TcG
W
(XKj
cGA
gat
GCT
033
TGA
PURINE BOX
-43
CAG
A A G GGA
AAA
GCT
CAG
AAG
GGA
AAA
aOC
GGG
73/63
cga
tct
cgG
GGA
AAA OCC
GGG
69/55
GCT
CAG
cga
Gat
ctc
GGG
57/47
OCC
WT
FIG. 2. Diagram of the linker-scanner mutations in the human HSP70 promoter. For each mutant, the numeric notation refers to the region in the promoer in which the indicated base changes have been made. The LSPN series contains 100 bases of the promoter, whereas the LSN series contains 188 bases 5' of the transcriptional start site. WT, wild type.
(Fig. 1; Morgan et al. 1987; Mosser et al. 1988). In the present study, we have examined whether the loss of expression from the basal promoter by mutation of CCAAT and the purine box could be complemented by extending the 5 ' boundary from - 100 to - 188. Our finding that extension of the 5' boundary restores full wild-type expression to CCAAT and purine box mutants suggests that multiple redundant cis-acting regulatory elements present in the human HSP70 promoter can function in Xenopus embryos after the onset of zygotic genome transcription. Materials and methods Embryo maintenance and microinjection Xenopus laevis eggs were obtained, fertilized, dejelled, and maintained in Steinberg's solution as previously described (Heikkila et al. 1985). Embryos were staged according to Nieuwkoop and Faber (1956). Circular or linearized plasmid DNA (400 pg in 20 nL) was microinjected into embryos within 2 h of fertilization using the apparatus designed by Hitchcock and Friedman (1980). Injected embryos were maintained in Steinberg's solution with 4% w/v Ficoll (Krieg and Melton 1985). The Ficoll content of Steinberg's solution was gradually decreased to 1% by the blastula stage. Only normally developing embryos were used for CAT activity and RNase protection assays. 5'-deletion and linker-scanner mutations Specific details describing the construction of the LSPN and LSN series mutations of the human HSP70 promoter that is fused to a CAT gene are described by Williams et al. (1989). Insertion of linker-scanners into the proximal 100 bases of the promoter generates the LSPN series, while the LSN series contains 188 bases of promoter. Linker-scanner mutations were constructed as described (Wu et al. 1987). Also, the microinjected plasmids contain a neomycin resistance (NEO) gene orientated in the opposite direction. The NEO gene is regulated by the proximal 74 bases of the human HSP70 promoter and is a constant on all of the microinjected constructs. CA T assays For each analysis, 10 embryos were homogenized in 100 pL of 0.25 M Tris (pH 7.8) and then centrifuged twice for 15 min at 8000 x g to remove yolk and cellular debris. The supernatants were stored at - 70°C. Enzyme assays were performed as described by
Gorman et al. (1982) using three embryo equivalents. Conversion of [14~]chloramphenicol to acetylated forms was monitored by TLC, followed by autoradiography using Kodak XAR-5 film. Radioactivity in the acetylated forms of chloramphenicol was measured by scintillation counting. RNA isolation and RNase protection assays The isolation procedure of total lithium chloride precipitable RNA from Xenopus embryos is outlined in Heikkila et a/. (1987). RNA samples were treated with 4 U of RQ1 DNase (Promega) in 40 mM Tris (pH 7.9) - 6 mM MgCI, for 1 h at 37"C, followed by extraction with phenol-chloroform and chloroform and subsequent ethanol precipitation. A 588-bp HindIII to EcoRI fragment from p delta H18 (Wu et al. 1985) containing 188 bp of HSP-CAT promoter sequences and 400 bp of transcribed sequences was cloned into the pGEM2 vector (Williams et al. 1989). Digestion of the resulting plasmid with HindIII and transcription with SP6 RNA polymerase results in an antisense riboprobe approximately 600 nt long, of which 400 nt are protected by correctly initiated HSP-CAT RNA. Labeled transcripts were passed through a Sephadex G-50 column. RNase protection assays were performed as described by Krieg and Melton (1987a, 1987b) using 10 pg of total RNA. RNase-protected transcripts were detected following electrophoresis on 4% polyacrylamide - 8 M urea gels and exposure to Kodak XAR-5 film at - 70°C.
Results In a previous study examining the basal expression of a human HSP70 promoter - CAT fusion gene in rnicroinjected Xenopus embryos, we found that 74 bp of promoter region was sufficient for expression after the midblastula transition. Additionally, linker-scanner mutations (LSPN series) in a number of proximal promoter elements (e.g., CCAAT box, purine box, GC box, ATF region) prevented expression (Ovsenek et al. 1990). The human HSP70 promoter also contained additional putative regulatory elements (e.g., CCAAT box, GC box) upstream of the basal promoter (Fig. 1). In the present study, we have examined whether the loss of expression in some of the LSPN series of linkerscanner mutations in the proximal promoter could be complemented by sequences upstream of the basal promoter.
34 1
Biochem. Cell Biol. 1992.70:339-342. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/21/15. For personal use only.
