USF2) Mrna in Hematopoietic Cells

Growth-dependent and PKC-mediated translational regulation of the upstream stimulating factor-2 (USF2) mRNA in hematopoietic cells

Zhao Cheng Zhang, Hovav Nechushtan, Jasmine Jacob-Hirsch, Dror Avni, Oded Meyuhas and Ehud Razin

Department of Biochemistry, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel

Upstream stimulating factor (USF2) is a basic helix ± (Blannar and Rutter, 1992; Sirito et al., 1994). USF2 loop ± helix leucine zipper transcription factor, which is sequence analysis reveals that the protein contains both found in most tissues. A critical role for USF2 in cellular the basic helix ± loop ± helix (HLH) and the leucine proliferation has been proposed based on its importance zipper (Zip) domains (Sirito et al., 1994). The bZip in the regulation of various cyclins and P53 and its domain is characterized by a heptad repeat of leucine capability to antagonize c-myc. In this paper we report residues to form an amphipathic a-helical structure; the that IL-3, which is a major growth factor for mast cells, bHLH domain consists of two amphipathic a-helices induces USF2 protein synthesis in murine mast cells separated by a variable amino acid loop, as found in (MC-9). Surprisingly, it does not signi®cantly aect the MyoD (Sirito et al., 1994). These functional domains level of USF2 mRNA in these cells at any of the time allow the formation of homodimers and heterodimers points tested. Using polysomal fractionation and RNA of proteins containing such elements. The role played analysis we then demonstrated that this translational by USF in the regulation of gene expression has just regulation is mostly the result of increased USF2 recently started to be investigated. One of the translational eciency. Moreover, protein kinase C assumptions is that USF1 and USF2 serve as (PKC) inhibitors prevented both the induction of USF2 transcription factors with general housekeeping tran- protein synthesis and the increase in USF2 translational scriptional regulatory roles (Gogswell et al., 1995; Luo eciency in IL-3-activated mast cells. Two other and Sawadogo, 1996; Reisman and Rotter, 1993; hematopoietic cell lines were used to determine whether Viollet et al., 1996). the translational regulation of USF2 is of a more general Most of the USF transcription complexes are nature: mouse lymphosarcoma cells whose proliferation composed of USF1-USF2 (Viollet et al., 1996). is inhibited by dexamethasone; and mouse erythroleuke- Surprisingly, the only protein from a HeLa cDNA mia cells that dierentiate upon exposure to hexamethy- expression library, besides Jun, that was able to len bisacetamide. In both cell types, USF2 translation associate with c-Fos, was USF2 (Blannar and Rutter, was repressed in the non-dividing cells. This strongly 1992). The relevance of this protein-protein association implies that USF2 is translationally repressed in was further supported by our observation that in vivo quiescent hematopoietic cells. Considering the proposed most of the c-Fos induced by aggregation of mast cell role of USF in proliferation it seems that translational FceRI binds to USF2 to form a DNA-binding complex regulation of USF2 might have an important role in (Lewin et al., 1993, 1996). Recently, we also observed cellular growth. that USF2 is encoded by an early response gene (Lewin et al., 1996) that produces a dimer with the Keywords: PKC; USF2; translational; regulation microphthalmia (mi) protein in activated mast cells (Nechushtan et al., 1997). The ability of c-Fos or mi to interact with USF2 may allow receptor-speci®c regulation of gene expression by increasing the Introduction diversity of DNA-binding protein complexes which regulate gene transcription. One of the obvious A vital role for cytokines and lymphokines in the interpretations regarding the intracellular function of regulation, maturation, activation, proliferation and USF2 is that it binds to c-Fos and mi, so preventing the speci®c functions of hematopoietic cells has been their association with other transcription factors. Other suggested (Nechushtan and Razin, 1996). Among the physiological functions of USF2, such as its role in the predominant cytokines that regulate the function of cell cycle, are currently being investigated. Sawadogo murine mast cells is the interleukin IL-3. IL-3 is a T- and coworkers reported that transfection of USF2 cell-derived growth factor which has been extensively genes resulted in the abrogation of the tumorogenic characterized in the mouse (Howard et al., 1982; Ihle et phenotype of c-myc transformed cells (Luo and al., 1983; Razin et al., 1984; Yung et al., 1981). Sawadogo, 1996). Our observations, that the induction Recently, we have extensively investigated the signal of USF2 synthesis occurs rapidly in cells triggered by transduction pathways induced by USF2 expression mast cell growth factors or by IgE-Ag, indicates that and DNA binding activity in murine mast cells (Lewin this transcription factor also plays a role in the et al., 1993, 1996). USF2 is a transcription factor that regulation of mast cell growth. Thus, this transcription was isolated on the basis of its identity to USF1 factor might have an important role in the control of cellular proliferation rates in many cell types. Correspondence: E Razin We have demonstrated that PKC is involved in the Received 22 May 1997; revised 22 September 1997; accepted 22 regulation of DNA synthesis in murine mast cells due September 1997 to their interaction with either IgE-Ag or IL-3 (Baranes PKC translational USF2 mast cell ZC Zhang et al 764 and Razin, 1991; Chaikin et al., 1994). Evidence for the detected in the polysomal fraction derived from the requirement of PKC activity in gene expression has IL-3-treated cells (Figure 3). Extraction of the RNA accumulated over the last twenty years. Studies from from the polysomal and subpolysomal fractions of IL-3- our laboratory also demonstrated the dependency on treated MC-9 cells revealed that IL-3 induced shifting of PKC of the induction of several transcription factors in USF2 mRNA from the subpolysomal fraction to the response to mast cell FceRI aggregation and mast cell- polysomal fraction. Thus, USF2 mRNA is relatively IL-3 interactions (Chaikin et al., 1994; Lewin et al., ineciently translated in untreated cells as only 42+4% 1996; Razin et al., 1994). In particular, these studies point to a role for PKC activity in the regulation of the transcription factor complex AP-1. In this work we further analyse the level at which kDa the expression of this protein is regulated in mast cells 98 — triggered by IL-3. We show that USF2, in contrast to USF1, is a translationally regulated early response gene 64 — and that this mode of regulation seems to be mediated by PKC. 50 — USF2

