©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the Transcription Factor, Spi-1 (PU.1), in Differentiating Murine Erythroleukemia Cells Is Regulated Post-transcriptionally
EVIDENCE FOR DIFFERENTIAL STABILITY OF TRANSCRIPTION FACTOR mRNAs FOLLOWING INDUCER EXPOSURE (*)

(Received for publication, July 13, 1995; and in revised form, November 9, 1995)

Jack O. Hensold (1) (2)(§) Carl A. Stratton (2) Diane Barth (1) Deborah L. Galson (3)

From the  (1)University/Ireland Cancer Center, Department of Medicine and Case Western Reserve University, Cleveland, Ohio 44106, the (2)Department of Veteran's Affairs Medical Center, Cleveland, Ohio 44106, and the (3)Arthritis Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Increased expression of the transcription factor Spi-1 (PU.1) results from retroviral insertion in nearly all Friend spleen focus-forming virus-transformed murine erythroleukemia cell lines and exposure of these cells to Me(2)SO, induces their differentiation and decreases Spi-1 mRNA level by 4-5-fold. While these results suggest that alterations in Spi-1 expression have significant effects on erythroblast growth and differentiation, neither the cause nor the effect of the decrease in Spi-1 expression that follows Me(2)SO exposure has been established. The experiments described here demonstrate that the effect of inducers on Spi-1 expression is regulated post-transcriptionally. Nuclear run-off transcriptions demonstrated that Spi-1 transcription was not decreased following Me(2)SO exposure. Additionally, expression of a recombinant Spi-1 mRNA under transcriptional control of a constitutively active Rous sarcoma virus promoter was regulated identically to endogenous Spi-1 mRNA. The ability of Me(2)SO to destabilize Spi-1 mRNA was selective, as the stability of the erythroid transcription factors GATA-1 and NF-E2 were not similarly effected. The effect of Me(2)SO on the stability of Spi-1 mRNA provides a novel means of altering gene expression in these cells and is likely to have significance for the differentiation of these cells.


INTRODUCTION

SFFV(^1)-transformed MEL cells are an established system for studying erythroid growth and differentiation. These cells are derived from mice infected with the Friend retrovirus complex, which includes the defective SFFV and a helper Friend murine leukemia virus(1) . Three discrete molecular events have been identified, which contribute to the transformation of these erythroblasts. An early, proliferative phase of the disease results from interaction of the mutant env gene product of the SFFV with the erythropoietin receptor, producing ligand-independent receptor activation(2) . These infected erythroblasts are still able to differentiate and withdraw from the cell cycle. The subsequent acute erythroleukemic phase of the disease is associated with accumulation of undifferentiated erythroblasts. For essentially all of these erythroleukemias, genetic changes can be demonstrated in two additional loci. These include mutations or deletions of p53 (3, 4) and retroviral insertion and transcriptional activation of the Spi-1 (PU.1) gene(5, 6) , which encodes a transcription factor(7, 8) . How these changes interact to transform these cells remains unknown.

Spi-1 encodes a protein related to the ets family of transcription factors (9) which has a demonstrated role in regulating gene expression in monocytes and B-lymphocytes(10, 11, 12) . Additional evidence suggests this transcription factor also plays a role in erythroid differentiation. Spi-1 is normally expressed in erythroid progenitors (CFU-E)(13) , and mice lacking this gene die in utero, displaying a general defect in hematopoiesis including defective maturation of developing erythroblasts(14) . In the erythroleukemic stage of Friend SFFV disease, overexpression of Spi-1 is a universal occurrence(6) , and overexpression of Spi-1 has also been shown to immortalize erythroblasts in long-term bone marrow cultures(15) . In addition, two related ets family genes, fli-1 and v-ets, have also been implicated in erythroleukemic transformation(16, 17) . These findings strongly suggest that increased expression of Spi-1 is significant for erythroblastic transformation and for blocking the normal differentiation of these cells.

MEL cells undergo terminal erythroid differentiation when exposed to Me(2)SO or a number of other inducing agents(18) , and the expression of Spi-1 decreases 4-5-fold in Me(2)SO-exposed cells(13, 19) . To understand how inducers cause differentiation of these cells, we investigated the regulation of Spi-1 expression in inducer-exposed cells. The data presented here demonstrate that transcription of Spi-1 is unaltered by Me(2)SO exposure, suggesting that the stability of this mRNA is decreased by inducer exposure. A Spi-1 cDNA transcribed by a constitutively active RSV promoter is regulated identically to endogenous Spi-1, confirming that expression of this mRNA is regulated post-transcriptionally and confining the required sequences for this regulation to the coding region plus 10 nucleotides of 5`- and 3`-UT sequences. In contrast, the stability of mRNAs encoding the erythroid transcription factors, GATA-1 and NF-E2, are unaffected by inducer exposure. These findings demonstrate that inducers can alter gene expression via post-transcriptional mechanisms. The ability of Me(2)SO to differentially affect transcription factor stability suggests that this mechanism plays a significant role in altering gene expression in cells exposed to this agent.


