INTRODUCTION

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The H19 gene, which was first identified a decade ago, has been suggested to belong to the category of polymerase II-driven genes that do not code for a protein product (1). Although the processed and polyadenylated transcript is highly conserved between mouse and man, it does not display a conserved open reading frame (2). Moreover, the mouse H19 transcript, which is localized in the cytoplasm, has been reported to be excluded from the polysomal fraction and to be unable to be translated in vitro (2). The only indication so far that the human H19 RNA may have any role derives from the observation that an H19 expression vector rescues the normal phenotype of rhabdomyosarcoma cells (3). This result would, however, appear to be at odds with the lack of a documented increased incidence of cancer in H19-deficient mice (4). Moreover, H19 expression is maintained at high levels in some human tumors and has been proposed to be selected for during the generation of choriocarcinoma (5). No consistent role (if any) has been established, therefore, for H19 in trans.

Both H19 and the insulin-like growth factor gene-2 (IGF2), which are close physical neighbors on chromosome 7 in mouse and on chromosome 11 in humans (6), were among the first genes to be identified as being genomically imprinted (7, 8). The generation of deletion mutants in the mouse has shown that the imprinting status of Igf2 and H19 is coordinated such that the deletion of H19 and a 10-kilobase upstream region up-regulates maternal Igf2 when the deleted region is maternally inherited (4, 9). Conversely, a deletion of the endodermal-specific H19 enhancers has shown that they are vital for the activity of paternally derived Igf2, at least in some cell types, when the deleted enhancer region is inherited paternally (10). It appears, therefore, that the region within and/or flanking the mouse H19 gene can regulate the allele-specific expression of Igf2 in cis.

The observations that many human tumors do not express H19, but express both parental IGF2 alleles, has prompted suggestions that the human H19 gene or its flanking sequences may also control the activity of IGF2 in cis (11, 12). Such notions have not, however, been substantiated at the cellular level. Indeed, it has been shown by examination at the cellular level that H19 can be biallelically expressed in a subpopulation of human placental cells that expresses IGF2 monoallelically (13). It has also been shown that the generation of Wilms' tumors involves a mosaic IGF2 imprinting status that does not correlate with the expression of H19 at the individual cellular level (14). Collectively, these data, although circumstantial, are not supportive with respect to a cis function of the human H19 gene.

Here we report that, in contrast to previous claims, the H19 transcript is associated with polysomes in a variety of cell types in both mouse and man. H19 expression appears to directly or indirectly modulate the cytoplasmic levels of IGF2 mRNAs without being genetically linked to the expressed IGF2 allele. We submit that the human H19 gene is an antagonist to IGF2 in trans.

	EXPERIMENTAL PROCEDURES

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Cell Samples and Extraction of Nucleic Acid-- The Wilms' tumor was collected at the Uppsala University hospital. Routinely processed formalin-fixed, paraffin-embedded tissues were used for in situ hybridizations. DNA was extracted from snap-frozen tissues and from peripheral leukocytes, as has been described previously (13). The mouse embryos, at 17 days postconception, were crosses between Mus mus musculus and Mus mus domesticus. The JEG-3 cell line was maintained as has been described (15). Pactamycin (a kind gift of Pharmacia-Upjohn) was administered to JEG-3 cells for 3.5 h before harvesting and used at a concentration of either 2.0 or 6.9 × 10⁶ M without any detectable difference in the effect. Genomic DNA and total cellular RNA were prepared according to routine protocols (13).