36 — Results

Kinetics of the induction of USF2 synthesis by IL-3 0 15 30 60 We have previously demonstrated that IgE-Ag induces Time (min) the accumulation of newly synthesized USF2 in murine Figure 1 Kinetics of USF2 protein expression in IL-3-stimulated mast cells (Lewin et al., 1996). We might have assumed mast cells. Immunoprecipitates of 35S-methionine labeled USF2 derived from IL-3-stimulated MC-9 cells, using anti mouse USF2 that IL-3 would similarly induce USF2 protein puri®ed antibodies. Duplicate samples are presented. Samples accumulation. Indeed, as shown in Figure 1, IL-3 were analysed by SDS-polyacrylamide gel electrophoresis. One induced USF2 protein synthesis with kinetics similar to representative experiment out of three is shown those of IgE-Ag induction, starting 15 min post- stimulation, reaching fourfold above control+0.2 s.e., n=3, at 30 min and exceeding the control levels for a more than 60 min. One explanation for the increase in the amount of the newly synthesized USF2 could be that it has a very short life under `starved' conditions which is signifi- USF2 — cantly increased after the addition of IL-3. We therefore used pulse chase experiments to show that USF2 is a stable protein over a period of 120 min post 35S-labeling (data not shown). Since the increase in the L-30 — level of [35S]USF2 observed in the activated cells occurred in the ®rst 15 ± 30 min, it seeems that this increase is due to induction of USF2 synthesis rather than an increase in its ability. — — — — 0153060 Regulation of the abundance of USF2 mRNA by IL-3 b To examine the possibility that the rate of USF2 Time (min) synthesis re¯ects the abundance of its mRNA, we monitored the levels of USF2 mRNA following IL-3 induction by Northern blot analysis (Figure 2). Quanti®cation of the radioactive signals by phosphorimager revealed that the ratios between the USF2 and c-fos — the ribosomal protein L30 band intensities were not signi®cantly altered by IL-3 treatment at any of the time points (Figure 2a and two additional experiments). In contrast, the abundance of c-Fos mRNA L-30 — was enhanced in these IL-3-stimulated cells (Figure 2b) (threefolds+0.2 s.e., n=3, of the control). It appears, therefore, that the rapid elevation (with

