Oncogene (2000) 19, 5810 ± 5816 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc H19 gene expression is up-regulated exclusively by stabilization of the RNA during muscle cell di€erentiation

Laura Milligan1, Etienne Antoine1, Catherine Bisbal1, MichaeÈ l Weber1, Claude Brunel1, Thierry Forne 1 and Guy Cathala*,1

1Institut de GeÂneÂtique MoleÂculaire, UMR 5535 CNRS-Universite Montpellier II, IFR 24, 1919 route de Mende, 34293 Montpellier Cedex 5, France

H19 is a paternally imprinted gene whose expression imprinting of H19 and/or Igf2 has also been described produces a 2.4 kb RNA in most tissues during in numerous cancers in adults (Cui et al., 1998; Hibi et development and in mammalian myoblastic cell lines al., 1996; Li et al., 1995; Singer et al., 1995; Verkerk et upon di€erentiation. Deletion of the active maternal al., 1997; Yun et al., 1996). allele of H19 and its ¯anking regions in the mouse leads Recent works showed that deletions of the active to biallelic methylation and loss of imprinting of the maternal allele of the H19 gene or its surrounding neighbouring Igf2 gene. The function of H19 RNA regions result in biallelic expression (Jones et al., 1998; remains unknown and, although polysome-associated, the Leighton et al., 1995a,b; Ripoche et al., 1997; absence of a conserved open reading frame suggests that Thorvaldsen et al., 1998) and changes in methylation it does not encode a protein product. We describe a novel patterns (Forne et al., 1997) of the Igf2 gene. These post-transcriptional regulation of H19 gene expression experiments suggest that the H19 gene and its ¯anking which, in spite of this lack of coding capacity, is sequences play important roles in the regulation of Igf2 dependent on translational activity. We show that imprinting in cis (Reik and Walter, 1998), in particular stabilization of the RNA is solely responsible for its a region located 2 kb upstream of the H19 gene was accumulation during in vitro muscle cell di€erentiation. shown to be the main imprinting control element in This conclusion is based on the ®nding that inhibition of this locus (Tremblay et al., 1997). protein synthesis results in a dramatic destabilization of The H19 gene codes for a primary transcript which H19 RNA in proliferating mouse C2C12 myoblastic cells is processed like a normal mRNA precursor to give a but not in di€erentiated cells, and on run-on experiments 2.4 kb mature transcript and, although localized in the which showed that the rate of transcription of H19 RNA cytoplasm, most likely remains untranslated (Brannan remains constant during muscle cell di€erentiation. This et al., 1990; Juan et al., 2000). The H19 transcript does mechanism could also be involved in H19 gene expression not contain any conserved open reading frames of during mouse development in addition to its transcrip- signi®cant length, and no corresponding protein has tional activation which we have shown to occur. been identi®ed in vivo (Brannan et al., 1990; Joubel et Oncogene (2000) 19, 5810 ± 5816. al., 1996), despite the demonstration that deletion of a large 5' region of the RNA enables in vitro translation Keywords: H19; parental imprinting; post-transcrip- (Joubel et al., 1996). Although the H19 transcript was tional regulation; muscle cell di€erentiation discovered some 15 years ago (Pachnis et al., 1984), and in spite of strong phylogenic evidence for its functionality (Hurst and Smith, 1999; Juan et al., Introduction 2000), very little is known about this RNA and its function. Apart from pathologies, H19 RNA is H19 belongs to a growing family of genes whose expressed in numerous tissues during mammalian expression is monoallelic and depends upon parental development and postnatal growth. In adult, H19 is origin of the alleles, a process known as genomic globally repressed but a signi®cant basal level of RNA imprinting (Barlow, 1997; Tilghman, 1999). Like its can still be detected in some tissues, especially cardiac close neighbour, the insulin-like growth factor-2 gene and skeletal muscle (Leibovitch et al., 1991; Pachnis et (Igf2), lying 90 kb usptream on mouse chromosome 7, al., 1988). Another early work described that the it was one of the ®rst imprinted genes discovered. expression of H19, called Myo H, is strongly induced While Igf2 is paternally expressed (DeChiara et al., during muscle cell di€erentiation (Davis et al., 1987). 