EXPERIMENTAL PROCEDURES

Materials

Tissue culture supplies were from Life Technologies, Inc., and fetal bovine serum was from Intergen (Purchase, NY). Me(2)SO was from Eastman Kodak Co., and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was obtained from Calbiochem. Biochemicals were from Sigma. Enzymes and reagents for molecular biology were from Boehringer Mannheim, New England Biolabs, Pharmacia Biotech Inc., and Promega. Radionucleotides were from DuPont NEN. Membranes for blotting were from Schleicher & Schuell.

Cell Culture

MEL cells were subclones 745-PC4-B1-2A17 m^1 and -2A17 m^3. These cells were derived from the original 745 cell line of Charlotte Friend and have been repeatedly subcloned to maintain high rates of differentiation when exposed to Me(2)SO. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 12% fetal bovine serum and 2 mML-glutamine and maintained at concentrations that ensured logarithmic phase of growth (0.5-10 times 10^5 cells/ml). Differentiation was induced by addition of 1.5% (v/v) Me(2)SO to the growth medium and assessed by accumulation of beta-globin mRNA.

Determination of Spi-1 mRNA Level

Cells were grown with or without Me(2)SO for the times described in the text. Cytoplasmic RNA was extracted from the cells and analyzed by Northern blot hybridization as described previously(20) . Hybridizations were in 5 times SSPE, 0.1% SDS, 200 µg/ml salmon sperm DNA, 5 times Denhardt's solution at 65 °C with a murine Spi-1 cDNA clone(13) , which had been labeled with P by random priming(21) . Washes were to a final stringency of 62 °C in 0.1 times SSC, 0.1% SDS. Blots were developed by autoradiography and quantitated by densitometry utilizing a Bio-Rad model 620 video densitometer. RNA loading was standardized for the amount of 18 S rRNA loaded per lane, as determined by hybridization with a labeled fragment of the mouse 18 S rRNA gene (22) followed by autoradiography and densitometry, as above.

Expression of the recombinant Spi-1 transcripts were determined by blot hybridization of RNAs separated by electrophoresis in 3.5% polyacrylamide gels, as described by Stoeckle and Guan(23) , or by RNase protection. For the RNase protections, a 271-base pair BamHI-XbaI fragment containing the bovine growth hormone 3`-UT sequences and polyadenylation signal from the pRC/RSV expression vector was used since RNA transcripts that included 3`-Spi-1 sequences were poorly digested with either RNase A and T1 or RNase One (Promega), presumably due to stable secondary structure in this GC-rich (62%) sequence(13) . The 3`-UT sequences were ligated between the BamHI-XbaI sites in pBlueScript II SK and P-labeled antisense RNAs transcribed with T7 RNA polymerase, as described by the manufacturer (Stratagene). 300,000-600,000 dpm of this radiolabeled RNA was hybridized with 15 µg of cytoplasmic RNA for 18 h at 60 °C in 0.4 M sodium chloride, 40 mM PIPES, 1 mM EDTA, 80% formamide. The unhybridized RNA was digested to completion with RNase One as described by the manufacturer (Promega), and the undigested fragments were separated by electrophoresis in denaturing 6% polyacrylamide gels and visualized by autoradiography.

Nuclear Run-off Transcriptions

Run-off transcriptions were performed on isolated nuclei as described previously(20) . The run-off transcripts were isolated by centrifugation through 5.7 M cesium chloride, resuspended in diethyl pyrocarbonate-treated water, and standardized for cpm/ml by scintillation counting. Equal amounts of radioactivity were hybridized at 65 °C in TES-buffered 1 M sodium chloride and 1% SDS for 48 h with 10 µg of linearized plasmid or 5 µg of single-stranded phagemid DNA, which had been bound to nitrocellulose membranes by slot blotting. Following hybridization, the blots were washed at 65 °C in 2 times SSC, 0.1% SDS with a final wash in 2 times SSC with RNase A added at 10 µg/ml. Results were visualized by autoradiography.

The DNAs used in these experiments included cloned DNAs encoding hsc70 (24) , beta-actin(25) , beta-globin(26) , Band 3(27) , GATA-1(28) , NF-E2 (29) , and Spi-1(13) . The cloning vectors for these cDNAs, pUC19, pBlueScript II, and pGEMM were used as negative controls. For determination of sense and antisense transcription, pBlueScript II SK(+) and pBlueScript II SK(-) phagemids containing the respective cDNAs were rescued by infection with VCS-M13 interference-resistant helper phage. Virus was isolated from the supernatants by precipitation with polyethylene glycol as described by Stratagene. Phage DNA was purified by phenol and chloroform extraction and ethanol precipitation. For the experiments requiring probes specific to the 3`-end of the Spi-1 transcript, a 250-base pair fragment of 3`-untranslated Spi-1 sequence was excised by digestion with ApaI and XhoI. The fragment was isolated by agarose gel electrophoresis and ligated into the polylinker of pBlueScript II SK(+) at the ApaI and XhoI sites.