15 ± 30 min) of the rate of synthesis of USF2 upon IL-3 — — — — stimulation re¯ects a control mechanism operative at 0153060 the translational level. To examine this hypothesis, Time (min) polysomal fractionation experiments were carried out Figure 2 Kinetics of USF2 and c-Fos mRNAs accumulation in IL-3 activated mast cells. Cytoplasmic RNA was extracted from (Figure 3). The absorbance pro®le of the total RNA of IL-3-stimulated MC-9 cells and Northern blot analysis was the polysomes and subpolysomes was changed in the performed as described in Materials and methods. (a) USF2, (b) 45 min IL-3-treated MC-9 cells. More RNA was c-fos. One representative experiment out of six is shown PKC translational USF2 mast cell ZC Zhang et al 765 of this mRNA is engaged in polysomes. In contrast, was seen both after calphostin C and after PMA induction by IL-3 treatment led to a signi®cant increase treatment in the activated cells (Figure 5a,b). Down- in the translational eciency of USF2 mRNA as judged regulation of PKC by either of these treatments did not by its recruitment into polysomes (69+8%). The eect the rate of USF2 synthesis in unstimulated cells apparent elevation in the translational eciency of (data not shown). However, USF2 mRNA accumula- USF2 mRNA is highly speci®c, as a mRNA encoding a tion was also not signi®cantly aected by PKC structurally related transcription factor, USF1, is inhibition in MC-9 cells activated by IL-3 (Figure 6). eciently translated regardless of the growth status of Again, the intensities of the band of mRNAs encoding the cells, as is the case for actin mRNA (Figure 3). USF2 and L30 were quanti®ed and results obtained in Interestingly, the translational eciency of rpL32 three separate experiments indicate that there was no mRNA, which is known to be upregulated by growth signi®cant dierences in the level of USF2 mRNA and stimulation (Falvey and Schibler, 1991) is twofold that of L30 in MC-9 cells at any of the time points. induced by 2 h treatment with IL-3. However, unlike Hence, these results indicate that PKC either directly USF2 mRNA, it is still mostly sequestered in or indirectly regulates the expression of USF2 in the subpolysomal fraction (Figure 3). MC-9 cells. An intriguing question is whether the translational Polysomal fractionation experiments revealed that upregulation of USF2 mRNA is also applicable to PKC inhibition by either calphostin (Figure 7) or 6 h other cell types undergoing transitions between resting treatment with PMA (data not shown) prevented the and growing states. To directly address this question, IL-3-induced shifting of USF2 mRNA from the we used two hematopoietic derived cell types: mouse subpolysomal fraction to the polysomal fraction. P1798 lymphosarcoma cell and mouse erythroleukemia cells (MEL). Results from both cell lines showed a shift of USF2 mRNA into the subpolysomal fraction in Discussion non-dividing cells (Figure 4). In this article we have presented evidence based on the use of dierent methodologies that translational Inhibition of PKC aborogates the up-regulation of USF2 control has a major role in the immediate response To further explore the mechanisms involved in the regulation of USF2. regulation of USF2 expression, we measured the Initially, we obtained our results by comparing expression of USF2 in IL-3-activated cells which were radioactive methionine incorporation into immunopre- either treated with the PKC inhibitor calphostin C, at a concentration shown to be speci®c for PKC inhibition (Gwilliam et al., 1993), or incubated for 6 h with PMA. A signi®cant decrease in USF2 protein synthesis a %mRNA dex con in polysomes P S P S con dex