1991), H19 is paternally imprinted and, therefore, Therefore, it seems reasonable to envisage a role for expressed only from the maternally inherited allele H19 RNA in development and muscle cell di€erentia- (Bartolomei et al., 1991). This reciprocal expression tion. In particular, it was proposed recently that, due pattern is frequently lost in Wilms' tumour, a child- to control of its expression by the p53 protein, H19 hood renal neoplasm, and in the Beckwith ± Wiede- could be involved in the cell cycle (Dugimont et al., mann overgrowth syndrome that predisposes to Wilms' 1998). tumour (Junien, 1992; Reik et al., 1994). Loss of It has been directly demonstrated that induced expression of a transfected copy of H19 suppresses cellular proliferation, clonogenicity and tumorigenicity *Correspondence: G Cathala in certain tumour cell lines (Hao et al., 1993). This Received 30 June 2000; revised 20 September 2000; accepted 25 point remains controversial as some groups obtained September 2000 results in agreement with this property (Cui et al., Stabilization of H19 RNA and muscle cell differentiation L Milligan et al 5811 1997; Moulton et al., 1994; Taniguchi et al., 1995), while others describe H19 RNA as a simple tumoral marker (Biran et al., 1994; Lustig-Yariv et al., 1997). However, in support of an anti-proliferative e€ect of H19 RNA, up-regulation of the H19 gene has frequently been demonstrated during in vitro cell di€erentiation and growth arrest (Davis et al., 1987; Eversole-Cire et al., 1995; Hayashida et al., 1997; Kim et al., 1994; Pachnis et al., 1984, 1988). More recently, Li et al. (1998) found H19 RNA to be polysome-associated, a result which is in contra- diction with previous data (Brannan et al., 1990). Their observations also led to the suggestion that H19 RNA could have an antagonistic trans e€ect on Igf2 translation. Here, we describe a novel post-transcriptional regulation of H19 gene expression in murine muscle cells which leads to stabilization of the RNA. We demonstrate that the accumulation of H19 RNA observed during in vitro muscle cell di€erentiation results exclusively from stabilization. In contrast, in muscle and liver during mouse perinatal growth, H19 RNA levels are primarily controlled by the transcrip- tional activity of the gene, even if a contribution by the post-transcriptional regulation we describe here re- mains plausible. Figure 1 Translation dependent levels of H19 RNA in prolifer- ating C2C12 cells. C2C12 cells were seeded at low density into multiple culture plates, cultured to 70% con¯uence and submitted to drug e€ects. (a) Northern blot analysis was performed on Results 15 mg of total RNA isolated on serial hours from individual plates after drug addition. Lanes 0, 1, 2 and 4 (indicated at the top of the ®gure) correspond to 0, 1, 2 and 4 h exposure of cells to Inhibition of protein synthesis reduces the level of H19 drugs. Two identical Northern blots were carried out simulta- RNA in proliferating C2C12 myoblasts neously and probed with H19 or c-Myc DNA probes. They were then probed for 18S rRNA. (b) The bar graphs depict the relative Recently, Li et al. (1998) provided results arguing that levels of H19 RNA (solid bars) and c-Myc RNA (open bars) after H19 RNA is associated with polysomes. This was normalizing for RNA loading using 18S rRNA levels contrary to what was currently admitted since H19 RNA was reported to remain in a free cytoplasmic particle (Brannan et al., 1990). As we felt that it was translational inhibition is e€ectively responsible for important to clarify this point, we performed sucrose the reduction in the level of H19 RNA or if H19 gene gradient centrifugations with extracts from C2C12 cells transcription is indirectly a€ected by the treatment. and, in agreement with Li et al. (1998), we found that Proliferating C2C12 cells were therefore treated for up H19 RNA is polysome-associated (not shown). During to 4 h with actinomycin D, an inhibitor of RNA the course of these experiments, we noticed that polymerases. This treatment had no e€ect whatsoever treatment of proliferating C2C12 cells with cyclohex- on the level of H19 RNA (Figure 2), whereas it led to a imide resulted in a decrease in the level of H19 RNA. dramatic fall in c-Myc mRNA due to its rapid As cycloheximide blocks translational elongation, this turnover (Dani et al., 1984). Therefore the half-life of led us to hypothesize that H19 RNA