Determination of Spi-1 mRNA Half-life

The transcriptional inhibitor, DRB, was utilized at a concentration of 30 µg/ml as described previously(30) . This concentration has been shown to inhibit transcription by greater than 90%(31) . MEL cells were grown with or without Me(2)SO, and, following 24 h of incubation, DRB was added to the cells and incubations continued for an additional 4 h. Aliquots of cells were removed at hourly intervals following the addition of DRB, and RNA was extracted as described above. 10 µg of RNA from each sample was analyzed for expression of Spi-1 by Northern blotting. Loading of RNA was standardized by hybridization with a radiolabeled 18 S rRNA probe. Autoradiographs were quantitated by densitometry with the results from each lane normalized to the amount of 18 S rRNA. Spi-1 mRNA half-life was calculated by curve-fitting assuming linear decay, using the program included in the graphics software (Cricket Graph, Philadelphia, PA).

Expression of Recombinant DNA Constructs

The cDNA clone encoding Spi-1 has been previously described(13) . For expression in MEL cells, a Spi-1 cDNA, which had truncations of both the 5`- and 3`-noncoding sequences, was prepared by polymerase chain reaction. Reactions were carried out in 50 mM potassium chloride, 1.5 mM magnesium chloride, 10 mM Tris, pH 7.6. The primers and linearized plasmid DNA were denatured at 94 °C for 1 min and annealed at 60 °C for 2 min, with extension at 72 °C for 3 min and denaturation at 94 °C for 1 min. This was repeated for 35 cycles in an MJ Research minicycler. The 5`-primer, GGATCTGACCAAACTAGTGCTCAGC, contained three point mutations at the underlined positions, creating a unique SpeI restriction site. The 3`-primer, GAGCCTGGCGGCCGCTGC, contained two point mutations at the underlined positions, creating a unique NotI site. When cut with SpeI and NotI, this cDNA contained 10 nucleotides of 5`- and 3`-untranslated sequences, proximal to the coding region. This 5`- and 3`-truncated cDNA was ligated into the SpeI and NotI sites in the pRC/RSV expression vector (Invitrogen), introduced into MEL cells by electroporation and stable transfectants selected in G418 (0.6 mg/ml, specific activity) as described previously (32) . Based on previous determinations of stable cloning efficiency with pRC/RSV (approximately 1 per 5 times 10^4 electroporated cells), cells were seeded in 24-well dishes at concentrations that resulted in the outgrowth of resistant cell ``pools,'' which represented the expansion of one to three original cell clones. The expanded pools were screened for expression of the recombinant mRNA by Northern blotting or RNase protection, and those expressing detectable levels were subjected to further analysis.


RESULTS

Post-transcriptional Regulation of Spi-1 Expression in Inducer-exposed Erythroleukemia Cells

Previous experiments have demonstrated that Me(2)SO exposure of MEL cells decreases expression of Spi-1(13, 15) . This effect was examined for the MEL cell clones used in these experiments. The decrease in expression of Spi-1 was detectable by 4 h of Me(2)SO exposure and continued to decline until reaching 25-30% of control levels at 20-36 h of inducer exposure (Fig. 1). This decrease entirely preceded the increase in expression of beta-globin mRNA, which was first apparent at 24-36 h. In contrast to the early decrease in Spi-1 expression and the subsequent increase in expression of beta-globin, the expression of two abundant ``housekeeping'' mRNAs, beta-actin and ribosomal protein L26, was not significantly altered following inducer exposure. Thus, the effect of Me(2)SO on Spi-1 expression appeared to be selective.


Figure 1: Me(2)SO exposure causes a selective decrease in Spi-1 mRNA abundance. MEL cells were grown in the presence of Me(2)SO, and cytoplasmic mRNA was extracted at the indicated times. Equal amounts of RNA were separated by gel electrophoresis, and mRNA levels were determined by Northern blot hybridization with P-labeled cDNAs encoding Spi-1, beta-actin, ribosomal protein S12, and beta-globin. Results were quantitated by densitometry and standardized for loading by normalizing to results obtained by rehybridizing the blots with a labeled fragment of the 18 S rRNA gene.



To determine if the decreased abundance of Spi-1 mRNA was due to a decrease in its transcription, run-off transcriptions were performed on isolated nuclei from cells grown under normal conditions or following 24 h of Me(2)SO exposure. Since previous studies had demonstrated that antisense transcription occurs across some genes in MEL cells (including c-myc(33) and hsc70(32) ), the labeled run-off transcripts were hybridized to single-stranded sense and antisense cDNA probes for Spi-1. As demonstrated in Fig. 2A, a low level of antisense transcription (detected by hybridization with the cDNA encoding the positive strand) is detected across the Spi-1 gene in these cells. This antisense transcription appears authentic, since no signal is detected hybridizing with the phagemid vector. The sense strand (hybridizing to the cDNA encoding the negative strand) is transcribed at approximately 10-fold the rate of the antisense transcript. However, as demonstrated here, neither sense nor antisense transcription rate is significantly altered following inducer exposure.