a actin 92 83

USF2 33 9

L32 42 5

b b %mRNA % mRNA con IL-3 in polysomes

con HMBA in polysomes P S P S con IL-3 P S P S con HMBA actin 88 86 actin 89 86

USF1 72±1(3) 77±1(3) USF2 75 40

USF2 48±4(4) 69±8(4) L32 81 32

Figure 4 Polysomal fractionation from non-dividing lymphosar- L32 ± ± 20 6(4) 42 4(4) coma and erythroleukemia cells. (a) Dexamethasone-treated mouse lymphosarcoma cell line (P1798.C7). (b) HMBA-treated Figure 3 Polysomal fractionation from IL-3 treated MC-9 cells. mouse erythroleukemia (MEL) cells. Size fractionation of Size fractionation of polysomes by sedimentation through sucrose polysomes by sedimentation through sucrose gradients, extrac- gradients, extraction of the RNA from the polysomal and tion of the RNA from the polysomal and subpolysomal fractions subpolysomal fractions (a) and Northern blot analysis (b) were and Northern blot analysis were performed as described in performed as described in Materials and methods Materials and methods PKC translational USF2 mast cell ZC Zhang et al 766 a kDa

98 — IL-3 con Cal+IL-3 Cal

64 —

50 — USF2 USF2 — 36 — IL-3 IL-3 L30 —

+PMA

b kDa Figure 6 The eect of down-regulation of PKC on USF2 98 — mRNA accumulation in IL-3-stimulated mast cells. MC-9 cells were incubated with or without the PKC inhibitor calphostin C and then stimulated with IL-3. Cytoplasmic RNA was extracted 64 — from the cells and Northern blot analysis was performed as described in Materials and methods. One representative experi- 50 — ment out of three is shown USF2

