DEVELOPMENTAL

BIOLOGY

148, 165-173 (1991)

The Transcription Factor, Egr-1 , Is Rapidly Modulated to Retinoic Acid in PI 9 Embryonal Carcinoma

in Response Cells

STEVENA. EDWARDS,**~TRISTANDARLAND,*RONALDSOSNOWSKI,~ MICHAEL SAMUELS,* AND EILEEN D. ADAMSON**’ *La Jullw Cuv~er Reseurch Foundutim, 10901 North Turrey Pines Roud, La Jolla, Cul(tin-nia 920.37; and tThe Cancer Cmter, livckersity of Cal~fcrniu, San Digrlo. Sm Dirqo, CalQbrnia 92037 Accepted April 4, 1991 The pluripotent murine embryonal carcinoma cell line, P19, differentiates along at least three main pathways under the inductive influence of retinoic acid (RA). The events most critical to the establishment of a particular differentiation pathway must occur early since P19 cells are committed to differentiation pathways after 30 min of exposure to RA (M. W. McBurney, personal communication and our unpublished results). We have, therefore, looked for genes that are induced (or repressed) within 30 min of RA addition and find that Egr-1 is one of these genes. Egr-1 is a transcription factor of the zinc-finger class and is known to transactivate genes after binding to specific oligonucleotide sequences. We describe here the extremely rapid and transient increase of Egr-1 transcript and protein levels in P19 cells after RA addition. Stable induction of Egr-1 transcripts occurred in the presence of protein synthesis inhibitors. Simultaneous addition of RA and cycloheximide did not result in an additive effect. The mechanism of induction with either drug appears to involve relief of a block to transcriptional elongation. The response was more rapid at high RA conrent,rations and this suggests that the Egr-1 transcription factor could play a role in initiation of differentiation pathways of P19 EC Ce&3.

cc‘ 1991 Academic

Press, Inc.

adult mouse brain, there is a gradient of Egr-1 expression with the highest levels found in the hippocampus and cerebral cortex (Christy et aZ., 1988; Saffen et al., 1988; Waters et al., 1990). Induction of Egr-1 (NGFlA) occurs after the induction of PC12 pheochromocytoma cells with NGF, a treatment that leads to differentiation into cells similar to sympathetic neurons. In most instances, Egr-1 is coregulated with the protooncogene c-fos including transient induction of expression with mitogens (Lemaire et al., 1988; Sukhatme et al., 1987) in various cell types, and induction by a variety of methods employed to stimulate neuronal cells (Sukhatme et ul, 1988; Saffen et al., 1988; Cole et al., 1989). Relatively stable expression of both Egr-1 and c-fos occurs at a number of sites in the developing embryo, particularly sites of chondrogenesis and ossification (McMahon et al., 1990) and extraembryonic tissues (Mtiller et al., 1983; Adamson et al., 1985 and our unpublished results). We have previously documented the induction of Egr1 and c-fos expression at relatively late stages of differentiation of P19 cells (Edwards and Adamson, 1986; Sukhatme et aZ., 1988; Darland et ab, 1991). The constitutive expression of c-fos and Egr-1 transcripts in fully differentiated cells is paralleled by the appearance of unusually stable forms of c-Fos and Egr-1 proteins that we hypothesize play roles in the establishment or the maintenance of the differentiated state (Darland et aZ., 1991). However, the events most critical to the establishment of a particular differentiation pathway must

INTRODUCTION

The early growth response gene, Egr-1 (zfp-6 in Standardized Genetic Nomenclature for Mice), also known as NGFlA (Milbrandt, 198’7), Krox 24 (Lemaire et ab, 1988), zif268 (Christy et al., 1988), and TlS-8 (Lim el ah, 1987), encodes a protein with three adjacent zinc-finger motifs, structures that are present in many DNA-binding transcription factors. Egr-1 has been shown to bind specifically to a GC-rich nine base pair consensus sequence (Christy and Nathans, 1989; Cao et ah, 1990; Lemaire et al., 1990). Egr-1 is a member of a family of mammalian genes which have some homology in their DNA binding domains to Krtippel, a segmentation gene of Drosophila. Some of these genes, including Egr-1, are expressed in a stage- and tissue-specific manner during development (McMahon et al, 1990; Chowdhury et al, 1988; Wilkinson et ah, 1989). Also related to Egr-1 is a gene that appears to be responsible for the suppression of Wilm’s tumor (Call et al, 1990; Gessler et ah, 1990) and which is also involved in the normal developmental processes of the urogenital tract (Pritchard-Jones et al., 1990). Egr-1 is ubiquitously expressed but accumulates to relatively high levels in only a few adult tissues including brain, heart, and lung (Sukhatme et al., 1988). In the i Current address: Dept. of Biochemistry, Meharry 1005 David B. Todd Blvd., Nashville, TN 37208. ‘To whom correspondence should be addressed.