Figure 2: Me(2)SO exposure does not affect transcription rate nor cause transcriptional attenuation of Spi-1. A, run-off transcriptions were performed on nuclei prepared from control and Me(2)SO-exposed cells as described in the text. Antisense Spi-1 transcripts were detected by hybridization of the labeled transcripts with single-stranded phagemid DNA containing the Spi-1 sense (+) strand. Sense transcripts were detected by hybridization with single-stranded phagemid containing the Spi-1 antisense(-) strand. The remaining DNAs used in this experiment were double-stranded. The plasmid DNAs pUC19 and pBlueScript II SK (+) were included as negative controls. B, run-off transcriptions were performed as above, and the labeled transcripts were hybridized with a cloned Spi-1 cDNA, which contained only the 3`-terminal 250 nucleotides of the mature transcript. Other plasmids included cDNAs with sequences encoding band 3, beta-globin, and beta-actin. The plasmid DNAs, pUC19 and pBlueScript II, were included as negative controls.



The early decrease in expression of c-myc mRNA that follows Me(2)SO exposure of MEL cells is due to transcriptional attenuation(34) . To determine if Me(2)SO caused attenuation of Spi-1 transcription, the run-off transcriptions were repeated and hybridized with a cDNA fragment of Spi-1, which included only the 3`-UT sequences of this mRNA. Transcripts extending to the 3`-UT of this gene were equally represented in the run-off transcripts from both control and Me(2)SO-exposed cells (Fig. 2B), demonstrating that attenuation of transcription had not occurred on this gene. These assays were sensitive to changes in transcription, since the transcription of beta-globin at this time had increased from 3 to 17% of the rate actin transcription. The transcription of band 3 also appeared to increase, although this could not be quantified due to the lack of a detectable signal in the control cells. These results demonstrate that the transcription of Spi-1 in MEL cells was unaffected by Me(2)SO exposure, and thus the decreased accumulation of this mRNA was mediated post-transcriptionally.

To determine the half-life of Spi-1 mRNA following Me(2)SO exposure, cells were exposed to DRB, a specific inhibitor of RNA polymerase II transcription, since actinomycin D has previously been shown to induce MEL cell differentiation(35) . We analyzed mRNA half-life following 24 h of inducer exposure, since the full extent of the decrease in expression of Spi-1 was evident by this time (see Fig. 1). Cells were grown in the presence or absence of Me(2)SO for 24 h, then DRB was added for an additional 4 h. Cytoplasmic RNA was extracted at hourly intervals following the addition of DRB, and Spi-1 mRNA levels were determined by Northern blotting. The results shown in Fig. 3are normalized for RNA loading as determined by rehybridizing the blots with an 18 S rRNA fragment and represent the average of two separate experiments. For control cells, the half-life of Spi-1 mRNA was determined to be 8.2 h, while following 24 h of Me(2)SO exposure the half-life was reduced to 3 h. The decay was noted to be biphasic for both control and Me(2)SO-treated cells, with the initial decrease (at 1-2 h of DRB exposure) accounting for the entire difference in half-life. Other investigators have also noted biphasic mRNA decay following exposure to actinomycin D(30) , suggesting that a secondary response to transcriptional inhibitors accounted for this latter rate. While the measured decrease in half-life difference was slightly less than the 3-4-fold decrease in Spi-1 mRNA abundance that followed inducer exposure, these results supported the conclusion that the stability of Spi-1 mRNA was decreased by inducer exposure.


Figure 3: Me(2)SO decreases Spi-1 mRNA half-life. MEL cells were grown in the presence or absence of Me(2)SO for 24 h; transcription was then inhibited by the addition of DRB, and the cells were incubated for an additional 4 h. RNA was extracted from the cells at 1-h intervals, and the Spi-1 mRNA level was determined by Northern blot hybridization. The experiments were performed in duplicate, and the results were quantitated by densitometry and standardized for loading by normalizing to results obtained by rehybridizing the blots with a cloned fragment of the 18 S rRNA gene. Representative blots are shown in panel A, and the results of the densitometric quantitation are shown in panel B. The results are expressed as a percentage of Spi-1 mRNA present at time 0, prior to the addition of DRB. The vertical bars in panel B represent the range for each determination.



Expression of a Recombinant Spi-1 cDNA Is Regulated Identically to Endogenous Spi-1

To provide independent confirmation of the preceding results, a Spi-1 cDNA, which included the entire coding sequence and 10 bases of 5`- and 3`-UT sequences, was ligated into an expression vector under the transcriptional control of the constitutively active RSV promoter. The terminal 130 nucleotides of 3`-UT and the polyadenylation signal were derived from the vector. This construct was introduced into MEL cells by electroporation, and stable cell lines were selected and analyzed for expression of the recombinant Spi-1 gene by RNase protection or Northern blotting of extracted RNAs. Clones with detectable expression of the recombinant construct were analyzed further.

The effect of Me(2)SO on expression of the recombinant transcript was determined on eight independent clones that expressed detectable levels of the recombinant transcript. The results of nuclease protection assays utilizing a probe derived from the 3`-UT sequences of the vector are shown for four of these clones (Fig. 4A). A fragment of 135 nucleotides, which corresponded to the expected length of the 3`-UT of the recombinant transcript, was detected in all the clones. The smaller fragment resulted from use of an alternative cleavage and polyadenylation site present in the 3`-UT(36) . Hybridizing sequences were not detected in control cells. Exposure to Me(2)SO (24 h) resulted in decreased expression of the recombinant transcript in all analyzed clones. To compare this decrease with that of endogenous Spi-1 mRNA, clone pRC/Spi-1(8) was analyzed by blot hybridization of RNA separated in 3.5% denaturing acrylamide gels. As demonstrated in Fig. 4B, following 24 h of Me(2)SO exposure the extent of the decrease in expression of the recombinant mRNA was identical to that of the endogenous mRNA. Similar behavior was observed on two other clones similarly screened (not shown).


