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. 1988 Dec;336(6201):744-9.
doi: 10.1038/336744a0.

Activation and repression of transcription by homoeodomain-containing proteins that bind a common site

J B Jaynes  1 , P H O'Farrell
Affiliations

Activation and repression of transcription by homoeodomain-containing proteins that bind a common site

J B Jaynes et al. Nature. 1988 Dec.

Abstract

The product of the fushi tarazu (ftz) gene is shown to be a site-dependent activator of transcription. In vitro-defined binding sites act as ftz-dependent enhancers in cultured cells. Another homoeodomain-containing protein, the engrailed gene product, competes for homoeodomain-binding sites and counteracts ftz activation.

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Figures

Fig. 1
Fig. 1
Plasmids. a, Homoeodomain protein producer plasmids (Ac-ftz and pAc-en) contain complete cDNA coding sequences inserted into Marc Krasnow's actin 5C promoter/polyadenylation signal vector, pPAc. Homoeodomains are the solid black regions. Ac-ftz (pPAcG1100) was provided by Gary Winslow and Matthew Scott. Stop codons were introduced into the coding sequences to produce pAc-ftzSTOP and pAc-enSTOP (see below). b, Responder plasmids 1 and 2 containing truncated distal ADH promoters (from −33 and −86 nucleotides, respectively, to +53 nt; −86dADH includes a binding site for a factor that appears to be important in regulating ADH expression in the embryo47) driving chloramphenicol acetyltransferase (CAT) gene expression (pD –33CAT, pD –86CAT) were provided by Bruce England and Robert Tjian. Six tandem copies of a consensus homoeodomain-binding site (NP6, see c below) were inserted immediately upstream of these promoters. Responders 3 and 4 contain the hsp70 promoter driving β-galactosidase (β-gal) expression; the latter includes cis-acting heat-shock transcription factor binding sites,. Various homoeodomain-binding sites were inserted just upstream by Jean-Paul Vincent (J.-P. Vincent and P.H.O'F., unpublished results). Responder 5 (pAF0) contains a proximal ADH promoter and structural gene. Again, NP6 was inserted at the 5'-end of the promoter. c, Sequences of homoeodomain-binding sites used in the responder constructs are shown. Tandem arrows show the relative orientations of the individual NP sites within the multimers. No orientation can be assigned to the palindromic RP and LP sites. d, Reference plasmids. pCOPiCAT contains a copia transposable element long terminal repeat (LTR) (including the copia promoter) fused to a CAT structural gene. hsp82LacZ (pLac82SU) was provided by Dale Dorsett (Sloan-Kettering Cancer Center, New York) and contains the hsp82 promoter driving a β-gal structural gene. Methods. pAc-en was made by inserting a blunt-ended EcoRI fragment, containing en cDNA leader, complete coding, and 180 nucleotides of 3′ non-translated sequences, into the unique BamHI site of pPAc. An XbaI nonsense codon linker (NEB) was inserted into the unique MluI site following en amino-acid 406 (out of 552) to yield pAc-enSTOP. A second nonsense insertion after en amino-acid 81 (NotI site) was also constructed and tested as a negative control (see text). The en homoeodomain (amino acids 453–512) should not be produced by these `STOP' plasmids. pAc-ftzSTOP was similarly constructed from Ac-ftz by insertion of a nonsense codon linker into the unique XhoI site after the 16th amino acid of the homoeodomain. A second nonsense insertion after the 33rd amino acid of the homoeodomain (EcoRV site) was also made and tested for activity (see text). Both of these `STOP' constructs are expected to produce a ftz protein truncated before the putative helix-turn-helix DNA-binding motif. To make the `+ sites' versions of responders 1 and 2, a SmaI-PstI fragment from an M13mp18 clone containing NP6 in the BamHI site was inserted into pD −33/−86CAT cut with XbaI (blunted) and PstI. Construction of responders 3 and 4 will be described in detail elsewhere. Briefly, they consist of a P-element vector (pSXhLac-7, obtained from Pieter Wensink, Brandeis University) with hsp70 promoter and leader sequences (−194 to +1,250 nucleotides) fused to the Escherichia coli LacZ structural gene, followed by hsp70 3′ non-coding sequences. Homoeodomain-binding sites were inserted into unique restriction sites immediately upstream of the promoter. Responder 5 contains the Drosophila melanogaster proximal ADH promoter and structural gene starting at −386 nucleotides. A PstI-EcoRI (blunted) fragment from the M13mp18 clone containing NP6 was inserted into the unique PstI site immediately upstream of the proximal ADH promoter. pCOPiCAT was constructed by fusing a bluescript vector (Stratagene; cut with EcoRI and BamHI) with both the copia LTR (EcoRI-HindIII fragment) from pCOPneo and the CAT gene/poly(A)-site region (HindIII-BamHI fragment) from pSV2CAT.