36 — IL-3 IL-3 % mRNA

+Cal IL-3+cal IL-3 in polysomes Figure 5 The eect of down-regulation of PKC on USF2 protein P S P S IL3+cal IL3 expression in IL-3-stimulated mast cells. MC-9 cells were incubated for 6 h with or without PMA (a) or for 20 min with actin 64 75 or without the PKC inhibitor calphostin C (b) and then stimulated with IL-3 for 45 min in DMEM methionine-free medium containing 50 mCi/ml [35S]methionine. Cells were lysed and soluble proteins immunoprecipitated with antibody to mouse USF2 14 43 USF2. Proteins were resolved by SDS ± PAGE. One representative experiment of three is shown Figure 7 Polysomal fractionation from calphostin C-treated IL-3-stimulated MC-9 cells. MC-9 cells were incubated with or without the PKC inhibitor calphostin C and then stimulated with IL-3. Size fractionation of polysomes by sedimentation through sucrose gradients, extraction of the RNA from the polysomal and cipitated USF2 protein after IL-3 induction, to the subpolysomal fractions and Northern blot analysis were levels of USF2 mRNA obtained following the same performed as described in Materials and methods stimuli. Our results demonstrated stable USF2 mRNA levels under these conditions with a profound increase in the levels of newly synthesized USF2 protein following IL-3 induction. It could be argued that the fraction in mast cells induced by IL-3. By analysing increase in protein half life is one of the causes of the the levels of USF2 mRNA associated with the stimuli-mediated elevation in USF2. However, since we polysomes compared to that not associated with have shown that USF2 is barely degraded even after polysomes, we obtained an estimate of USF2 transla- 2 h in unstimulated mast cells, and considering the tional rates. This assessment was not made using short half life of USF2 protein accumulation, it would antibodies, but rather a well-characterised cDNA seem that indeed stimulation with the cytokine probe. While USF2 mRNA was found mostly in the increases the rate of USF2 protein synthesis. subpolysomal fraction in unstimulated cells, it was The ®nding that USF2 protein is regulated in found mainly in the polysomal fraction in the IL-3- response to IL-3 only at the translational level came induced cells. In contrast, USF1 and actin mRNAs somewhat as a surprise in light of the regulation of were nearly entirely in the polysomal fraction both in related transcription factors. It was logical to have control and stimulated cells. These results veri®ed our assumed that USF2, like its heterodimerization partner initial observations and furthermore pointed towards c-Fos, its bHLH-Z family member c-Myc and most the initial translation steps as those probably respon- other early response transcription factors that have sible for the alteration in USF2 translational rate. been studied (Falvey and Schibler, 1991), is regulated Sequencing of the 5' region of USF2 mRNA mainly on the mRNA accumulation level in response revealed structural similarity to other translationally to mitogenic stimuli. regulated mRNAs (Lin et al., 1994). eIF4E is an Further con®rmation for the observation that USF2 initiation factor which is part of an initiation complex is translationally regulated was obtained by using a that binds to the mRNA cap. Since activation of eIF4E dierent methodology. We performed sucrose gradient has been suggested to lead to increased expression of of RNA from mast cells before and after IL-3 mRNAs containing long untranslated 5' regions induction. Using this system, we clearly demonstrated (similar to USF2 5' mRNA) and USF was shown to a signi®cant change in the amount of polysomal regulate the expression of eIF4E, it is possible that a PKC translational USF2 mast cell ZC Zhang et al 767 positive feedback regulation occurs in which activation mented with 2 mML-glutamine, 2 mM nonessential amino of USF translation by eIF4E leads to the increased acids, 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco), expression of that protein. 50 mM b-mercaptoethanol (Fisher Scienti®c, Medford, It is interesting to notice that c-Myc, which is MA), 10% fetal calf serum (FCS) (Bio-Lab, Jerusalem), usually regulated at the mRNA accumulation level in and IL-3 and IL-4 (kindly provided by I Clark-Lewis, Vancouver, Canada). response to mitogenic stimuli, was shown to be more Mouse lymphosarcoma cells (P1798.C7) were grown in eciently translated in cells derived from Bloom suspension. Cell division was arrested by treatment with syndrome patients (West et al., 1995). Previous studies 0.1 mM dexamethasone (Sigma) for 24 h as previously have demonstrated that, in certain situations, sequences described (Avni et al., 1994). Mouse erythroleukemia in the 5' of myc mRNA