Figure 4: Me(2)SO exposure decreases the expression of a Spi-1 mRNA transcribed by the RSV promoter. A Spi-1 cDNA containing 10 nucleotides of 5`- and 3`-untranslated sequence under the transcriptional control of the RSV promoter was introduced into MEL cells, and stable clones expressing the recombinant transcript were selected in G418. A, the effect of Me(2)SO on the expression of the recombinant transcript in four of these clones was determined by RNase protection, utilizing a probe that hybridized with the unique 3`-UT sequences derived from the vector. Each lane represents the results of a hybridization with 20 µg of cytoplasmic RNA from either the parental cell line (MEL) or from the cloned cells (indicated by name above each paired set of lanes). Nuclease protections were performed with RNA from uninduced cells (C) and following 24 h of Me(2)SO exposure (D). A protected fragment of the correct predicted size (135 nucleotides) was detected in the selected clones but not in the parental cell line. The smaller fragment results from the use of an alternative cleavage and polyadenylation site in the vector. B, the effect of Me(2)SO on the expression of both recombinant and endogenous Spi-1 mRNAs was determined by electrophoresis of RNAs in 3.5% acrylamide gels and blot hybridization as described in the text. The results for clone pRC/Spi-1(8) are shown in the panel. Endogenous Spi-1 transcripts (wt) and the recombinant transcripts (Delta5`/3`) are indicated by the arrows to the right of the autoradiograph. Expression of the recombinant transcripts in two other analyzed clones exhibited a similar change.



The conclusion that expression of the recombinant Spi-1 transcript was regulated post-transcriptionally was inferred from evidence that the RSV promoter is constitutively active under these conditions, as determined by transient assays. (^2)However, to confirm this, the effect of Me(2)SO on transcription of the recombinant Spi-1 gene in clone pRC/Spi-1(8) was determined by run-off transcriptions. The vector (pRC/RSV) without inserted cDNA sequences was used to detect the 3`-sequences uniquely present in the recombinant transcripts. Run-off transcripts that hybridized to vector sequences were easily detected in the transfected clone, and their transcription was unaffected by Me(2)SO (Fig. 5). Similar results were found with analysis of an additional clone. There was no significant hybridization detected with the pRC/RSV vector sequences in the parental cell line (see Fig. 6C). A weak signal was detected hybridizing to the plasmid DNA, pUC19, in the electroporated cells. This was detected in repeated experiments and was also noted for pGEMM plasmid DNA (data not shown). Since this was not detected in the nuclear run-off transcriptions performed on untransfected cells (see Fig. 1and Fig. 2), this may represent hybridization with transcripts from the expression vector that extended into the plasmid backbone, since transcription past the cleavage and polyadenylation site would be an expected occurrence. These experiments confirmed that transcription from the vector was constant and unaffected by Me(2)SO exposure. Therefore, the decreased expression of the recombinant Spi-1 mRNAs detected in the preceding experiments must be mediated post-transcriptionally.


Figure 5: Transcription of the recombinant Spi-1 transcript is unaffected by Me(2)SO exposure. Transcriptional activity of the RSV promoter was determined for the clone shown in Fig. 4B by nuclear run-off transcription in cells grown with and without Me(2)SO (1.5%) for 30 h as described in the text. Labeled transcripts were hybridized with 10 µg of linearized plasmid DNAs. Plasmids containing cDNA inserts encoding beta-globin and the ribosomal protein L26 or the plasmid pUC 19, without inserted sequences, were included as controls. Spi-1 transcripts derived from the expression vector were detected by hybridization with 3`-UT sequences present in the pRC/RSV vector to avoid detection of endogenous Spi-1 transcripts. Hybridization with pRC/RSV sequences was not detected in run-off transcriptions done on the parental cell line MEL cells (see Fig. 6C).




Figure 6: The stability of GATA-1 and NF-E2 mRNAs are unaffected by Me(2)SO exposure. A, RNA was extracted from MEL cells at the times of Me(2)SO exposure indicated, and expression of GATA-1 and NF-E2 mRNA assessed by Northern blot hybridization is shown. beta-globin mRNA expression was similarly assessed. B, the results were quantitated by densitometry and normalized for loading by hybridization with an 18 S rRNA gene fragment. The results are presented graphically at the right of the figure. The increase in beta-globin gene expression was not quantified, since expression in uninduced cells was below the limits of detection by densitometry. C, nuclear run-off transcriptions were performed on uninduced cells and at 30 h of Me(2)SO exposure. The preparation of nuclei and P labeling of run-off transcripts were performed as described in the text. The labeled transcripts were hybridized with 10 µg of slot-blotted plasmid DNAs with inserted cDNA sequences encoding r-protein L26 and beta-globin or with single-stranded phagemids with inserted sequences corresponding to GATA-1 and NF-E2 antisense strands to detect hybridization with the sense transcripts of these genes. Phagemid DNA without inserted sequences (pBSSK) was included as control. The plasmid pRC/RSV was also included as a control for the run-off transcriptions performed on the electroporated cell lines shown in Fig. 5.