turnover could be H19 RNA is much longer than 4 h and, as such, a€ected by inhibition of protein synthesis. cannot be determined using transcriptional inhibitors. To explore this possibility, we used three drugs, We can conclude from these experiments that the cycloheximide, hygromycin and puromycin, known to H19 RNA detected in proliferating C2C12 cells block translational elongation. Treatment of proliferat- appears to be very stable and that inhibition of protein ing C2C12 myoblasts with each of these drugs resulted synthesis leads to its destabilization. in a noticeable decrease in the level of H19 RNA with very similar kinetics (Figure 1). The average half-life of Inhibition of protein synthesis does not affect H19 RNA H19 RNA following translational inhibition was levels in differentiated C2C12 cells approximately 4 h (Figure 1, see also Figure 3a). In this experiment we used c-Myc mRNA, rather than Upon serum starvation, C2C12 cells di€erentiate other generic transcripts, as the positive control of drug spontaneously into myotubes and steady state levels e€ects. c-Myc mRNA has been extensively studied in of H19 RNA rise considerably. We carried out the this system and it is known to be highly unstable (Dani same experiments as above to inhibit the translational et al., 1984), except after inhibition of protein synthesis activities on di€erentiated C2C12 cells. As found in where accumulation is observed (Yeilding et al., 1998). proliferating cells, the inhibition of transcription had As expected, all three drugs resulted in a spectacular no e€ect on H19 RNA levels (Figure 3b, compare TC- increase in the level of c-Myc mRNA (Figure 1). with C). Surprisingly, inhibition of protein synthesis no Although all three drugs had the same e€ect, it was longer e€ects H19 RNA levels after di€erentiation still necessary to establish with certainty whether (Figure 3b, compare TL- with C) in spite of the large

Oncogene Stabilization of H19 RNA and muscle cell differentiation L Milligan et al 5812

Figure 3 Comparison of drug e€ects on H19 RNA levels between proliferating and di€erentiated C2C12 cells. C2C12 cells were seeded at low density into multiple culture plates and cultured to 70% con¯uence. Half of the cultured plates were used for the analysis on proliferating cells as in Figures 1 and 2, the second half were induced to di€erentiate by 3 days of serum starvation and used for the analysis on di€erentiated cells. The e€ects of each drug (cycloheximide, hygromycin and puromycin for translational inhibition, and actinomycin D for transcriptional inhibition) were tested on duplicate cultured plates. Total RNA was extracted from each cultured plate and 15 mg was analysed on Northern blot using an H19 DNA probe, and subsequently an 18S probe in order to normalize the H19 RNA levels for RNA loading. The bar graphs depict the relative levels of H19 RNA in non-treated control cells (C), and after 4 h of translational (TL-) or transcriptional (TC-) inhibition in proliferating (a) and di€erentiated (b) cells. For translational inhibition, the values shown are the mean of the values obtained for each of the three drugs

tional activity of the H19 gene in nuclei from the same cells as above and we found that the rate of transcription of H19 RNA remains constant during C2C12 cell di€erentiation (Figure 4b, left panel and c, left panel, open bars). Therefore, transcriptional activation is not responsible for the rise observed in Figure 2 H19 RNA levels in proliferating C2C12 cells are not the level of H19 RNA during in vitro muscle cell a€ected by 4 h of trancriptional inhibition. C2C12 cells were di€erentiation, this accumulation is exclusively due to cultured and analysed as in Figure 1 after actinomycin D post-transcriptional regulation. In order to check for treatment. (a) Northern blots: lanes 0, 0.5, 1, 2 and 4 correspond proper di€erentiation in these cells and to normalise to 0, 0.5, 1, 2 and 4 h of exposure of the cells to actinomycin D. the activity of the di€erent nuclei in this experiment, (b) The bar graphs depict the relative levels of H19 RNA (solid bars, average of three di€erent experiments) and c-Myc RNA the transcriptional activities of several genes were used (open bars) after normalizing for RNA loading using 18S rRNA as controls. As expected, myogenin, a-actin, and p21 levels are transcriptionally activated during myogenic di€er- entiation whereas