Fig. 2
Fig. 2
Ftz induces transcription in cultured cells. Drosophila cells (Schneider line 2, cultured as previously described54) were co-transfected with (1) either (+ftz) `producer' plasmid expressing ftz cDNA, or (–ftz) a control plasmid (either pAc-ftzSTOP or pPAC), and (2) the indicated `responder' plasmid (# 1–# 5, see Fig. 1), either with (upper panel) or without (lower panel) the NP6 homoeodomain-binding sites. Responder activity was ascertained by quantitating the appropriate enzymatic activity in whole-cell extracts. Activity was determined relative to the activity of a co-transfected reference gene plasmid; the results were essentially the same when expressed per μg of total protein in the extract (determined by the method described55). Activity is normalized to the basal level of each responder, without binding sites and `–ftz' (set at 1.0). Error bars indicate the range of values in duplicate transfections within the same experiment. Equivalent results were obtained in at least two separate experiments for each responder. Methods. Cells were grown in Schneider's Drosophila medium supplemented with 12% fetal bovine serum. Transfection was by the calcium phosphate precipitation method, done essentially as described. Cells were collected two days after transfection by scraping, rinsed once with phosphate-buffered saline and lysed by three cycles of freeze-thaw (−70 °C to room temperature). Extracts were prepared by centrifugation (15,000g, 5 min), and supernatants were either stored at −70 °C or assayed immediately. CAT activity (from responders 1 and 2, and pCOPiCAT reference gene used with responders 3 and 4) was determined as described. Relative activities were the same whether or not extracts were heated (68 °C, 5 min) to destroy potential de-acetylase activity. High activity extracts were diluted to keep determinations within the linear range of the assay, as shown by standard curves. CAT reaction time courses and extract mixing experiments indicated that little or no inhibitory activity was present in low activity extracts (data not shown). β-galactosidase (β-gal) activity (from responders 3 and 4 and hsp82LacZ reference gene used with responders 1, 2 and 5) was determined by fluorimetric assay. Drosophila ADH activity was determined as described. There was a detectable background activity in cells from control transfections (without responder plasmid) for β-gal and ADH (but not CAT). The β-gal background was at most 10% of the smallest plasmid-dependent activity, and so did not affect the results presented. The ADH background was relatively higher, about equal to the uninduced activities shown (responder 5), so the induction value for the −386pADH promoter (60-fold) is a minimum estimate. Amounts of plasmid DNA used per transfection (60-mm Petri dish) were as follows: 5 μg responder 1 (+/− sites) with 1 μg Ac-ftz (+/− STOP codon), 1 μg hsp82LacZ (reference gene), and 3 μg pAc-ftzSTOP; 1 μg responder 2 (+/− sites) with either 1 μg Ac-ftz and 8 μg pPAc, or 9 μg pPAc; 0.1 μg responder 3 (+/− sites) with 3 μg Ac-ftz (+/− STOP codon), 1 μg pCOPiCAT (reference gene), and 3 μg pPAc; 0.05 μg responder 4 (+/− sites) with 3 μg Ac-ftz (+/− STOP codon), 1 μg pCOPiCAT (reference gene), and 6 μg pAc-ftzSTOP; or, 7 μg responder 5 (+/− sites) with 3 μg Ac-ftz (+/− STOP codon), and 0.01 μg hsp82LacZ (reference gene). Amounts of responder DNA were chosen to give a readily quantifiable basal (uninduced) activity level, except responder 5 (see above). Small amounts of responders 3 and 4 were used because much larger amounts (1−2 μg) gave somewhat reduced induction ratios, consistent with either a titration of activating factor(s) or a `ceiling' on stable β-gal accumulation in the cells.
Fig. 3
Fig. 3