Erythroid Transcription Factor mRNAs Are Not Destabilized by Me(2)SO Exposure

To determine if Me(2)SO had a similar effect on stability of other transcription factor mRNAs we analyzed the expression of two erythroid transcription factors, GATA-1 and NF-E2. As determined by Northern blot hybridization, Me(2)SO had a minimal effect on the expression of GATA-1, although a transient decrease in mRNA level was noted at early times of inducer exposure (Fig. 6). NF-E2 also demonstrated a transient decrease in mRNA level followed by a modest induction, most evident at 72 h of inducer exposure (Fig. 6B). A similar early, transient decrease in expression of other mRNAs has been noted in inducer-exposed cells, including mRNAs encoding c-myc(41) and c-myb(42) as well as beta(min)-globin (see Fig. 1). (^3)However, the failure to observe a persistent decrease in expression of GATA-1 and NF-E2 mRNAs suggested that their stability was unaffected by inducer exposure.

The transient decrease in GATA-1 and NF-E2 mRNA levels that followed Me(2)SO exposure suggested that a decrease in the stability of these mRNAs could have been offset by a subsequent increase in their transcription. To determine if this was the case, we assessed the transcription of these genes in control cells and at 30 h of Me(2)SO exposure by nuclear run-off transcription. At this time of inducer exposure, GATA-1 and NF-E2 mRNAs were expressed at approximately 110 and 125% of their respective levels in uninduced cells. Run-off transcriptions demonstrated that at 30 h of Me(2)SO exposure, the transcription of these genes was unchanged relative to their transcription rates in the control cells. In contrast, an increase in transcription of beta-globin was clearly evident at this time. These results demonstrate that the stability of these two mRNAs was not significantly altered by Me(2)SO exposure. Therefore, the decrease in Spi-1 mRNA stability that occurs following inducer exposure is not the result of a global decrease in stability but exhibits specificity since neither GATA-1 nor NF-E2 mRNAs are similarly affected.


DISCUSSION

Exposure of MEL cells to the inducer of differentiation, Me(2)SO, causes a 3-5-fold decrease in accumulation of mRNA encoding the transcription factor Spi-1(13, 19) . The data presented here demonstrate that post-transcriptional regulatory mechanisms are responsible for this decrease. In contrast, the stability of mRNAs encoding the transcription factors, GATA-1 and NF-E2, is unaffected by inducer exposure, demonstrating specificity to this regulation. These findings provide insight into the regulation of Spi-1 expression and suggest that differential stability of transcription factor mRNAs following inducer exposure may play a role in establishing the pattern of gene expression in the differentiating cells.

Exposure of MEL cells to Me(2)SO results in a 70-75% decrease in accumulation of Spi-1 mRNA. Nuclear run-off transcriptions demonstrated that this decrease was mediated post-transcriptionally. Since increased expression of Spi-1 in Friend virus-transformed cells is due to retroviral insertional activation of transcription, these results suggest that the transcriptional activity of the retroviral long terminal repeat is unaffected by Me(2)SO, and results of transient transfection assays have reached a similar conclusion. (^4)Thus, the decreased expression of Spi-1 is regulated post-transcriptionally. While the data presented here do not exclude that Me(2)SO blocked nucleocytoplasmic transport of this mRNA, this appears unlikely since Schuetze et al.(19) detected a similar decrease in Spi-1 expression when whole cell mRNA was analyzed.

Determination of Spi-1 mRNA half-life by transcriptional inhibition demonstrated that Me(2)SO exposure decreased the stability of Spi-1 mRNA. Since the expression of a recombinant Spi-1 cDNA transcribed by the constitutively active RSV promoter was regulated similarly to endogenous Spi-1, this limits the sequence required for this regulated change in stability to the coding region plus the adjoining 10 nucleotides of 5`- and 3`-untranslated sequences. The coding region of a number of other mRNAs, including beta-tubulin, c-fos, and c-myc, has also been shown to play a role in determining stability(43, 44, 45, 46) . The mechanisms by which these sequences regulate mRNA stability remain to be established. However, the ability of inducers of differentiation to regulate the effect of these elements provides an additional means to investigate these mechanisms.

The degradation of mRNAs is frequently preceded by their deadenylation(30, 37, 38, 39, 40) , and this is consistent with our recent observation that the inducers Me(2)SO, hypoxanthine, and A23187 all cause substantial deadenylation of mRNAs. (^5)Spi-1 is one of the mRNAs so affected. In contrast, for two ``housekeeping'' mRNAs (specifically ribosomal protein L26 and S12), inducer exposure affected neither abundance nor poly(A) tail length. Although the mechanisms resulting in this selectivity of deadenylation are unknown, this likely plays a role in the differential stabilities of these mRNAs following inducer exposure. Current studies are assessing the effect of Me(2)SO on the adenylation of GATA-1 and NF-E2 mRNAs.