b-actin, GAPDH, and c-Myc main- tain constant transcription as previously reported amount of H19 RNA present compared to proliferat- (Wisdom and Lee, 1990; Hayashida et al., 1997). ing cells (compare relative levels in Figure 3a,b. Activity of the Igf2 gene, known to be coregulated Therefore, taking these results together with those with the H19 gene (Leighton et al., 1995b), was also obtained in proliferating cells, we can conclude that analysed and a transcriptional activation was observed post-trancriptional regulation contributes to the ex- in di€erentiated cells after 3 days (Figure 4b, left panel, pression of H19 RNA during muscle cell di€erentia- lane 3 days). This indicates that these two genes, tion. although sharing certain regulatory elements (Leighton et al., 1995b), can be independently regulated at the transcriptional level at least in C2C12 cells. Igf2 is Post-transcriptional regulation leads to H19 RNA transcriptionally induced, whereas H19 undergoes post- accumulation during muscle cell differentiation transcriptional RNA stabilization. In order to determine the contribution of post- transcriptional regulation to H19 RNA expression During mouse development, H19 RNA is mainly regulated during muscle cell di€erentiation, we compared H19 at the transcriptional level RNA levels with the transcriptional activity of the gene. Northern blot analysis of H19 RNA levels in H19 RNA is highly expressed in numerous tissues proliferating C2C12 cells and during di€erentiation is during the perinatal period of mouse development, shown in Figure 4a (left panel). As expected, there is a than drastically falls to very low levels in adults. sevenfold increase in the level of H19 RNA between Apart from the parental imprint, which leads to proliferating and di€erentiated C2C12 cells (compare monoallelic expression of the maternal allele, little is lanes P and 3 days in a and in c, solid bars). Run-on known about H19 gene regulation during develop- experiments were carried out to measure the transcrip- ment. Given the results we obtained during muscle cell

Oncogene Stabilization of H19 RNA and muscle cell differentiation L Milligan et al 5813

Figure 4 Transcriptional analysis of H19 gene activity. (a) Northern blots: 15 mg of total RNA extracted at serial times from cells or tissues were analysed by Northern blot using an H19 DNA probe, and subsequently an 18S DNA probe in order to normalize the H19 RNA levels. (b) Nuclear run-on assays: Nuclei were isolated from cells or tissues at the same times as in (a). The a-32P-UTP labelled transcripts, synthesized by 2.106 nuclei for cells and liver and 1.106 for muscle, were hybridized to ®lters on which had been bound denatured plasmids containing the appropriate insert DNA for each gene tested, H19 containing plasmids were loaded in duplicate, therefore the transcriptional activity was taken as the sum of the two. (c) Bar graph comparison of H19 steady state RNA levels with the transcriptional activity of the gene. The relative levels of H19 RNA are depicted by solid bars and the relative transcriptional activity by open bars. The run-on values for H19 were expressed relative to those of c-Myc and GAPDH. b-actin was not taken into account in the normalization of the data as it was shown that changes in the transcriptional activity of this gene occur during myogenesis (DePonti-Zilli et al., 1988). An empty plasmid (line `plasmid') was used to detect non-speci®c hybridization. Except for the run-on value in muscle, which represents the average of the two normalizations using c-Myc or GAPDH, all other values were from three di€erent experiments di€erentiation, it seemed important to us to investi- gene also appears to be transcriptionally regulated in gate H19 gene regulation during mouse development. both tissues. We chose the appropriate developmental stages, between 15 days post coitum and 18 days post partum, in muscle and liver and carried out the same Northern Discussion blot and run-on analyses as in C2C12 cells (Figure 4, middle and right panels). In both tissues we observed In addition to the known monoallelic expression and that the level of H19 RNA correlates with the transcriptional regulation of the gene, we show here transcriptional activity of the gene. The transcriptional that H19 RNA is subject to considerable stabilization, activity of the H19 gene is maximal at 2 days post which is dependent