Sucrose Gradient Analysis-- Postmitochondrial supernatants of both mouse embryos and cultured tumor cells were prepared according to Brannan et al. (2). Supernatants without added EDTA were layered onto 10.6 ml of 10-50% sucrose gradient containing 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl₂, and 15 units of RNasin/ml. The supernatant with added EDTA (30 mM final concentration) was layered onto a 10.6-ml 10-50% sucrose gradient containing 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 30 mM EDTA. The gradients were centrifuged in an LKB 2331 ultracentrifuge with a 40T768 rotor at 38,000 rpm for 1.5 or 6 h, as indicated in the legend to Fig. 1. Following division into 18-20 fractions, RNA was phenol-extracted from 100-µl aliquots and analyzed by Northern blot, RNase protection assay, and RT-PCR.¹

Probes-- DdeI-linearized human H19 cDNA cloned in Bluescript (a kind gift from Dr. Wolf Reik, Babraham, Cambridge, UK) was used to generate an antisense riboprobe (253 bases; T7 polymerase). A 558-base pair HinfI-PstI human IGF2 cDNA insert, cloned in pGem-3 and encompassing exon 7 to the 5'-region of exon 9, was cut with XhoI and used as the template for generation of an antisense riboprobe (145 bases; SP6 polymerase) (16). A 756-base pair antisense H19 RNA probe (17) was used to detect mouse H19 transcripts.

Northern Blot Hybridization and RNase Protection Analysis-- Northern blot hybridization analysis on 1% agarose gels was performed as described (18). RNase protection analysis was performed using the RPA II^TM ribonuclease protection assay kit (Ambion).

Analysis of Allele-specific IGF2 Transcripts-- The overall functional IGF2 imprinting status was determined by thermocyclic amplification of cDNA, produced by priming of IGF2 mRNA, and by diagnostic digestion with ApaI as has been described (19, 20) with the following exception. To ensure a linear amplification of low abundance IGF2 cDNAs in the sucrose gradient analyses, the oligo primers were 5'-labeled and the number of cycles were reduced to 25. This approach was verified by mixing experiments (not shown) as has been described (21). The resulting PCR products were analyzed on 8% polyacrylamide-urea sequencing gels, as has been described (21).

	RESULTS

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The H19 Transcript Co-sediments with Polysomes-- In initial attempts to identify any ribonucleoprotein particles containing human H19 RNA, we investigated the distribution of H19 RNA in the cytoplasm of JEG-3 choriocarcinoma cells. Our approaches included sucrose density gradient centrifugation analysis of postmitochondrial cell lysates. Fig. 1A shows an RNase protection analysis which documents that H19 RNA distributes along a 10-50% sucrose gradient, with a peak that appeared to partially overlap with polysomes containing IGF2 mRNAs. In EDTA-treated samples, the collapse of the polysome to generate ribosomal subunits appears to be accompanied by a similar shift of the H19 RNA-protein complexes to the upper portion of the sucrose gradient. To examine the extent of sedimentation overlap of H19 RNA-protein complexes with polysomes containing the different types of IGF2 mRNA transcripts derived from the three major promoters, we repeated the experiment and analyzed the sucrose gradient fractions by Northern blot analysis. Fig. 1B shows that there is an extensive similarity in the distribution of H19 RNA with the promoter 3-derived 6.0-kilobase IGF2 transcript. Because this similarity extends to the samples treated with EDTA, we reasoned that H19 might be associated with polysomes of a size similar to those containing IGF2 mRNA.