NOTES
LSPNLSN wt
L S P e S N LSPNLSN L S P ~ S N 73/63 69/55 57/47
FIG. 3. Expression of LSN linker-scanner mutations in eno opus embryos. CAT activity in extracts (three embryo equivalents) from gastrula embryos that had been microinjected at the one-cell stage with 400 pg of LSNwt, LSN73163, or LSN57/47 were compared with the activity of extracts from gastrulae transformed with equal amounts of the corresponding LSPN constructs, i.e., LSPNwt, LSPN73/63. LSPN69/55, and LSPN57/47. The position of the labeled chloramphenicol is indicated by CM, while its acetylated forms are indicated by arrows. LSN69/559
TABLE1. CAT activity in gastrula stage embryos microinjected with LSPN and LSN HSP70-CAT fusion gene mutants CAT enzyme activity (Yo)*
LSPN
LSN
*Percent acetylation of chloramphenicol by equivalent amounts of embryo extract. +WT, wild type.
This was done by microinjecting DNA from a separate series of LSN linker-scanner mutants that contain the same substitutions in the proximal region as the LSPN vectors, but differ by having a 5' boundary extended to - 188 bp (Fig. 2). In these experiments, the LSPN and LSN vectors were microinjected into fertilized eggs and assayed for CAT activity at the gastrula stage. A representative autoradiogram is presented in Fig. 3. Quantitation of CAT activity displayed in Fig. 3 was carried out by scintillation counting of acetylated chloramphenicol spots cut out of the TLC plate and is presented in Table 1. The basal level of CAT activity from the LSPN and LSN wild-type vectors is similar. However, the LSPN linker-scanner mutants 73/63 and 69/55 which alter the CCAAT box and 57/47 which modifies the purine box were not expressed in gastrula stage embryos. Additionally, we utilized an internal control, namely, the expression of the NEO gene which is present on the LSPN plasmids to demonstrate the presence of these vectors in Xenopus embryos (data not shown). The lack of expression of these LSPN mutants in gastrula-stage embryos agrees with our previous results (Ovsenek et al. 1990). Interestingly, the LSN mutants which contain the identical promoter muta-
wt
73/63 69/55 57/47
FIG, 4. RNaSe protection analysis of human HSp70-CAT RNA in embryosmicroinjectedwith LSN promoter mutants. Ten micrograms of total RNA was isolated from gasmla stage embryos
that had been rnicroinjectedwith either the wild-type LSN promoter or LSN mutants outlinedin the captionto ~ i3. ~h~ ~ RNA . was subjectedto RNase protectionanalysisand separated on a 4% po~yacry~ami~e-urea denaturing gel. The arrow indicates the position of the 400-nt protected HSp70-CAT transcript. tions found in the LSPN mutants but have an extended 5' boundary ( - 188 bp) were fully expressed (Fig. 3; Table 1). The level of CAT activity in the 73/63,69/55, and 57/47 LSN mutants were similar to that found with the LSN wild type. The constitutive expression of the LSN mutants was detected immediately after the midblastula transition, as well as in postgastrula stage embryos (data not shown). RNase protection analysis was performed to ensure that the transcription of microinjected LSN mutants was initiated correctly. As shown in Fig. 4, a labeled antisense riboprobe spanning the transcriptiona1 start site of the HSP70-CAT fusion gene protects a correctly initiated 4004 transcript present in rota1 RNA from gastrula stage embryos that had been injected with either the LSN wild-type vector or the 73/63, 69/55, and 57/47 LSN mutants. Since alteration of the same promoter sequences that inactivated the - 100 deletion mutant LSPN did not have an affect on the expression of the - 188 deletion mutant LSN, it is tenable that sequences between - 100 and - 188 of the HSP70 promoter can restore transcriptional activity.
Discussion The role of promoter sequences upstream of the basal human HSP70 promoter during early Xenopus embryogenesis was investigated by microinjecting a set of LSN linker-scanner mutants that included 188 bp of the human HSP70 promoter. Base alterations that inactivated the basal promoter had no effect on HSP70-CAT expression when promoter sequences between - 100 and - 188 were present (Fig. 3). It is possible that the promoter elements in the upstream region of the promoter act alone or in concert with intact elements in the basal promoter to restore transcriptional activity to the LSN mutants. It is tenable that the upstream CCAAT element may substitute for the downstream CCAAT sequence. Interestingly, we found that a mutation in the purine box could also be rescued by additional upstream promoter sequences, even though no additional purine boxes were present. This result suggests that other sequences such as the upstream GC, CCAAT, ATF, or HSE elements may compensate for a nonfunctional
Biochem. Cell Biol. 1992.70:339-342. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/21/15. For personal use only.