Regardless of the mechanism(s) that account for the differential stability of Spi-1 and GATA-1 and NF-E2 mRNAs in inducer-exposed MEL cells, these differences are likely to be significant for the regulation of gene expression in differentiating cells. While the Me(2)SO-induced decrease in expression of Spi-1 has not yet been demonstrated to play a role in MEL cell differentiation, the consistent overexpression of Spi-1 during leukemogenesis of these cells suggests that this decrease is unlikely to be without effect. Further, the ability of Me(2)SO to destabilize mRNAs is unlikely to be limited to Spi-1. It is noteworthy that expression of a recombinant Id transcript in MEL cells has been reported to be decreased by Me(2)SO(48) , suggesting that Me(2)SO also destabilizes this mRNA. The stability of GATA-1 and NF-E2 following inducer exposure has increased significance in light of evidence suggesting that the activity of GATA-1 containing promoters can be regulated by competition for binding with other transcription factors(49, 50) . Thus, the ability of Me(2)SO to destabilize Spi-1 or other, yet to be identified transcription factor mRNAs may lead to a functional increase in activity of those mRNAs unaffected by this change. Further knowledge of how inducers affect mRNA stability should provide additional insights into the mechanisms by which these agents cause differentiation of these cells.


FOOTNOTES

*
This work was supported by Grant DK43414 from the National Institutes of Health and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: UCRC 2, Suite 200, 11001 Cedar Ave., Cleveland, OH 44106. Tel.: 216-844-8245; Fax: 216-844-8230.

(^1)
The abbreviations used are: SFFV, spleen focus-forming virus; MEL, murine erythroleukemia; RSV, Rous sarcoma virus; DRB, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole; PIPES, piperazine-N,N`-bis(2-ethanesulfonic acid); TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; UT, untranslated.

(^2)
D. L. Galson, unpublished data.

(^3)
J. O. Hensold, C. A. Stratton, D. Barth, and D. L. Galson, unpublished observations.

(^4)
D. L. Galson, unpublished observations.

(^5)
J. O. Hensold, D. Barth, and C. A. Stratton, submitted for publication.


ACKNOWLEDGEMENTS

-We thank Fritz Rottman for helpful discussions and the following individuals for the cloned DNA fragments used for these experiments: L. Giebl, B. Spiegelman, S. Alper, S. Orkin, and I. Wool.