have in¯uence on the (MEL) cells were also grown in suspension. Dierentiation translational regulation of this gene. However, in of these cells was induced by 72 h treatment with 5 mM contrast to the regulation of USF2, c-Myc regulation hexamethylene bisacetamide (HMBA) for 72 h (Shama et al., in response to mitogenic stimuli was shown to be 1995). mainly at the mRNA accumulation levels and therefore is dierent from that of USF2. Growth factor-mediated activation We can only postulate a reason for the dierent MC-9 cells were transferred to serum-free medium. A mechanism used for USF2 regulation compared with suspension of 2.5 ± 56106 cells was washed and incubated USF1 and most other similar early response transcrip- with 15 ml serum-free RPMI medium for 4 h at 378C. The tion factors. The possible involvement of USF2 in cells were then washed and incubated in RPMI medium translational regulation through its regulation of eIF4E containing 100 U IL-3 for 15, 30 or 60 min. The induction might provide us with a clue as to the usefulness of reaction was stopped by centrifugation. Trypan blue translational regulation in the regulation of this gene, exclusion showed over 95% cell viability after each since many genes involved in translation have been experiment. found to be translationally regulated proteins (Math- ews et al., 1996). PKC inhibition We have also shown that USF2 is translationally Two dierent methods of PKC inhibition were used: 6 h repressed in two other hematopoietic derived cell lines incubation with PMA (20 ng/ml) which causes degradation (Figure 7). Combined with the results from the of PKC (Lewin et al., 1996) or 20 min incubation with the experiments with IL-3-induced mast cells, these results PKC inhibitor calphostin C (4 nM) which binds to the PKC strongly imply that USF2 is generally translationally regulatory domain (Gwilliam et al., 1993). The cells were repressed in quiescent cells. Considering the importance incubated in the absence or presence of PMA or calphostin of USF as a controller of genes such as P53, cyclin B C either prior to or during cell activation. and D and its antagonism of c-Myc, it seems logical to assume that translational regulation of USF2 has an Isolation of Cytosolic RNA (Sambrook et al., 1989) important role in cell cycle control. Further study is At the end of each experiment 107 cells were pelleted and needed in order to investigate this hypothesis further. resuspended in lysis buer containing heparin, spermidine, Since USF2 is ubiquitiously expressed and has such and NP-40 (Sigma), immediately centrifuged to remove the an important role in the regulation of many genes, it nuclei, and the RNA extracted with phenol and chloro- would also seem that further research is warranted for form : isoamylalcohol. The RNA was precipitated over- the better understanding of the role of USF2 night at 7208C in ethanol and sodium chloride, and translational regulation and its mechanisms in differ- washed with 70% ethanol. The concentration was then ent tissues. determined spectrophotometrically. Previously we have shown that PKC is an essential component of the signaling pathway from mast cell Northern blot analysis (Sambrook et al., 1989) surface to the nucleus (Baranes and Razin, 1991; Lewin et al., 1993, 1996; Razin et al., 1994). Here we have Samples of 5 ± 10 mg RNA derived from MC-9 cells were further elucidated the level of PKC involvement in the concentrated and denatured in a mixture of formaldehyde regulation of USF2 expression by studying the eects (BDH, United Kingdom) and formamide (Fluka) for 10 min at 658C. They were then separated on an of two PKC inhibitors on its induction. We have agarose/MOPS gel (SIGMA) which was blotted onto a clearly shown that PKC inhibition did not in¯uence the NY 13N Nytran (Amersham) in 106SSC for 48 h and steady state levels of USF2 mRNA, whereas the then exposed to U.V. for 4 min. After 2 ± 3 h of 428C induction of USF2 protein synthesis and the inhibition prehybridization in a solution of 66SSPE, 50% forma- of the IL-3-induced shifting of USF2 mRNA from the mide, 1% SDS, 56Denhart solution and 100 mg/ml subpolysomal fraction to the polysomal fraction was salmon sperm DNA, hybridization was carried out at prevented. Thus, PKC is involved in mediating the 428C for 24 h using 20 ng of heat denatured 32P-labeled translational response of USF2 to various external DNA probe. After an initial wash in 26SSC, 0.1% SDS stimuli. at room temperature followed by 25 min at 568C, the ®lter was exposed to ®lm (Kodak Curix RP2) for up to 2 days at 7708C. Quanti®cation of the radioactive signals on the blots was carried out by phosphorimager (Fujix BAS 1000, Fuji, Japan). Materials and methods