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purine box in the basal promoter. In fact, Xenopus transcription factors have been shown to interact with human CCAAT, ATF, GC, and HSE elements (Ovsenek et al. 1990; Ovsenek and Heikkila 1990, 1991). Our finding that distal elements may also play a role in the expression of the parental human HSP70-CAT fusion gene in developing Xenopus embryos supports previous work demonstrating that the complex pattern of HSP70 regulation involves several cis-acting regulatory elements (Wu et al. 1986; Greene et al. 1987; Morgan et al. 1987; Wu et al. 1987). Expression of the human HSP70 gene is regulated during normal cell growth and differentiation (Kao et al. 1985; Wu and Morimoto 1985; Milarski and Morimoto 1986) and in response to various stresses (Wu et al. 1986; Watowich and Morimoto 1988). The multiple cis-acting regulatory elements that are involved in the complex transcriptional regulation of the human HSP70 gene appear to be redundant in the heterologous Xenopus system. There may be a number of differences between the array of transcription factors present in human cells and Xenopus embryos that account for the activity of the human HSP70 promoter under nonstress conditions. The recent observation that a microinjected Xenopus HSP70-CAT fusion gene is only expressed in a heat-inducible manner in postmidblastula stage embryos (Krone and Heikkila 1989) strengthens this hypothesis. Comparison of the human and Xenopus HSP7O (Bienz 1984) promoters indicates that the ATF binding site and the purine-rich region present in the human promoter are absent in the Xenopus HSP70 promoter. The human and Xenopus HSP70 promoters are otherwise quite similar in terms of the spatial organization of cis-regulatory elements such as CCAAT, TATA, HSE, and GC elements. These results reveal that mutations in the basal HSP70 promoter that affect embryonic can be complemented by a distal promoter region in which multiple redundant promoter elements are located. Acknowledgements This research has been supported by a Natural Sciences and Engineering Research Council of Canada grant to J.J.H. We would like to thank R.I. Morimoto, Northwestern University, Chicago, Ill., for kindly supplying the LSN and LSPN constructs. Bienz, M. 1984. Xenopus hsp 70 genes are constitutively expressed in injected oocytes. EMBO J. 3: 2477-2483. Chase, J.W., and Dawid, I.B. 1972. Biogenesis of mitochondria during Xenopus laevis development. Dev. Biol. 27: 504-5 18. Gorman, C.M., Moffat, L.F., and Howard, B.H. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2: 1044-1051. Greene, J.M., Larin, Z., Taylor, I.C.A., et al. 1987. Multiple basal levels of a human hsp70 promoter function differently in human and rodent cell lines. Mol. Cell. Biol. 7: 3646-3655. Heikkila, J.J., Kloc, M., Bury, J., et al. 1985. Aquisition of the heat shock response and thermotolerance during early development of Xenopus laevis. Dev. Biol. 107: 483-489. Heikkila, J.J., Ovsenek, N., and Krone, P. 1987. Examination of heat shock protein mRNA accumulation on early Xenopus laevis embryos. Biochem. Cell Biol. 65: 87-94. Hitchock, M.J.M., and Friedman, R.M. 1980. Microinjection of Xenopus oocytes. An automated device for volume control in the nanoliter range. Anal. Biochem. 109: 338-344. Kao, H.T., Capasso, O., Heintz, N., and Nevins, J.R. 1985. Cell
1992
cycle control of the human HSP70 gene: implications for the role of a cellular E1A-like function. Mol. Cell. Biol. 5: 628-633. Kimelman, D., Kirschner, M., and Scherson, T. 1987. The events of the midblastula transition in Xenopus are regulated by changes in the cell cycle. Cell, 48: 399-407. Krieg, P., and Melton, D. 1985. Developmental regulation of a gastrula specific gene injected into fertilized Xenopus eggs. EMBO J. 4: 3463-3471. Krieg, P.A., and Melton, D.A. 1987a. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155: 397-415. Krieg, P., and Melton, D. 1987b. An enhancer responsible for activating transcription at the mid-blastula transition in Xenopus development. Proc. Natl. Acad. Sci. U.S.A. 84: 2331-2335. Krone, P.H., and Heikkila, J.J. 1989. Expression of hsp 70/CAT and hsp30/CAT chimeric genes in developing Xenopus embryos. Development (Cambridge. U.K.), 106: 271-281. Milarski, K.L., and Morimoto, R.I. 1986. Expression of human HSP70 during the synthetic phase of the cell cycle. Proc. Natl. Acad. Sci. U.S.A. 83: 9517-9521. Morgan, W.D., Williams, G.T., Morirnoto, R.I., et al. 1987. Two transcriptional activators. CCAAT-box binding transcription factor and heat shock transcription factor, interact with a human hsp7O gene promoter. Mol. Cell. Biol. 7: 1129-1 138. Mosser, D.D., Theodorakis, N.G., and Morimoto, R.I. 1988. Coordinate changes in heat shock element binding activity and HSP70 gene transcription rates in human cells. Mol. Cell. Biol. 8: 4736-4744. Newport, J., and Kirschner, M. 1982a. A major developmental transition in early Xenopus embryos. I. Characterization and timing of cellular changes at the midblastula stage. Cell, 30: 675-686. Newport, J., and Kirschner, M. 1982b. A major developmental transition in early Xenopus embryos. 11. Control of the onset of transcription. Cell, 30: 687-696. Nieuwkoop, P.D., and Faber, J. 1956. Normal table of Xenopus laevis (Daudin). North Holland Publishing Company, Amsterdam. Ovsenek, N., and Heikkila, J.J. 1990. DNA sequence-specific binding activity of the heat-shock transcription factor is heatinducible before the midblastula transition of early Xenopus development. Development (Cambridge, U.K.), 110: 427-433. Ovsenek, N., and Heikkila, J.J. 1991. Analysis of CCAAT box transcription factor binding activity during early Xenopus laevis embryogenesis. Dev. Biol. 145: 323-327. Ovsenek, N., Williams, G.T, Morirnoto, R.I., and Heikkila, J.J. 1990. Cis-acting sequences and trans-acting factors required for constitutive expression of a microinjected HSP70 gene after the midblastula transition of Xenopus laevis embryogenesis. Dev. Genet. 11: 97-109. Watowich, S.S., and Morimoto, R.I. 1988. Complex regulation of heat shock- and glucose-responsive genes in human cells. Mol. Cell. Biol. 8: 393-405. Williams, G.T., McClanahan, T.K., and Morirnoto, R.I. 1989. E l a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex. Mol. Cell. Biol. 9: 2574-2587. Wu, B.J., and Morimoto, R.I. 1985. Transcription of the human hsp70 gene is induced by serum stimulation. Proc. Natl. Acad. Sci. U.S.A. 82: 6070-6074. Wu, B.J., Hunt, C., and Morimoto, R. 1985. Structure and expression of the human gene encoding major heat shock protein HSP70. Mol. Cell. Biol. 5: 330-341. Wu, B.J., Kingston, R.E., and Morimoto, R.I. 1986. Human hsp70 promoter contains at least two distinct regulatory domains. Proc. Natl. Acad. Sci. U.S.A. 83: 629-633. Wu, B.J., Williams, G.T., and Morimoto, R.I. 1987. Detection of three protein binding sites in the serum-regulated promoter of the human gene encoding the 70-kDa heat shock protein. Proc. Natl. Acad. Sci. U.S.A. 84: 2203-2207.
Biochem. Cell Biol. 1992.70:339-342. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/21/15. For personal use only.
Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis NICKOVSENEK' AND JOHNJ. HEIKKILA Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1 Received December 4, 1991 N., and HEIKKILA, J. J. 1992. Compensatory effect of distal promoter sequences on the basal expression OVSENEK, of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis. Biochem. Cell Biol. 70: 339-342. The promoter sequences involved in the basal expression of a human 70-kilodalton heat shock protein (HSP70) gene during Xenopus embryogenesis were analyzed by microinjection of mutant promoters of a HSP70 - chloramphenicol acetyltransferase fusion gene into fertilized eggs and following their expression during early development. While deletion of the HSP70 gene promoter to - 100 base pairs (bp) did not affect basal transcription in postmidblastula stage embryos, linker-scanner mutations in the CCAAT and purine box elements blocked expression. However, extension of the 5' boundary to - 188 bp restored full wild-type expression to these mutants. These results suggest that multiple redundant cis-acting regulatory elements present in the human HSP7O gene promoter can function during Xenopus embryogenesis. Key words: heat shock protein gene, Xenopus embryos, microinjection, linker-scanner mutations, transcription.