REFERENCES

  1. Ben-David, Y., and Bernstein, A. (1991) Cell 66, 831-834 [Medline] [Order article via Infotrieve]
  2. Li, J.-P., D'Andrea, A., Lodish, H., and Baltimore, D. (1990) Nature 343, 762-764 [CrossRef][Medline] [Order article via Infotrieve]
  3. Lavigeur, A., Cheong, G., and Bernstein, A. (1990) New Biol. 2, 1015-1023 [Medline] [Order article via Infotrieve]
  4. Peacock, J., and Benchimol, S. (1990) Mol. Cell. Biol. 10, 3307-3313 [Medline] [Order article via Infotrieve]
  5. Moreau-Gachelin, F., Tavitian, A., and Tambourin, P. (1988) Nature 331, 277-280 [CrossRef][Medline] [Order article via Infotrieve]
  6. Moreau-Gachelin, F., Ray, D., Mattei, M.-G., Tambourin, P., and Tavitian, A. (1989) Oncogene 4, 1449-1456 [Medline] [Order article via Infotrieve]
  7. Goebl, M., Moreau-Gachelin, F., Ray, D., Tambourin, P., Tavitian, A., Klemsz, M., McKercher, S., Celada, A., van Beveren, C., and Maki, R. (1990) Cell 61, 1165-1166 [Medline] [Order article via Infotrieve]
  8. Galson, D., and Housman, D. (1988) Mol. Cell. Biol. 8, 381-392 [Medline] [Order article via Infotrieve]
  9. Klemsz, M., McKercher, S., Celada, A., van Beveren, C., and Maki, R. (1990) Cell 61, 113-124 [Medline] [Order article via Infotrieve]
  10. Kominata, Y., Galson, D., Waterman, W., Webb, A., and Auron, P. (1995) Mol. Cell. Biol. 15, 59-68 [Abstract]
  11. Pongubala, J., Nagulapalli, S., Klemsz, M., McKercher, S., Maki, R., and Atchison, M. (1992) Mol. Cell. Biol. 12, 368-378 [Abstract]
  12. Zheng, D.-E., Hetherington, C., Chen, H.-M., and Tenen, D. (1994) Mol. Cell. Biol. 14, 373-381 [Abstract]
  13. Galson, D., Hensold, J., Bishop, T., Schalling, M., D'Andrea, A., Jones, C., Auron, P., and Housman, D. (1993) Mol. Cell. Biol. 13, 2929-2941 [Abstract]
  14. Scott, E., Simon, M., Anastasi, J., and Singh, H. (1994) Science 265, 1573-1577 [Medline] [Order article via Infotrieve]
  15. Schuetze, S., Steinberg, P., and Kabat, D. (1993) Mol. Cell. Biol. 13, 5670-5678 [Abstract]
  16. Ben-David, Y. E. G., Letwin, K., and Bernstein, A. (1991) Genes & Dev. 5, 908-918
  17. Nunn, M., Seeburg, P., Moscovici, C., and Duesberg, P. (1983) Nature 306, 391-395 [Medline] [Order article via Infotrieve]
  18. Marks, P., and Rifkind, R. (1978) Annu. Rev. Biochem. 47, 419-448 [CrossRef][Medline] [Order article via Infotrieve]
  19. Schuetze, S., Paul, R., Gliniak, B., and Kabat, D. (1992) Mol. Cell. Biol. 12, 2967-2975 [Abstract]
  20. Hensold, J., Dubyak, G., and Housman, D. (1991) Blood 77, 1362-1370 [Abstract]
  21. Feinberg, A., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13 [Medline] [Order article via Infotrieve]
  22. Wilson, G., Hollar, B., Waterson, J., and Schmekel, R. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 5367-5371 [Abstract]
  23. Stoeckle, M., and Guan, L. (1993) BioTechniques 15, 227-229 [Medline] [Order article via Infotrieve]
  24. Giebel, L., Dworniczak, B., and Bautz, E. (1988) Dev. Biol. 125, 200-207 [Medline] [Order article via Infotrieve]
  25. Spiegelman, B., Frank, M., and Green, H. (1983) J. Biol. Chem. 258, 10083-10089 [Abstract/Free Full Text]
  26. Tilghman, S., Tiemeier, D., Polsky, F., Edgell, M., Seidman, J., Leder, A., Enquist, L., Norman, B., and Leder, P. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 4406-4410 [Abstract]
  27. Kopito, R., and Lodish, H. (1985) Nature 316, 234-238 [Medline] [Order article via Infotrieve]
  28. Tsai, S-F., Martin, D., Zon, L., D'Andrea, A., Wong, G., and Orkin, S. (1989) Nature 339, 446-451 [CrossRef][Medline] [Order article via Infotrieve]
  29. Andrews, N., Erdjument-Bromage, H., Davidson, M., Tempst, P., and Orkin, S. (1993) Nature 362, 722-728 [CrossRef][Medline] [Order article via Infotrieve]
  30. Laird-Offringa, I., de Wit, C., Elfferich, P., and van der Eb, A. (1990) Mol. Cell. Biol. 10, 6132-6140 [Medline] [Order article via Infotrieve]
  31. Laub, O., Jakobovits, E., and Aloni, Y. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3297-3301 [Abstract]
  32. Hensold, J., Hunt, C., Calderwood, S., Housman, D., and Kingston, R. (1990) Mol. Cell. Biol. 10, 1600-1608 [Medline] [Order article via Infotrieve]
  33. Kindy, M., McCormack, J., Buckler, A., Levine, R., and Sonenshein, G. (1987) Mol. Cell. Biol. 7, 2857-2862 [Medline] [Order article via Infotrieve]
  34. Nepveu, A., Marcu, K., Skoultchi, A., and Lachman, H. (1987) Genes & Dev. 1, 938-945
  35. Ebert, P., Wars, I., and Buell, D. (1976) Cancer Res. 36, 1809-1813 [Abstract]
  36. Woychik, R., Lyons, R., Post. L., and Rottman, F. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3944-3948 [Abstract]
  37. Bernstein, P., and Ross, J. (1989) Trends Biochem. Sci. 14, 373-377 [CrossRef][Medline] [Order article via Infotrieve]
  38. Decker, C., and Parker, R. (1993) Genes & Dev. 7, 1632-1643
  39. Sachs, A. (1993) Cell 74, 413-421 [Medline] [Order article via Infotrieve]
  40. Shyu, A-B., Belasco, J., and Greenberg, J. (1991) Genes & Dev. 5, 221-231
  41. Lachman, H., and Skoultchi, A. (1984) Nature 310, 592-594 [Medline] [Order article via Infotrieve]
  42. Ramsey, R., Ikeda, K., Rifkind, R., and Marks, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6849-6853 [Abstract]
  43. Kabnick, K., and Housman, D. (1988) Mol. Cell. Biol. 8, 3244-3250 [Medline] [Order article via Infotrieve]
  44. Shyu, A., Greenberg, M., and Belasco, J. (1989) Genes & Dev. 3, 60-72
  45. Wisdom, R., and Lee, W. (1991) Genes & Dev. 5, 232-243
  46. Yen, T., Gay, D., Pachter, J., and Cleveland, D. (1988) Mol. Cell. Biol. 8, 1224-1235
  47. Deleted in proof
  48. Shoji, W., Yamamoto, T., and Obinata, M. (1994) J. Biol. Chem. 269, 5078-5084 [Abstract/Free Full Text]
  49. Fischer, K., Haese, A., and Nowock, J. (1993) J. Biol. Chem. 268, 23915-23923 [Abstract/Free Full Text]
  50. Aird, W., Parvin, J., Sharp, P., and Rosenberg, R. (1994) J. Biol. Chem. 269, 883-889 [Abstract/Free Full Text]

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