Medical College,

165

0012.1606/91 $3.00 Copyright All rights

(0 1991 by Academic Press, Inc. of reproduction in any form rexwed.

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DEVELOPMENTALBIOLOGY

“Zinc fingers” ATG

2456 nt lntron 664 nt I

-

3 3769 nt

peptlde

I 3’ probe 190 nt

5’ probe 302 nt

FIG. 1. Diagram of the mouse Egr-1 gene to show the location of the 5’ and 3’ probes used for ribonuclease protection assays. The peptide (-) used to prepare a rabbit antibody was derived from sequences overlapping the third zinc finger. The latter is part of the DNA-binding domain. T, TATA box; ATG, two predicted translation start sites of which the first is the one used in mouse 3T3 cells (Ragona et al., 1991).

occur much earlier. P19 cells may be fully committed to differentiation pathways after 30 min (our unpublished results) to 4 hr (Berg and McBurney, 1990) of exposure to RA. We now report that there is an extremely rapid and transient induction of Egr-1 in P19 cells within 20 min of RA addition. We have made some preliminary observations of the nature of the regulatory mechanism and hypothesize that the regulation of Egr-1 expression by RA may be due in part to rapid changes in rates of transcriptional elongation. The rapid production of an increased level of a transcription factor that has been implicated in developmental processes has important implications to the subsequent events of differentiation.

SDS were then added and samples incubated for an additional 15 min at 37°C. Samples were phenol/chloroform extracted, then ethanol precipitated using glycogen as a carrier. Protected fragments, 302 and 190 bp for 5’ and 3’ probes, respectively, were analyzed on denaturing acrylamide/urea gels (Maniatis et ab, 1982). Sizes were determined by comparison to end-labeled MspI fragments of pBR322 (BRL). The dried gels were exposed to XAR film for l-3 days, and the film was subjected to signal quantification using an LKB laser densitometer. As an invariant control, a riboprobe that detects L32, a ribosomal protein gene transcript, was included in all assays (Dudov and Perry, 1984). Immunoprecipitations

MATERIALS

RNAse Protection

AND METHODS

Assays

This procedure was performed essentially as described by Melton et al. (1984). Two RNA probes for Egr1 sequences were used (Fig. 1). One was complementary to the first 302 bases of clone OC3.1 (Sukhatme et ah, 1988) a nearly full length cDNA clone of Egr-1. The other was complementary to 206 base pairs at the 3’end. DNA fragments were subcloned into pGEM4 (Promega Biotec) and the RNA probes synthesized with T7 polymerase (Stratagene) using [32P]UTP as the labeled nucleotide. Total cellular RNA was isolated as previously described (Edwards and Adamson, 1986). Fifty micrograms of RNA was hybridized to probe for each sample. Hybridization was performed at 45°C in 30-40 ~1 of buffer containing 80% deionized formamide, 40 mM Pipes, pH 6.7,0.4 M NaCl, and 1 mM EDTA. Hybridization was continued for 24-48 hr. After hybridization, samples were diluted with 300 ~1 of RNAse digestion buffer (10 mM Tris-HCl, pH 7.4,0.3 M NaCl, and 5 mM EDTA) containing 40 pg/ml RNAse A (Sigma) and ‘700 U/ml RNAse Tl (BRL), and incubated at 30°C for 45 min. Two micrograms of proteinase K and 20 ~1 of 10%