OVSENEK, N., et HEIKKILA, J. J. 1992. Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis. Biochem. Cell Biol. 70 : 339-342. Les stquences du promoteur permettant l'expression basale du gtne humain d'une prottine de choc thermique de 70 kilodaltons (kDa) (PCT70) durant I'embryogentse chez le Xenopus ont t t t analyskes en micro-injectant, dans des oeufs fertilids, des promoteurs mutants du gtne de fusion PCT7O - chloramphtnicol acktyltransftrase, puis en suivant leur expression au dtbut du dheloppement. Alors que la dtlttion d'une portion du promoteur du gtne de la PCT70, jusqu'h - 100 paires de bases, n'affecte pas le taux basal de la transcription dans les embryons ayant dtpasst le milieu du stade blastula, des mutations de liaison-lecture (linker-scanner) des tlkments de la skquence CCAAT et de la stquence riche en purines bloquent la transcription. Cependant, I'extension de la frontitre 5' du gtne a - 188 paires de bases permet l'expression de ces gtnes mutts au meme t a w que le gtne non mutt. Ces rtsultats suggtrent que de multiples sequences cis-rtgulatrices redondantes, prtsentes dans le promoteur du gtne humain de la PCT70, peuvent avoir une fonction durant l'embryogentse chez le Xenopus. Mots elks : gene de prottine de choc thermique, embryons du Xenopus, micro-injection, mutations de liaison-lecture (linker-scanner), transcription. [Traduit par la rtdaction]
Introduction During Xenopus Iaevis embryogenesis, zygote genome transcription is not activated until the midblastula stage (Newport and Kirschner 1982a, 19828; Kimelman et al. 1987). This point in development, which has been termed MBT is also characterized by a desynchronization of subsequent cell divisions and the onset of cell motility. Prior to MBT, the only detectable RNA synthesis in Xenopus embryos is from mitochondria1 genes at a low rate (Chase and Dawid 1972). Activation of the zygotic genome results in an abrupt 200-fold increase in the rate of RNA synthesis (Kimelman et al. 1987). The mechanism by which Xenopus embryos remain transcriptionally dormant until MBT and then activate selected genes is not fully understood. It has been suggested that the lack of transcriptional activity during cleavage stages is a result of an overabundance of either cytoplasmic M-phase-promoting factor or initiation factor, ABBREVIATIONS: HSP70, 70-kilodalton heat shock protein; bp, base pair(s); MBT, midblastula transition; CAT, chloramphenicol acetyltransferase; NEO, neomycin resistance; TLC, thin-layer chromatography; nt, nucleotide(s). 'present address: Center for Developmental Biology, Department of Zoology, University of Texas, Austin, TX 78712, U.S.A. Printed in Canada / Imprime au Canada
which prevents a G2phase in the cell cycle by rapidly triggering the onset of mitosis after S phase. Among the set of zygotic genes that are selectively transcribed or inducible at MBT are a number of the HSP genes including HSP70. Recently, we found that a microinjected human HSP70 promoter - CAT chimeric gene was strongly expressed under nonstress conditions at MBT, indicating that it shared common regulatory features associated with endogenous genes expressed at MBT (Ovsenek et al. 1990). The availability of various promoter mutants permitted the examination of the cis-acting sequences required for expression at MBT. Deletion mutant analysis revealed that only 74 bp of the 5' promoter region was required for constitutive expression. Also, we examined the role of specific sequences in the basal promoter by injecting HSP70-CAT vectors containing linker-scanner mutations in the basal elements such as CCAAT, purine-rich element, GC element, ATF/AP 1, and TATA. Interestingly, the alteration of any one of these elements in the basal promoter prevented expression at MBT. In the human HSP70 promoter there are a number of potentially functional cis-acting elements between - 100 and - 188 including CCAAT, AP-2, GC box, and HSE, some of which have been previously shown to interact with trans-acting factors
BIOCHEM. CELL
GC
CCAAT
AP2
BIOL. VOL. 70, 1992
CCAAT Purine Box GC
HSE
ATF/AP 1
TATA
AP2
...................... ...................... ......................
Biochem. Cell Biol. 1992.70:339-342. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/21/15. For personal use only.
FIG. 1. Diagram of the human HSP7O promoter. Major established promoter elements are indicated. LSPNILSN SERIES CCAAT BOX
-75 GCT
033
TGA TTG
GCc
gaG
atc
TcG
W
(XKj
cGA
gat
GCT
033
TGA
PURINE BOX
-43
CAG
A A G GGA
AAA
GCT
CAG
AAG
GGA
AAA
aOC
GGG
73/63
cga
tct
cgG
GGA
AAA OCC
GGG
69/55
GCT
CAG
cga
Gat
ctc
GGG
57/47
OCC
WT
FIG. 2. Diagram of the linker-scanner mutations in the human HSP70 promoter. For each mutant, the numeric notation refers to the region in the promoer in which the indicated base changes have been made. The LSPN series contains 100 bases of the promoter, whereas the LSN series contains 188 bases 5' of the transcriptional start site. WT, wild type.