Cell culture Polysomal fractionation MC-9 cells (Nabel et al., 1981), obtained from the Harvesting, lysis of cells and size fractionation of American Type Culture Collection (Rockville, Maryland), polysomes by sedimentation through sucrose gradients were maintained at 378C in RPMI 1640 medium supple- were performed as described (Meyuhas et al., 1996). PKC translational USF2 mast cell ZC Zhang et al 768 DNA probes 50 mCi/ml [35S]methionine (Amersham) and incubated for 1 h at 378C. The cells were then either activated with DNP- The USF2 probe, a 300 bp oligonucleotide fragment BSA or with each lymphokine alone for various periods. derived from the 3'-region of the mouse USF2 cDNA The immunoprecipitation procedure previously described generated by PCR methods, was used for the analysis of (Kovary and Bravo, 1991) was modi®ed as follows: 2.56106 speci®c mRNA levels on Northern blots. The primers used cells either labeled or not with 35S-methionine were lysed by for the PCR were: (5' primer) 5'-AGGAGAAGAGCC- the addition of 500 ml cold lysis buer (0.01 M Tris-HCl CAG CACAAC-3' residue=766 and (3' primer) 5'- pH 7.4, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, TCGAAGCAGGGCGTTCTCATTCTT-3' residue=1026. 0.15 M NaCl and 0.25 mM phenylmethyl-sulfonyl¯uoride). Ac-Fos-speci®c probe (a 3.2 kb EcoRI ± BamHI fragment These cells were then homogenized and the supernatants isolated from a l c-Fos (mouse)-2 clone (Curran et al., collected after a 30 min spin in a microcentrifuge at 48C. To 1983) was used for the analysis of speci®c mRNA level on normalize samples before immunoprecipitation, 2 mlofeach Northern blots. A 0.97 kb fragment bearing the ribosomal mixture were removed, the 35S-labeled proteins were TCA- protein L-30 processed gene (Meyuhas et al., 1987), linking precipitated and the radioactivity determined. Equal counts the 5' and 3' regions of the p3A fragment, was used as a from each mixture were then added to the anti-mouse USF2 control for loading an equivalent amount of total RNA. A antibody which was either preincubated with USF2 peptide 2.2 kb fragment of mouse b-actin (Avni et al., 1994) and a or buered (control). After overnight incubation at 48C, 1.9 kb fragment of USF1 (Meier et al., 1994) were also 10 mg of protein-A sepharose beads (Sigma) were added to used as probes for this study. The DNA fragments were the incubated mixtures and agitated for 3 h at 48C. The labeled with 32P-dCTP (Amersham) using the random protein A-sepharose beads were washed three times with primed labeling technique (Feinberg and Vogelstein, lysis buer and then washed again with Tris-EDTA buer. 1983) up to a speci®c activity of 36108 c.p.m./mg. Labeled After boiling in Laemmli sample buer containing 0.5% probes were used at the ®nal concentration of SDS, the 35S-labeled and unlabeled precipitates were 56106 c.p.m./ml hybridization mixture. separated by discontinuous 10% acrylamide-bisacrylamide SDS slab gels, along with molecular weight markers. For Preparation of rabbit anti-mouse USF2 antibody the 35S-labeled materials the gels were dried and exposed to Kodak X-Omat AR ®lm at 7708C for various periods. Rabbit polyclonal anti-mouse USF2 antibodies were Quantitation of the autoradiographic bands was performed prepared against a synthetic 14 amino acid peptide, by densitometric analysis. corresponding to the sequence encoded by the base pairs 180 ± 216 of the open reading frame of the mouse USF2 cDNA (Lewin et al., 1996; Sirito et al., 1994), as previously Pulse-chase experiments described by our group (Lewin et al., 1996). Cells were washed once with DMEM methionine-free medium (Biological Industry, Beth Haemek, Israel), Immunoprecipitation resuspended in a DMEM-methionine-free medium contain- Cells, which were either sensitized or not with IgE, were ing 50 mCi/ml [35S]methionine (Amersham) and incubated washed once with DMEM methionine-free medium for 30 min at 378C. The cells were then washed with RPMI (Biological Industries, Beth Haemek, Israel), resuspended medium and incubated for 120 min in RPMI medium and in DMEM-methionine-free medium either with or without then the [35S]USF2 was immunoprecipitated.