Alterations in binding sites change responsiveness to ftz. Different amounts of ftz producer (see below) were co-transfected with the −50hsp70 responder containing, a, either zero, two, four, or six NP binding sites (see Fig. 1), and, in b, four tandem copies of either the consensus (NP), or single-nucleotide alterations (LP, RP; see Fig. 1), or no homoeodomain-binding sites. Activity of each responder was determined as in Fig. 2, and was normalized to the basal level without ftz, set at 1.0, to give `Responder induction'. Error bars indicate the range of values of duplicate transfections. Similar results were obtained with the −194hsp70 responder. Amounts of plasmid DNA used per transfection were: 0.1 μg each responder with 3 μg pPAC, 1 μg pCOPiCAT (reference gene), and the amounts of ftz producer (Ac-ftz) shown. Total DNA was made constant by adding pAc-ftzSTOP. Control experiments using a plasmid with an actin 5C promoter driving CAT showed that expression levels were proportional to the amount of added DNA from 0.1 to 10 μg (data not shown).
Fig. 4
Fig. 4
Repression by en. Error bars indicate the range of values from duplicate transfections in the same experiment. Equivalent results were obtained in at least two separate experiments. a, Cells were co-transfected (see Fig. 2 legend) with (1) producer plasmids Acftz (`with ftz'), pAc-en (+en), pAc-ftzSTOP (`without ftz'), and/or pAc-enSTOP (-en), and (2) the indicated responder (see Fig. 1) with the NP6 homoeodomain-binding sites. Without homoeodomain-binding sites, there was no response to either ftz (Fig. 2) or en (data not shown). Amounts of plasmid DNA used per transfection were as follows: 5 μg responder 1 with 1 μg hsp82LacZ (reference gene) and either (1) (−ftz, −en) 4 μg pAc-ftzSTOP, (2) (+ftz, −en) 1 μg Ac-ftz and 3 μg pAc-enSTOP, or (3) (+ftz, +en) 1 μg Ac-ftz, 1 μg pAc-en, and 2 μg pAc-enSTOP; 0.1 μg responder 3 with 1 μg pCOPiCAT (reference gene) and either (1) (−ftz, −en) 3 μg pAc-ftzSTOP and 3 μg pPAc, (2) (+ftz, −en) 3 μg Ac-ftz and 3 μg pAc-enSTOP, or (3) (+ftz, +en) 3 μg Ac-ftz and 3 μg pAc-en; 0.05 μg responder 4 with 0.2 μg pCOPiCAT (reference gene), 3 μg Ac-ftz (+/−STOP codon, = `without/with ftz'), and 6 μg pAc-en (+/−STOP codon, = `−/+en'); 7 μg responder 5 with 0.01 μg hsp82LacZ (reference gene) and either (1) (−ftz, −en) 3 μg pAc-ftzSTOP, (2) (−ftz, +en) 2 μg pAc-ftzSTOP and 1 μg pAc-en, (3) (+ftz, −en) 1 μg Ac-ftz, 1 μg pAc-ftzSTOP, and 1 μg pAc-enSTOP, or (4) (+ftz, +en) 1 μg Ac-ftz, 1 μg pAc-ftzSTOP, and 1 μg pAc-en. `ND', response with en alone not determined in this experiment; several other experiments with responder 1 showed that en had a small negative effect with binding sites (see text). b, Different amounts of en producer were co-transfected with a constant amount of ftz producer and the −194hsp70 responder containing either no homoeodomain-binding sites, or the NP4, LP4, or RP4 binding site array (see Fig. 1). Amounts of plasmid DNA per transfection were: 0.05 μg each responder with 3 μg Ac-ftz, 1 μg pCOPiCAT (reference gene), and the amounts of en producer (pAc-en) shown. Total DNA was made constant with pAc-enSTOP. Responder activities for both a and b were determined as described in the Fig. 2 legend.
Fig. 5
Fig. 5
Multiple patterns of gene expression can be generated by competition among regulators for binding to related but different cis-acting sites. Beginning with overlapping patterns of expression of three regulatory proteins with related DNA-binding specificities, three novel patterns are produced by differential competition. A is an activator, whereas R1 and R2 are repressors. Related cis-acting sites in the responding genes have different relative affinities for the regulators. The first responding gene has a binding site with relative affinities R1 > A > R2, so that it is activated by A and repressed by R1, but not by R2. The second responding gene has a site with relative affinities R2 > A > R1, whereas the site in the third gene has a higher affinity for both R1 and R2 than for A. Such a scheme could operate in the embryo to refine spatial patterns of regulatory gene expression and to subdivide domains of expression. This simple example assumes that regulator concentrations are equal and constant in space and time. Similar competitive interactions could also generate multiple domains of responder expression from a gradient of regulator concentration.
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