(Fig. 1; Morgan et al. 1987; Mosser et al. 1988). In the present study, we have examined whether the loss of expression from the basal promoter by mutation of CCAAT and the purine box could be complemented by extending the 5 ' boundary from - 100 to - 188. Our finding that extension of the 5' boundary restores full wild-type expression to CCAAT and purine box mutants suggests that multiple redundant cis-acting regulatory elements present in the human HSP70 promoter can function in Xenopus embryos after the onset of zygotic genome transcription. Materials and methods Embryo maintenance and microinjection Xenopus laevis eggs were obtained, fertilized, dejelled, and maintained in Steinberg's solution as previously described (Heikkila et al. 1985). Embryos were staged according to Nieuwkoop and Faber (1956). Circular or linearized plasmid DNA (400 pg in 20 nL) was microinjected into embryos within 2 h of fertilization using the apparatus designed by Hitchcock and Friedman (1980). Injected embryos were maintained in Steinberg's solution with 4% w/v Ficoll (Krieg and Melton 1985). The Ficoll content of Steinberg's solution was gradually decreased to 1% by the blastula stage. Only normally developing embryos were used for CAT activity and RNase protection assays. 5'-deletion and linker-scanner mutations Specific details describing the construction of the LSPN and LSN series mutations of the human HSP70 promoter that is fused to a CAT gene are described by Williams et al. (1989). Insertion of linker-scanners into the proximal 100 bases of the promoter generates the LSPN series, while the LSN series contains 188 bases of promoter. Linker-scanner mutations were constructed as described (Wu et al. 1987). Also, the microinjected plasmids contain a neomycin resistance (NEO) gene orientated in the opposite direction. The NEO gene is regulated by the proximal 74 bases of the human HSP70 promoter and is a constant on all of the microinjected constructs. CA T assays For each analysis, 10 embryos were homogenized in 100 pL of 0.25 M Tris (pH 7.8) and then centrifuged twice for 15 min at 8000 x g to remove yolk and cellular debris. The supernatants were stored at - 70°C. Enzyme assays were performed as described by
Gorman et al. (1982) using three embryo equivalents. Conversion of [14~]chloramphenicol to acetylated forms was monitored by TLC, followed by autoradiography using Kodak XAR-5 film. Radioactivity in the acetylated forms of chloramphenicol was measured by scintillation counting. RNA isolation and RNase protection assays The isolation procedure of total lithium chloride precipitable RNA from Xenopus embryos is outlined in Heikkila et a/. (1987). RNA samples were treated with 4 U of RQ1 DNase (Promega) in 40 mM Tris (pH 7.9) - 6 mM MgCI, for 1 h at 37"C, followed by extraction with phenol-chloroform and chloroform and subsequent ethanol precipitation. A 588-bp HindIII to EcoRI fragment from p delta H18 (Wu et al. 1985) containing 188 bp of HSP-CAT promoter sequences and 400 bp of transcribed sequences was cloned into the pGEM2 vector (Williams et al. 1989). Digestion of the resulting plasmid with HindIII and transcription with SP6 RNA polymerase results in an antisense riboprobe approximately 600 nt long, of which 400 nt are protected by correctly initiated HSP-CAT RNA. Labeled transcripts were passed through a Sephadex G-50 column. RNase protection assays were performed as described by Krieg and Melton (1987a, 1987b) using 10 pg of total RNA. RNase-protected transcripts were detected following electrophoresis on 4% polyacrylamide - 8 M urea gels and exposure to Kodak XAR-5 film at - 70°C.
Results In a previous study examining the basal expression of a human HSP70 promoter - CAT fusion gene in rnicroinjected Xenopus embryos, we found that 74 bp of promoter region was sufficient for expression after the midblastula transition. Additionally, linker-scanner mutations (LSPN series) in a number of proximal promoter elements (e.g., CCAAT box, purine box, GC box, ATF region) prevented expression (Ovsenek et al. 1990). The human HSP70 promoter also contained additional putative regulatory elements (e.g., CCAAT box, GC box) upstream of the basal promoter (Fig. 1). In the present study, we have examined whether the loss of expression in some of the LSPN series of linkerscanner mutations in the proximal promoter could be complemented by sequences upstream of the basal promoter.
34 1
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NOTES
LSPNLSN wt
L S P e S N LSPNLSN L S P ~ S N 73/63 69/55 57/47
FIG. 3. Expression of LSN linker-scanner mutations in eno opus embryos. CAT activity in extracts (three embryo equivalents) from gastrula embryos that had been microinjected at the one-cell stage with 400 pg of LSNwt, LSN73163, or LSN57/47 were compared with the activity of extracts from gastrulae transformed with equal amounts of the corresponding LSPN constructs, i.e., LSPNwt, LSPN73/63. LSPN69/55, and LSPN57/47. The position of the labeled chloramphenicol is indicated by CM, while its acetylated forms are indicated by arrows. LSN69/559
TABLE1. CAT activity in gastrula stage embryos microinjected with LSPN and LSN HSP70-CAT fusion gene mutants CAT enzyme activity (Yo)*
LSPN
LSN
*Percent acetylation of chloramphenicol by equivalent amounts of embryo extract. +WT, wild type.