References

Avni D, Shama S, Loreni F and Meyuhas O. (1994). Mol. Lin Q, Luo X and Sawadogo M. (1994). J. Biol. Chem., 269, Cell. Biol., 14, 3822 ± 3833. 23894 ± 23902. Baranes D and Razin E. (1991). Blood, 78, 2354 ± 2360. Luo X and Sawadogo M. (1996). Proc. Natl. Acad. Sci. USA, Blannar MA and Rutter WJ. (1992). Science, 256, 1014 ± 93, 1308 ± 1312. 1017. Mathews MB, Sonberg N and Hershey JWB. (1996). Origins Chaikin E, Hakeem I and Razin E. (1994). J. Biol. Chem., and targets of translational control. Translational Control. 269, 8498 ± 8503. Mathews MB, Sonberg N and Hershey (eds). Cold spring CogswellJP,GodlevskiMM,BonhamM,BisiJandBabiss Harbor Laboratory Press, pp 1 ± 8. L. (1995). Mol. Cell. Biol., 15, 2782 ± 2789. Meier JL, Lio X, Sawadogo M and Straus SE. (1994). Mol. Curran TMW, Van Straaten F and Verma IM. (1983). Mol. Cell. Biol., 14, 6896 ± 6906. Cell. Biol., 3, 914 ± 920. Meyuhas O, Thompson EA and Perry RP. (1987). Mol. Cell. Falvey E and Schibler U. (1991). FASEB J., 5, 309 ± 314. Biol., 7, 2691 ± 2699. Feinberg AP and Vogelstein B. (1983). Anal. Biochem., 132, Meyuhas O, Biberman Y, Pierandrei-Amaldi P and Amaldi 6 ± 20. F. (1996). A laboratory guide to RNA: isolation, analysis, Gwilliam DJ, Jones PM, Persaud SJ and Howell SL. (1993). and synthesis, Krieg P (ed) Wiley Liss: New York, pp 65 ± Acta Diabetol., 30, 99 ± 104. 81. Howard M, Farrar J, Hil®ker M, Johnson B, Takatsu K, Nabel G, Galli SJ, Dvorak AM, Dvorak HF and Cantor H. Hamaoko T and Paul WE. (1982). J. Exp. Med., 155, (1981). Nature, 291, 332 ± 335. 914 ± 930. Nechushtan H and Razin E. (1996). Crit. Rev. Oncol. Ihle JN, Keller J, Oroszlan S, Henderson LE, Capeland YD, Hematol., 23, 131 ± 150. Prystowsky MB, Goldwaser E, Schrader JW, Palaszynski Nechushtan H, Zhang ZC and Razin E. (1997). Blood, 89, E, Dy M and Label B. (1983). J. Immunol., 131, 282 ± 295. 2999 ± 3008. Kovary K and Bravo R. (1991). Mol. Cell. Biol., 11, 2451 ± Razin E, Ihle JN, Seldin E, Mencia-Huerta JM, Katz HR, 2461. Leblanc P, Hein A, Caul®eld JP, Austen KF and Stevens Lewin I, Nechushtan H, Qingen K and Razin E. (1993). RL. (1984). J. Immunol., 132, 1479 ± 1491. Blood, 82, 3745 ± 3751. Razin E, Szallasi Z, Kazanietz MG, Blumberg PM and Lewin I, Jacob-Hirsch J, Zhang ZC, Kupershtein V, Szallasi Rivera J. (1994). Proc. Natl. Acad. Sci. USA, 91, 7722 ± Z, Rivera J and Razin E. (1996). J. Biol. Chem., 271, 7726. 1514 ± 1519. PKC translational USF2 mast cell ZC Zhang et al 769 Reisman D and Rotter V. (1993). Nucl. Ac. Res., 21, 345 ± Viollet B, Lefrancois-Martinez AM, Henrion A, Kahn A, 352. Raymondjean M and Martinez A. (1996). J. Biol. Chem., Sambrook J, Fritsch EF and Maniatis T. (1989). Molecular 271, 1405 ± 1410. Cloning Cold Spring Harbor Laboratory, Cold Spring West MJ, Sullivan NF and Willis AE. (1995). Oncogene, 11, Harbor, NY. 2515 ± 2524. Shama S, Avni D, Frederickson RM, Sonenberg M and Yung YP, Eger R, Tertian G and Moore MAS. (1981). J. Meyuhas O. (1995). Gene Expression, 4, 241 ± 252. Immunol., 127, 794 ± 807. Sirito M, Lin Q, Maity T and Sawadogo M. (1994). Nuc. Ac. Res., 22, 427 ± 429.