Preparation of anti-Egr-1 antisera was as previously described for c-fos (Adamson et al., 1985), and the gene location of the peptide is shown in Fig. 1. The specificity of the anti-Egr-1 peptide is demonstrated in Fig. 2. The antibody recognizes Egr-1 in human and mouse cells. Cells were incubated for 30-min time periods as indicated in the Fig. 4 legend in methionine-free DMEM containing 0.5 to 1.0 mCi/ml [35S]methionine and cysteine (ICN). Cells were washed in PBS then lysed in 1 ml RIPA buffer (1% deoxycholate, 1% Nonidet-P40, 0.1% SDS, 50 mM Tris, pH 8.0, 0.4 M NaCl). Equal quantities of TCA precipitable counts were used for each sample. Antibody complexes were collected on fixed Staphylococcus aureus. Precipitated products were analyzed by 7.5% SDS-PAGE and fluorography. Films were exposed for l-2 days. Nuclear Run-On Transcription

Assays

P19 cells were cultured and treated with drugs as indicated in the Fig. 5 legend. Nuclei were prepared, and nascent RNA transcripts labeled with [32P]UTP (NEN) and isolated essentially as described (Greenberg and Ziff, 1984). Denatured linearized plasmids (10 pg DNA)

EDWARDS ET AL.

Rapd Response to RA in ECC

-116 -97 Egr -66

1Fos

HF-QIJ

HF-Stim.

FIG. 2. Test of anti-Egr antibodies. Metabolic labeling (%-met) of quiescent and serum-stimulated (60 min of stimulation) human fibroblasts for 30 min was followed by lysis of cells and analyses of aliquots containing equal TCA-precipitable radioactivity. Anti-c-fos antibodies detect a broad band of labelled protein at 50-60 kDa, which is present in increased levels in serum-stimulated cells (right). Similarly, anti-Egr antibodies detect a major protein at 82 kDa much increased in stimulated cells. This result is typical of immediate early growth response genes. The heavy bands at the top of the gel are matrix proteins that bind to Staphylococcus A independently of the antibody added.

were fixed to nitrocellulose strips using a “slot blot” manifold (BRL). Plasmid probes used were a full length Egr-1 cDNA clone, (OC3.1, Sukhatme et ah, 1988), an Egr-15’ probe of 340 nts from l-340, an Egr-13’ probe of 3448 nts from 341 to 3789 (see Fig. l), a full length c-fos probe, and a human P-actin cDNA clone (Ponte et ah, 1984), or L32 ribosomal protein gene (Dudov and Perry, 1984) for standardization. pGEM-1 (Promega Biotech) was used as a control plasmid for nonspecific signal. Nitrocellulose strips were prehybridized for 16 hr in hybridization buffer (5~ SSPE, 50% formamide). Hybridization was performed in fresh buffer with labeled RNA for 4 days at 42°C. Strips were washed and treated with RNAse (10 pg/ml) to remove background as described (Ausubel et al., 1989). RESULTS

RA Induces a Transient Increase in Egr-1 Transctipts Unstimulated P19 cells express a fairly low steadystate level of Egr-1 mRNA and this level is greatly and rapidly increased by treatment with retinoic acid. By RNAse protection experiments (Fig. 3a) using a probe for the extreme 3’terminus of Egr-1, we found that Egr1 transcripts rapidly accumulated in aggregate cultures of P19 cells after stimulation with RA, to reach fivefold greater levels by 10 min. This was followed by a period