This was done by microinjecting DNA from a separate series of LSN linker-scanner mutants that contain the same substitutions in the proximal region as the LSPN vectors, but differ by having a 5' boundary extended to - 188 bp (Fig. 2). In these experiments, the LSPN and LSN vectors were microinjected into fertilized eggs and assayed for CAT activity at the gastrula stage. A representative autoradiogram is presented in Fig. 3. Quantitation of CAT activity displayed in Fig. 3 was carried out by scintillation counting of acetylated chloramphenicol spots cut out of the TLC plate and is presented in Table 1. The basal level of CAT activity from the LSPN and LSN wild-type vectors is similar. However, the LSPN linker-scanner mutants 73/63 and 69/55 which alter the CCAAT box and 57/47 which modifies the purine box were not expressed in gastrula stage embryos. Additionally, we utilized an internal control, namely, the expression of the NEO gene which is present on the LSPN plasmids to demonstrate the presence of these vectors in Xenopus embryos (data not shown). The lack of expression of these LSPN mutants in gastrula-stage embryos agrees with our previous results (Ovsenek et al. 1990). Interestingly, the LSN mutants which contain the identical promoter muta-
wt
73/63 69/55 57/47
FIG, 4. RNaSe protection analysis of human HSp70-CAT RNA in embryosmicroinjectedwith LSN promoter mutants. Ten micrograms of total RNA was isolated from gasmla stage embryos
that had been rnicroinjectedwith either the wild-type LSN promoter or LSN mutants outlinedin the captionto ~ i3. ~h~ ~ RNA . was subjectedto RNase protectionanalysisand separated on a 4% po~yacry~ami~e-urea denaturing gel. The arrow indicates the position of the 400-nt protected HSp70-CAT transcript. tions found in the LSPN mutants but have an extended 5' boundary ( - 188 bp) were fully expressed (Fig. 3; Table 1). The level of CAT activity in the 73/63,69/55, and 57/47 LSN mutants were similar to that found with the LSN wild type. The constitutive expression of the LSN mutants was detected immediately after the midblastula transition, as well as in postgastrula stage embryos (data not shown). RNase protection analysis was performed to ensure that the transcription of microinjected LSN mutants was initiated correctly. As shown in Fig. 4, a labeled antisense riboprobe spanning the transcriptiona1 start site of the HSP70-CAT fusion gene protects a correctly initiated 4004 transcript present in rota1 RNA from gastrula stage embryos that had been injected with either the LSN wild-type vector or the 73/63, 69/55, and 57/47 LSN mutants. Since alteration of the same promoter sequences that inactivated the - 100 deletion mutant LSPN did not have an affect on the expression of the - 188 deletion mutant LSN, it is tenable that sequences between - 100 and - 188 of the HSP70 promoter can restore transcriptional activity.
Discussion The role of promoter sequences upstream of the basal human HSP70 promoter during early Xenopus embryogenesis was investigated by microinjecting a set of LSN linker-scanner mutants that included 188 bp of the human HSP70 promoter. Base alterations that inactivated the basal promoter had no effect on HSP70-CAT expression when promoter sequences between - 100 and - 188 were present (Fig. 3). It is possible that the promoter elements in the upstream region of the promoter act alone or in concert with intact elements in the basal promoter to restore transcriptional activity to the LSN mutants. It is tenable that the upstream CCAAT element may substitute for the downstream CCAAT sequence. Interestingly, we found that a mutation in the purine box could also be rescued by additional upstream promoter sequences, even though no additional purine boxes were present. This result suggests that other sequences such as the upstream GC, CCAAT, ATF, or HSE elements may compensate for a nonfunctional
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purine box in the basal promoter. In fact, Xenopus transcription factors have been shown to interact with human CCAAT, ATF, GC, and HSE elements (Ovsenek et al. 1990; Ovsenek and Heikkila 1990, 1991). Our finding that distal elements may also play a role in the expression of the parental human HSP70-CAT fusion gene in developing Xenopus embryos supports previous work demonstrating that the complex pattern of HSP70 regulation involves several cis-acting regulatory elements (Wu et al. 1986; Greene et al. 1987; Morgan et al. 1987; Wu et al. 1987). Expression of the human HSP70 gene is regulated during normal cell growth and differentiation (Kao et al. 1985; Wu and Morimoto 1985; Milarski and Morimoto 1986) and in response to various stresses (Wu et al. 1986; Watowich and Morimoto 1988). The multiple cis-acting regulatory elements that are involved in the complex transcriptional regulation of the human HSP70 gene appear to be redundant in the heterologous Xenopus system. There may be a number of differences between the array of transcription factors present in human cells and Xenopus embryos that account for the activity of the human HSP70 promoter under nonstress conditions. The recent observation that a microinjected Xenopus HSP70-CAT fusion gene is only expressed in a heat-inducible manner in postmidblastula stage embryos (Krone and Heikkila 1989) strengthens this hypothesis. Comparison of the human and Xenopus HSP7O (Bienz 1984) promoters indicates that the ATF binding site and the purine-rich region present in