on protein synthesis, as use of partum in muscle as is the level of the RNA, whereas pharmacological inhibitors of translation led to a in liver both maxima are attained slightly later at 8 decrease in H19 RNA levels in proliferating C2C12 days post partum. Transcription is shut down in both cells. Therefore, stabilization of this RNA could be tissues by 18 days post partum. It is clear that during coupled to translation by the requirement of a mouse development, contrary to in vitro muscle cell relatively short-lived trans-acting stabilizing factor. di€erentiation, transcriptional regulation plays a major This was con®rmed when transcriptional inhibition role in H19 RNA levels. One can note that the Igf2 had no e€ect whatsoever on the level of H19 RNA

Oncogene Stabilization of H19 RNA and muscle cell differentiation L Milligan et al 5814 over the same period of time. We conclude that after suggested that NMD (nonsense-mediated decay) could inhibition of transcription by actinomycin D, H19 be involved (Smith and Steitz, 1998). However, RNA appears to be very stable but after inhibition of contrary to GAS5, inhibition of protein synthesis protein synthesis its half-life is reduced to 4 h in results in destabilization of H19 RNA, indicating that proliferating C2C12 cells. it is unlikely that the same mechanism is involved. In di€erentiated C2C12 cells, it was somewhat H19 RNA is also extremely abundant in numerous surprising to ®nd that, even though there is approxi- tissues during mouse perinatal growth. Based on mately seven times more H19 RNA, inhibition of nuclear run-on analysis of trancriptional activity and protein synthesis no longer a€ected H19 RNA levels. Northern blot analysis of steady state mRNA levels, Therefore, during di€erentiation, H19 RNA acquires a we demonstrate that, in mouse liver and muscle, H19 resistance to destabilization by translational inhibition. gene regulation is primarily transcriptional since steady This suggests that H19 RNA stabilization is dependent state RNA levels are proportional to the transcrip- on a translation product (or products) which has been tional activity of the gene. From these experiments, we modi®ed either in quantity or activity by di€erentia- are unable to rigorously quantify the contribution of tion. transcriptional and post-transcriptional regulation to Nuclear run-on analysis was performed and, as the H19 RNA expression, however it seems reasonable to trancriptional activity of the H19 gene remained assume that stabilization of H19 transcripts also constant during muscle cell di€erentiation, we conclude occurs. Since di€erentiation and proliferation are that the accumulation of H19 RNA during myogenic concomitant during development, the question arises di€erentiation is exclusively due to post transcriptional whether or not the putative trans-acting H19 RNA regulation. stabilizing factor could be ubiquitously induced during The simplest explanation for these results is that H19 di€erentiation. Finally, the stabilization that we RNA is directly stabilized by a relatively unstable describe here could be sucient to explain the trans-acting factor present in limiting quantity in frequently observed up-regulation of the H19 gene proliferating muscle cells but in relative excess after associated with in vitro cell di€erentiation and growth di€erentiation. The 4 h half-life of H19 RNA, after arrest (Davis et al., 1987; Eversole-Cire et al., 1995; translational inhibition in proliferating cells, would Hayashida et al., 1997; Kim et al., 1994; Pachnis et al., then re¯ect the stability of the trans-acting factor. As to 1984, 1988), suggesting that the anti-proliferative the stability of the unprotected H19 RNA molecules, activity of H19 RNA could be used to promote which exist at least in proliferating cells, their decay di€erentiation. must be extremely rapid, since no fall in the level of Post-transcriptional down-regulation of H19 RNA H19 RNA is observed following transcriptional by the product of the Raf gene was reported, some inhibition. Kinetics of this type bring to mind nuclear time ago, to be involved in the repression of H19 gene post-transcriptional mechanisms such as transport or expression after birth in the mouse (Vacher et al., splicing, where precursors and intermediates are 1992). This down-regulation would appear to be tissue practically undetectable. Recent evidence indicates that