167

during which levels declined substantially below basal levels, a decrease at 90 min after RA addition, of as much as 20-fold from peak levels (Fig. 3b). No response to RA was seen in the accumulation of L32 transcripts, which encode a ribosomal protein (Fig. 3a). Figure 3a also shows the signal detected by the 5’ probe in ribonuclease protection assays. The absolute level of signal detected cannot be directly compared between the two probes because the UTP label incorporates better into the 3’ compared to the 5’ probe which is composed of 70% G + C bases. However, the 5’ probe is longer, and they may be approximately equally labeled since the exposure times are similar at about 2 days. Notice that the signal detected by the 5’ probe varies relatively little over the RA induction period. The induction of differentiation of P19 cells (McBurney et al., 1982; Edwards and McBurney, 1983) requires two signals: RA (or DMSO) and the use of aggregate cultures of cells, readily formed by P19 cells when they are seeded in Petri dishes. Cultures exposed to RA at >0.5 PM for 2-3 days in aggregate culture followed by 3-6 days as outgrowths in tissue culture dishes differentiate into neural and glial cells, RA at 10 nM (or DMSO at 1%) for 4-5 days followed by 2-7 days in tissue culture produce mostly skeletal muscle, while RA at 1 nM (or DMSO at 0.5%) stimulates the differentiation into predominantly cardiac muscle and visceral endoderm cells. Monolayers at all RA concentrations differentiate into a mixture of fibroblast/epithelial/endodermal cells; muscle and nerve are never seen. It was therefore of interest to determine if the response to RA differed under these different conditions. Relative to aggregate cultures we found that monolayer cultures responded more slowly and required higher concentrations of RA. Treatment of monolayers of P19 cells with high concentrations of RA (3 PM) produced fourfold higher levels of Egr-1 transcripts after 20 min (compared to fivefold in 10 min for aggregate cultures, Fig. 3b). One hundredfold lower levels of RA (30 nM) on aggregate cultures gave Egr-1 transcript levels fivefold higher than basal at 20 min, while monolayers peaked at 40 min with only a twofold rise. Thus there appears to be a correlation between the extent and timing of the response and the nature of the inducing signal to differentiate. There were significant differences in the amplitude (but not the kinetics) of the Egr-1 response as measured with a 5’ probe. Figure 3c illustrates the difference between the two measurements for treatment of a monolayer culture with RA, and similar divergence was seen for the aggregate cultures (see Fig. 3a). Such a discrepancy could be explained if the the 5’ end of the gene is constitutively transcribed at a higher rate than the 3’ end, a proposition that is consistent with other data we have obtained (see below).

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FIG. 3. Transient increase in Egr-1 transcript levels was observed after RA stimulation. P19 cells were cultured either as aggregates (a and b) or as monolayers (b and c). Cells were treated with 3 PLMRA or 30 nM RA (low concentrations in b only) for the time periods indicated, total RNA was prepared, and RNAse protection experiments were performed using probes for either the 5’or 3’ends of the Egr-1 message, or a probe for L32, a message encoding a ribosomal protein. Figure 3a is a comparison of autoradiographs of RNAse protection assays for the 3’ and 5’ probes at various time points (minutes) after RA stimulation of P19 aggregates. Figure 3b shows a kinetic comparison of monolayers versus aggregate in their reaction to RA at the two different concentrations using the 3’ probe. Figure 3c is a graphic comparison of Egr-1 transcripts detected using 5’ versus 3’ probes on monolayer cultures, at various times in minutes after RA addition. Similar divergence was observed for aggregate cultures. Figures 3b and 3c are normalized such that values obtained for untreated cultures equal one.

synthesis inhibitors (Sukhatme et al., 1987; Lau and Nathans, 198’7),we were also interested in the effects of Egr-1 mRNA accumulation as observed with the 3’ these inhibitors on transcription. probe is matched by corresponding changes in the rate P19 cells were treated with cycloheximide alone or in of Egr-1 protein synthesis. P19 cells were metabolically combination with RA before the analysis of transcriplabeled with [35S]methionine in 30-min increments after tion rates by run-on assays. In untreated P19 monolayer RA (1 PM) treatment. Cell lysates were immunoprecipicultures, the transcription rates for Egr-1 and actin tated with an anti-peptide antiserum specific for Egr-1 were similar. After 10 min of treatment with either RA (Fig. 2) and analyzed by SDS-PAGE (Fig. 4). Egr-1 is (3 PM), cycloheximide (10 pg/ml) or both drugs, the represented by bands of 82 and 88 kDa, consistent with transcription rate of Egr-1 had increased to almost the mobilities reported by other investigators in other three times that of actin. By 20 min of treatment, the cell types (Christy et al., 1988; Cao et ah, 1990). The 88transcription rate had returned to near basal levels. The kDa protein is initiated at a site 5’ to the two shown in induction of transcription with RA or cycloheximide Fig. 1 (Lemaire et al, 1990). There was a burst of Egr-1 was not additive, indicating that the two drugs might synthesis between 30 and 60 min leading to a fivefold work through a common mechanism. increase over basal levels. Synthesis of Egr-1 decreased The results in Figure 3 suggest that the transcripts significantly from 60 to 90 min and was essentially undedetected with a 5’ Egr-1 probe vary little compared to tectable from 90 to 120 min. Similar results were obthe transcripts detected with a 3’ Egr-1 probe. This tained for both aggregate and monolayer cultures (Fig. could indicate that the 5’ half of the message is tran4, lower panel). Egr-1 protein is, therefore, one of the scribed at a constant rate while complete mRNA is more earliest gene products induced in response to RA and selectively achieved. The implication is that transcripsince this protein is a transcription factor, it presumtion is blocked at some point after nt 340, possibly in the ably alters the expression of other genes, in turn. intron (nts 588-1153). Accordingly, we performed runon transcription assays using 5’ (nts l-340) to compare RA Increases the Rate of Transcription of the Egr-1 Gene with one encoding the rest of the gene (3448 nts). Nuclei In order to determine the mechanisms of Egr-1 gene were isolated from P19 cells treated with 1 PM RA for a regulation following RA addition, we measured changes time course up to 90 min and processed as described. Figure 5b shows that the rate of transcription increases in gene transcription using nuclear run-on transcription assays (Fig. 5). Since Egr-1 transcript levels are 1.8-fold 20 min after RA addition when measured with a superinduced by mitogens in combination with protein 3’ probe and then falls 3.3-fold by 90 min. The changes RA Rapidly