the human promoter are absent in the Xenopus HSP70 promoter. The human and Xenopus HSP70 promoters are otherwise quite similar in terms of the spatial organization of cis-regulatory elements such as CCAAT, TATA, HSE, and GC elements. These results reveal that mutations in the basal HSP70 promoter that affect embryonic can be complemented by a distal promoter region in which multiple redundant promoter elements are located. Acknowledgements This research has been supported by a Natural Sciences and Engineering Research Council of Canada grant to J.J.H. We would like to thank R.I. Morimoto, Northwestern University, Chicago, Ill., for kindly supplying the LSN and LSPN constructs. Bienz, M. 1984. Xenopus hsp 70 genes are constitutively expressed in injected oocytes. EMBO J. 3: 2477-2483. Chase, J.W., and Dawid, I.B. 1972. Biogenesis of mitochondria during Xenopus laevis development. Dev. Biol. 27: 504-5 18. Gorman, C.M., Moffat, L.F., and Howard, B.H. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2: 1044-1051. Greene, J.M., Larin, Z., Taylor, I.C.A., et al. 1987. Multiple basal levels of a human hsp70 promoter function differently in human and rodent cell lines. Mol. Cell. Biol. 7: 3646-3655. Heikkila, J.J., Kloc, M., Bury, J., et al. 1985. Aquisition of the heat shock response and thermotolerance during early development of Xenopus laevis. Dev. Biol. 107: 483-489. Heikkila, J.J., Ovsenek, N., and Krone, P. 1987. Examination of heat shock protein mRNA accumulation on early Xenopus laevis embryos. Biochem. Cell Biol. 65: 87-94. Hitchock, M.J.M., and Friedman, R.M. 1980. Microinjection of Xenopus oocytes. An automated device for volume control in the nanoliter range. Anal. Biochem. 109: 338-344. Kao, H.T., Capasso, O., Heintz, N., and Nevins, J.R. 1985. Cell
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cycle control of the human HSP70 gene: implications for the role of a cellular E1A-like function. Mol. Cell. Biol. 5: 628-633. Kimelman, D., Kirschner, M., and Scherson, T. 1987. The events of the midblastula transition in Xenopus are regulated by changes in the cell cycle. Cell, 48: 399-407. Krieg, P., and Melton, D. 1985. Developmental regulation of a gastrula specific gene injected into fertilized Xenopus eggs. EMBO J. 4: 3463-3471. Krieg, P.A., and Melton, D.A. 1987a. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155: 397-415. Krieg, P., and Melton, D. 1987b. An enhancer responsible for activating transcription at the mid-blastula transition in Xenopus development. Proc. Natl. Acad. Sci. U.S.A. 84: 2331-2335. Krone, P.H., and Heikkila, J.J. 1989. Expression of hsp 70/CAT and hsp30/CAT chimeric genes in developing Xenopus embryos. Development (Cambridge. U.K.), 106: 271-281. Milarski, K.L., and Morimoto, R.I. 1986. Expression of human HSP70 during the synthetic phase of the cell cycle. Proc. Natl. Acad. Sci. U.S.A. 83: 9517-9521. Morgan, W.D., Williams, G.T., Morirnoto, R.I., et al. 1987. Two transcriptional activators. CCAAT-box binding transcription factor and heat shock transcription factor, interact with a human hsp7O gene promoter. Mol. Cell. Biol. 7: 1129-1 138. Mosser, D.D., Theodorakis, N.G., and Morimoto, R.I. 1988. Coordinate changes in heat shock element binding activity and HSP70 gene transcription rates in human cells. Mol. Cell. Biol. 8: 4736-4744. Newport, J., and Kirschner, M. 1982a. A major developmental transition in early Xenopus embryos. I. Characterization and timing of cellular changes at the midblastula stage. Cell, 30: 675-686. Newport, J., and Kirschner, M. 1982b. A major developmental transition in early Xenopus embryos. 11. Control of the onset of transcription. Cell, 30: 687-696. Nieuwkoop, P.D., and Faber, J. 1956. Normal table of Xenopus laevis (Daudin). North Holland Publishing Company, Amsterdam. Ovsenek, N., and Heikkila, J.J. 1990. DNA sequence-specific binding activity of the heat-shock transcription factor is heatinducible before the midblastula transition of early Xenopus development. Development (Cambridge, U.K.), 110: 427-433. Ovsenek, N., and Heikkila, J.J. 1991. Analysis of CCAAT box transcription factor binding activity during early Xenopus laevis embryogenesis. Dev. Biol. 145: 323-327. Ovsenek, N., Williams, G.T, Morirnoto, R.I., and Heikkila, J.J. 1990. Cis-acting sequences and trans-acting factors required for constitutive expression of a microinjected HSP70 gene after the midblastula transition of Xenopus laevis embryogenesis. Dev. Genet. 11: 97-109. Watowich, S.S., and Morimoto, R.I. 1988. Complex regulation of heat shock- and glucose-responsive genes in human cells. Mol. Cell. Biol. 8: 393-405. Williams, G.T., McClanahan, T.K., and Morirnoto, R.I. 1989. E l a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex. Mol. Cell. Biol. 9: 2574-2587. Wu, B.J., and Morimoto, R.I. 1985. Transcription of the human hsp70 gene is induced by serum stimulation. Proc. Natl. Acad. Sci. U.S.A. 82: 6070-6074. Wu, B.J., Hunt, C., and Morimoto, R. 1985. Structure and expression of the human gene encoding major heat shock protein HSP70. Mol. Cell. Biol. 5: 330-341. Wu, B.J., Kingston, R.E., and Morimoto, R.I. 1986. Human hsp70 promoter contains at least two distinct regulatory domains. Proc. Natl. Acad. Sci. U.S.A. 83: 629-633. Wu, B.J., Williams, G.T., and Morimoto, R.I. 1987. Detection of three protein binding sites in the serum-regulated promoter of the human gene encoding the 70-kDa heat shock protein. Proc. Natl. Acad. Sci. U.S.A. 84: 2203-2207.