speci®c as it functions in liver but not in muscle or an increasing number of RNA binding proteins seem heart (Pachnis et al., 1988), and consequently, the up- to play a role between transcription and these post- regulation we describe during muscle cell di€erentiation transcriptional processes (Ladomery, 1997). Di€erences would be unrelated. Therefore, H19 RNA is subject to in the proteins recruited onto nascent H19 transcripts complex post-transcriptional regulation in addition to has already been proposed to explain the absence in the trancriptional regulation of the gene. This should the cytoplasm of H19 RNA produced by leaky be kept in mind when attempting to correlate RNA expression from the paternal allele (Jouvenot et al., levels with H19 gene deregulation in tumours. 1999), and it is conceivable that the nascent H19 RNA A hallmark of past studies has been the diculties particle also contains factors which regulate its turn- encountered when attempts were made at expressing over. For example, hnRNP D and elav-like proteins H19 RNA after cell transfection (Yoo-Warren et al., are known to have antagonistic e€ects on the stability 1988) and transgenesis in mouse (Ainscough et al., of diverse ARE containing mRNA (for review see 1997). The requirement for the trans-acting stabilizing Mitchell and Tollervey, 2000), and their association factor postulated in the present work is perhaps with nascent transcripts in the nucleus has been sucient to explain these problems, consequently its suggested (Fan and Steitz, 1998). identi®cation and an understanding of its regulation Alternatively other nuclear or cytoplasmic mechan- will be useful for future studies of H19 RNA function. isms could account for this post-transcriptional up- regulation of H19 RNA. Firstly, compartmentalization could confer stability to this RNA, therefore a factor active in its transport would be indirectly involved in Materials and methods its stabilization. Secondly, inhibition of processing could lead to rapid decay, similar to the exosome Cell culture mediated decay of unspliced pre-mRNA in yeast (Mitchell and Tollervey, 2000) and, inversely, stimula- The mouse skeletal muscle cell line C2C12 was grown in 50% DMEM, 50% HAM'S F12 containing 10% foetal calf serum tion of processing to stabilization. Finally, other non- (FCS). Myoblastic di€erentiation was induced by serum coding RNA have been found to be polysome- starvation. When cells reached 70% con¯uence, FCS was associated, such as GAS5 (Smith and Steitz, 1998). reduced to 1%, and then di€erentiated cells were recovered The rapid decay of this RNA is strongly regulated by after 72 h. Cycloheximide at a ®nal concentration of 10 mg/ translocational activity and as, like H19 RNA, it ml, puromycin at 50 mg/ml, hygromycin at 1000 units/ml, and contains multiple short open reading frames, it was actinomycin D at 5 mg/ml were added to growth or

Oncogene Stabilization of H19 RNA and muscle cell differentiation L Milligan et al 5815 di€erentiation medium for the times indicated in the legends NaCl before spotting onto a nitro-cellulose (BA83, Schleicher to ®gures. and Schuell) ®lter presoaked in 26SSC. The ®lters were then washed with 26SSC, dried in a vacuum oven at 808C for 2 h and prehybridized at 428C in 1 ml of hybridization bu€er RNA extraction and analysis (50% formamide, 50 mM NaPO4 (pH 6.5), 0.8 mM NaCl, Total RNA was isolated by the guanidinium thiocyanate 1mM EDTA, 2.56Denhardt's bu€er, 250 mg/ml salmon procedure. Routinely, subsequent RNA extraction was sperm DNA, 500 mg/ml yeast tRNA). carried out using acidic phenol/chloroform followed by Nuclei in 100 ml of Bu€er C were added to an equal isopropanol precipitation and ®nally re-extracted with phenol volume of reaction bu€er (10 mM Tris-HCl (pH 8.0), 5 mM (pH 7.5), chloroform and ethanol precipitation. Tissue MgCl2, 0.3 M KCl, 1 mg/ml Heparin, 1 mM ATP, 1 mM samples were immediately disrupted in guanidinium thiocya- CTP, 1 mM GTP, 100 mCi [a-32P]UTP at 800 Ci/mmol) and nate solution using a Dounce Homogeniser. shaken at 308C for 30 min then treated with DNase Q1 and RNA was electrophoresed on a 1.1% agarose formalde- Proteinase K. Labelled RNA (1 c.p.m. per nucleus for C2C12 hyde gel. Transfer to a positively charged nylon membrane cells and liver or 0.2 c.p.m. per nucleus for muscle) were (Appligen Oncor) was carried out in 150 mM ammonium extracted with an equal