Induces Egr-1 Protein

Synthesis

EDWARDS ET AL

Rapid Response to RA

a) P19 monolayers I

aEar

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by measuring the remaining message at various times after treatment of the cells with a-amanitin, an inhibitor of polymerase II transcription. In both untreated and in differentiated P19 cells the half-life is about 90 min (Darland et al., 1991). However, when RA was added to EC cells there was a change in the stability of the mRNA for Egr-1. Figure 6 shows that the message becomes more unstable in the presence of RA and that the half-life decreases to about 30 min but this change occurs after the peak of transcript accumulation. In contrast there was no change in the level of L32 transcripts over the course of the experiment (data not shown). The increase in the instability of Egr-1 mRNA after 30 min may account, in part, for the rapid fall in accumulated levels after the initial peak. Accumulation of Egr-1 Transcripts Protein Synthesis Inhibitors

100

0

Time

Period

After

RA

Additton

(min)

FIG. 4. Pulse of Egr-1 biosynthesis in response to RA. Cells were labeled for 30 min intervals following RA administration with %S-labeled amino acids (see Materials and Methods). Lane 1, No RA, labeled 30 min; lane 2, with RA, labeled 30 min; lane 3, with RA, labeled 30-60 min after RA addition; lane 4, with RA, labeled 60-90 min after RA addition; lane 5, with RA, labeled 90-120 min after RA addition. Cell extracts in RIPA buffer were immunoprecipitated with either antiEgr-1, or preimmune sera. The top pane1 shows a fluorograph of immunoprecipitated products. Bands of 82 and 88 kDa corresponding to Egr-1 encoded proteins are indicated. The band at 130-140 kDa has not been identified; it may represent another member of the Egr family. The bottom pane1 shows a graphic comparison of the response of monolayers versus aggregates. Densitometric tracing of the 82-kDa bands were normalized to the maximal response. The value preceding 0 is for the 30-min period before RA addition and so on. The peak is at 30-60 min after RA addition.

measured by the 5’ probe are much smaller and in three experiments appear to be rather invariant compared to the 3’ probe signals. These results support a mode of Egr-1 regulation at least partly based on transcription termination. The Effect of RA on Egr-1 Message Stability

The increase in accumulated Egr-1 transcripts after RA addition, observed in Fig. 3, could also be explained if RA increased the stability of the message. The degradation rate of Egr-1 mRNA in P19 cells was determined

in the Presence of

In Figs. 3a and 3c there was a noticeable difference in the measurements of Egr-1 transcripts using probes that hybridized with the extreme 3’ and 5’ ends of the message. This effect was particularly evident in the presence of cycloheximide. A number of immediate early growth response gene transcripts accumulate in quiescent fibroblasts treated with cycloheximide, even in the absence of mitogenic stimulation (Chen and Allfrey, 1987; Almendral et ab, 1988). Similarly, Egr-1 transcripts accumulated rapidly in P19 cells in response to cycloheximide treatment (Fig. ?‘a).The increase was lofold as measured by the S’probe; very little increase was seen in the concentration of 5’ ends. The increased accumulation was essentially complete by 1 hr, as very little difference was seen between 1 and 3 hr of treatment with cycloheximide (Fig. 7b). As with transcription rate measurements (Fig. 5a), the effect of RA and cycloheximide on transcript accumulation was not additive (data not shown). There was no superinduction as is observed in serum stimulated fibroblasts simultaneously treated with cycloheximide (Sukhatme et al., 1987; Lau and Nathans, 1987). Cycloheximide treatment did, however, prevent the decline in Egr-1 transcripts normally observed at later times (a60 min) after RA treatment (Fig. 7b). Treatment with puromycin, which causes premature termination of nascent polypeptide chains, had effects similar to cycloheximide (data not shown). The increase in transcription and transcript levels in response to protein synthesis inhibitors argues for a repressive mode of regulation involving an unstable protein. DISCUSSION

We have shown that the expression of Egr-1 in P19 cells is rapidly and transiently induced by retinoic acid in a dose-dependent manner that is most marked in ag-

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FIG. 5. Transient increase in Egr-I transcription rates in response to RA. (a) P19 monolayers were treated with 3 FM RA or 10 pg/ml cycloheximide or both drugs for the indicated times. Nuclei were isolated and nuclear run-on transcription assays were performed as described under Materials and Methods. Changes in Egr-1 transcription were quantified by densitometry relative to actin, which was considered invariant during the short time course. The negative control (ctrl) was noncoding plasmid DNA. (b) P19 aggregates were treated with 1 FM RA for the indicated times before processing to isolate nuclei. Run-on transcription assays were performed as described under Materials and Methods. =P-labeled transcripts were hybridized to DNA probes on nitrocellulose. Egr-15’ DNA consists of the first 340 nts and the 3’ probe contains the rest of the cDNA. L32 is a ribosomal protein gene which is invariant and used to normalize the signals detected with the other probes. One result of three similar ones is shown after laser densitometry to quantify the autofluorographic signals.

gregated cells. The response correlates roughly with the subsequent differentiative pathway and therefore indicates that this surge of Egr-1 protein may play a role in the specificity of cellular responses. The rise in accumulated Egr-1 mRNA peaks at 10 to 20 min after RA addi1.2 Y m

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0

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,15

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30

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+RA

FIG. 6. Half life of Egr-1 message decreases when cells are treated with RA. P19 cells were pretreated with 2 pg/ml ru-amanitin for 30 min, then RA was added and the cells were incubated for the times indicated. RNA was isolated and assayed as before using a 3’ probe, which protects a 190-bp fragment. The lower panel shows an autoradiograph from such an experiment. The upper panel is a graphic representation of the average of three determinations.

tion and these transcripts are actively translated into protein, which rises to a maximum at 30 to 60 min after stimulation. The changes described are transient and after 90 min Egr-1 transcript levels are lower than the basal (but detectable) levels in unstimulated cells. A similar effect of RA on Egr-1 in preosteoblastic cells was described recently and the effect was shown to be restricted to cells that differentiate in response to RA (Suva et al., 1991). Changes in accumulated message in P19 cells in response to RA or cycloheximide appeared greater when measured with 3’ rather than 5’ probes suggesting regulation at the level of elongation of transcription. We have never detected antisense transcripts in ribonuclease protection assays with an appropriate RNA probe; therefore, this does not explain the differences in 3’ versus 5’ measurements (Nepveu and Marcu, 1986). Transcription rates increased, very transiently, approximately threefold relative to actin using the complete cDNA sequences as a probe (Fig. 5a). Since cycloheximide was also able to elicit a similar change in transcription rate, both drugs may act by a common mechanism. Good evidence for transcriptional blocking of Egr-1 is provided by showing that a fairly constant rate of transcription is measured with a 5’ Egr-1 probe after RA addition to P19 cells, whereas the 3’ end of the gene shows a small but clear increase after 20 min that declines to below normal values by 90 min (Fig. 5b). In Figure 5b, the average level of the signal using the 3’ probe is 3-fold that of the 5, but the 3’ probe is about lo-fold longer, therefore the relative rate of transcription of the 3’ portion of the gene is lower than the 5’ end. This supports the notion that transcription of the gene starts but does not finish for a proportion of the tran-

EDWARDSETAL.