volume of phenol/chloroform, acetate for 8 h. The membrane was then dried for 30 min at ethanol precipitated, dissolved in 100 ml of TE containing 808C in a vacuum oven and then exposed to weak UV light 0.1% SDS and hybridized to ®lters for 48 h. The ®lters were (0.07 J/cm2) using an Ultraviolet Crosslinker (Amersham). then washed for 10 min at room temperature in 26SSC, Membranes were hybridized overnight at 428C in 50% 30 min at 658C in 0.26SSC, and treated with 10 mg/ml formamide, 56SSC, 56Denhardt's solution, 10% dextran RNase A in 26SSC for 15 min at 378C. Finally, ®lters were sulfate, 20 mM sodium phosphate bu€er (pH 7.0) containing washed twice more in 26SSC for 30 min at 378C. 100 mgml71 denatured herring sperm DNA (DNA typing grade, Gibco BRL). Final wash of membranes was twice Quantification of RNA levels and transcriptional activity of the 15 min at 658C in 0.1 SSC, 0.1% SDS. gene Northern blots and run-on assays were analysed on a Isolation of nuclei Molecular Dynamics PhosphorImager and the relative levels Nuclei were isolated from proliferating and con¯uent murin were determined by normalizing for RNA loading using the C2C12 cells in growth medium and after 1 and 3 days in level of 18S rRNA for Northern blots and various RNA di€erentiation medium as follows: 2.107 cells were washed Polymerase II transcripts for run-on assays as indicated in twice with cold PBS, resuspended in cold bu€er A (10 mM the legends to ®gures. All experiments were performed at Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% NP40) least twice (except for the run-on assays on muscle) and and disrupted using a Dounce Homogeniser. The disrupted representative results are displayed in the ®gure presented. cells were then deposited on a cushion of Bu€er B (30% Apart from Figure 2, the relative levels were calculated by sucrose, 50 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 0.1 mM according 100 to the highest value in each experiment. EDTA) and centrifugated for 9 min at 1200 g. The pellet of nuclei were resuspended in 100 ml of Bu€er C (40% glycerol, Probes used on Northern blot 50 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 0.1 mM EDTA), aliquoted, and stored frozen until use in run-on assays. The H19 DNA probe was the gel puri®ed ApaI±EcoRI Mouse livers were homogenized in 2.1 M sucrose, 10 mM restriction fragments of an H19 cDNA construct (positions HEPES (pH 7.6), 2 mM EDTA, 15 mM KCl, 10% glycerol, 1019 to 2343 from the transcriptional start). The c-Myc DNA 0.15 mM spermine, 0.5 mM DTT, 0.5 mM PMSF and 7 mg/ml probe was the gel puri®ed HindIII cDNA fragment (gift from aprotinin using a Potter. The homogenate was then deposited Jean-Marie Blanchard, Montpellier). The 18S ribosomal on a cushion of the same bu€er and centrifugated at DNA probe was a gift from Jean-Pierre Bachellerie, Toulouse 100 000 g for 40 min. The pellet was then washed in 10 mM (Raynal et al., 1984). Probes were labelled by random Tris-HCl (pH 7.4), 15 mM NaCl, 60 mM KCl, 0.15 mM priming using the Nonaprimer kit (Appligene Oncor) and spermine, 0.5 mM spermidine, before being resuspended in [a-32P]dCTP at 3000 Ci/mmol (New England Nuclear). Bu€er C. Nuclei were isolated from mouse muscle in the same manner except that the tissue was ®rst disrupted using an ultra-turrax before homogenization. Using 24 15-dpc (day post coõÈ tum) embryos, 19 2-day-old, 15 8-day-old and 10 18-day-old individuals, we obtained Acknowledgments approximately 2.108 nuclei from liver whereas 39 15-dpc We thank Dr D Tollervey, Edinburgh, for helpful com- embryos, 23 2-day-old, 12 8-day-old and 8 18-day-old ments on the manuscript and Dr M Silhol for technical individuals only provided 106 nuclei from muscle. assistance on run-on experiments. This work was sup- ported by grants from the Association pour la Recherche contre le Cancer given to Claude Brunel (contrat 2908), Run-on assays from the Ligue National contre le Cancer given to Claude Filters were prepared as follows: 5 mgofa-Actin, b-Actin, Brunel and Thierry Forne and the Ligue Nationale contre GAPDH, H19 Igf2, c-Myc, Myogenin, p21 and control le Cancer (comite de l'herault) given to Claude Brunel. plasmid DNA (per ®lter) was denatured by boiling for 10 min Laura Milligan was supported by the Fondation pour la in 0.3 M NaOH and neutralized with 10 volumes of cold 5 M Recherche Me dicale.

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