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Rapid Respmse to RA in ECC

b

a Egr-1 mRNA accumulation with cycloheximide treatment

5’

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lh

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FIG. 7. (a) Egr-1 mRNA accumulates with cycloheximide treatment. P19 cells were treated with cycloheximide for 30 or 60 min, RNA was isolated, and RNAse protection experiments were performed using either a 3’ probe (190 bp) or a 5’probe (302 bp) and analyzed on the same gel. Right panel shows an autoradiograph of the gel and the left panel shows a graphic representation of a densitometric scan in which values are normalized such that the untreated sample equals one. No superinduction of transcripts with RA and cycloheximide. P19 cells were cultured as aggregates for 2 days, then treated with either RA (3 &f) or cycloheximide (10 pg/ml) or both, for 1 or 3 hr. Total RNA was isolated and analyzed as before with either a 3’ or 5’ probe. The lower panel shows the autoradiograph of a typical RNAse protection assay and the upper panel shows a graphic representation of the densitometric scan. (b) No superinduction of transcripts with RA and cycloheximide. P19 cells were cultured as aggregates for 2 days, then treated with either RA (3 PM) or cycloheximide (10 Fg/ml) or both, for 1 or 3 hr. Total RNA was isolated and analyzed as before with either a 3’ or 5’ probe. The lower part shows the autoradiograph of the RNAse protection assay, the upper panel shows a graphic representation of the densitometric scan

scripts. The simplest explanation is that a labile protein, possibly an RA receptor, is responsible for a block to elongation and that this is removed by both RA and cycloheximide. Transcriptional pausing has been described for c-fos (Fort et al., 198’7; Lamb et al, 1990) and c-myc (Nepveu and Marcu, 1986), both coding for early growth response genes. This may be a device to change the production of important regulatory factors in the most rapid fashion. An alternative explanation would be that separate transcription start sites are used for constitutive transcription and RA or cycloheximide induce transcription and that the induced site is downstream of the 5’ probe. We cannot distinguish between the two explanations by the results described here. Further work is underway and will be reported separately. Two transcription initiation sites have been described for Egr-2 (Krox-20) a related gene, one site which is induced by serum and one site which is used constitutively (Cortner and Farnham, 1990).

The rapid fall in the level of Egr-1 transcripts after the initial activation by RA may be accounted for by a combination of several processes. First, RA-induced transcription returns to basal levels very quickly (Fig. 5). Second, lability of the transcript increases after 30 min of treatment with RA (Fig. 6). Third, translation of the transcripts could be linked to message instability, as is the case for c-fos (Wilson and Treisman, 1988). This is supported by the timing of the increase in protein synthesis and the fact that protein synthesis inhibitors interfere with the drop in Egr-1 transcript levels (Fig. 7b). Increased production of Egr-1 gene product is not in itself sufficient to stimulate the differentiation of P19 cells. Each time the culture medium containing 10% FBS is changed there are transient stimulatory effects in immediate early genes which contain serum response elements. We have observed this for Egr-1, especially in trypsinized P19 cells (data not shown). The addition of fresh serum or trypsinization does not lead to differentiation. Differentiation of P19 cells is most effectively

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brought about by RA or DMSO. The specificity of the differentiative pathway must be dictated largely by particular levels and combinations of transcription factors and temporal changes in transcription factor activity. The early production of Egr-1 could be an important effector in the start of the differentiation program. The most logical accessory mediator of the RA response in P19 cells would be one of the nuclear RA receptors, of which there are at least two present in EC cells (Hu and Gudas, 1990; Pratt et al., 1990). These proteins bind DNA in the absence of RA but do not transactivate unless the ligand is present (Graupner et al., 1989; Sukov et al., 1990). We are currently seeking evidence for the interaction of the RA receptors with Egr-1 gene sequences that would explain how differentiation is initiated and how further signals are transmitted.

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