View larger version (64K): [in this window] [in a new window]   Fig. 1.   Sedimentation properties of H19 RNA in sucrose gradients. Postmitochondrial lysates were analyzed on sucrose gradients as described under "Experimental Procedures." RNA was extracted from gradient fractions and subjected to RNase protection (A, C, and D) or Northern blot hybridization (B) analysis, as indicated. A-C, distribution of H19 and IGF2 transcripts in JEG-3 cells. D, sedimentation of H19 and -actin transcripts in liver cells of a mouse fetus (17 days postconception) in sucrose gradients. Sucrose density gradients analyzed in (A and B) and (C and D) were centrifuged for 4 and 1 h, respectively. The UV-recorded polysomes are displayed in panel C. The positions of the 40 S and 60 S subunits were determined by agarose gel analyses of extracted RNA of aliquotted sucrose density gradient fractions. The position of the polysomes in the gradients is inferred from UV-recorded sucrose density gradients, as exemplified in panel C. The amplitude of the right-hand panel (+EDTA) is reduced to allow comparison with the left-hand panel (EDTA). The images are scanned x-ray films with the exception for panel B, which is derived from a Fuji phosphoimager file. The control lane indicates that yeast tRNA was used to assess any unspecific band pattern, whereas the +control lane shows the specific bands that could be obtained when examining the input RNA with each individual labeled RNA probe.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   The sedimentation property of the H19 transcript is pactamycin-sensitive. Postmitochondrial lysates of JEG-3 cells, control, and pactamycin-treated were analyzed on sucrose gradients as described under "Experimental Procedures." RNA was extracted from every second fraction and subjected to RNase protection (A). The analysis of the sedimentation properties of the H19 transcript was performed separate from the analysis of the GAP and IGF2 mRNAs. The H19- (B) and GAP- (C) specific protected bands were quantitated by PhosphorImager analysis. The filled and unfilled staples display relative levels of transcripts in control and pactamycin-treated cells, respectively.

Absence of H19 Transcripts Correlates with Increased Cytoplasmic Levels of IGF2 mRNA in a Wilms' Tumor-- We have previously documented a Wilms' tumor that is unique in the sense that it expresses IGF2 and H19 at high levels in almost identical patterns and displays a postneoplastic loss of IGF2 imprinting, where only a subpopulation of the tumor cells express both parental alleles, as determined by allele-specific in situ hybridization (Fig. 3A) (14). Fig. 3B shows a summary of these results: where subpopulations of cells with no H19 expression but monoallelic IGF2 expression are marked red, and cells with no H19 expression and biallelic IGF2 expression are marked green. Magnified views of these areas can be seen in Cui et al. (14). A closer look at the IGF2 expression levels revealed that the hybridization signal was 2-3-fold higher in the H19-negative cells when compared with the neighboring H19-positive cells (Fig. 3B). This observation could be documented in each of the H19-negative areas, suggesting an inverse correlation between H19 expression and cytoplasmic levels of IGF2 mRNA.

View larger version (90K): [in this window] [in a new window]   Fig. 3.   Inverse correlation of IGF2 and H19 transcripts in a Wilms' tumor as analyzed by in situ hybridization. Panel A, shows x-ray images (inverted in the computer using Adobe Photoshop 4.0) of allelic IGF2 expression patterns, as determined by oligo DNA-based in situ hybridization analysis (frames marked IGF2 allele A and B) (14). The frame marked as H19 is the result of an antisense riboprobe hybridized to a section immediately adjacent to those hybridized to allele-specific IGF2 probes. Red arrows pinpoint just a few of the cell populations that do not express H19 but expresses increased levels of the allele A of the IGF2 gene. Green arrows identify the subpopulation of cells that is H19-negative and expresses IGF2 biallelically. Panel B summarizes these results and shows relationships between H19 and IGF2 expression patterns in closer detail. The left-most frame provides a bright-field overview of cells that lack H19 expression but express IGF2 monoallelically (red) and cells that lack H19 expression and express IGF2 biallelically (green). All the other frames of panel B are dark-field views to show IGF2 or H19 expression patterns in adjacent sections, as indicated in the figure. The magnified images are color-coded to identify the boxed areas. Magnifications are (2.2-fold (red-lila-encoded image magnified in panel B) and 44-fold (yellow and blue-encoded images magnified in panel B). Images in panel A are magnified 1.3-fold. The brightfield view of the left-most frame of panel B shows actual size.

H19 Expression Correlates Inversely with Translatability of IGF2 mRNAs in a Wilms' Tumor-- We next addressed whether or not the pattern of H19 expression correlates with a difference in the polysome profile of IGF2 mRNAs. This approach was made possible by the strategy outlined in Fig. 4, where the parental alleles (A and B) are distinguishable by a sequence polymorphism. The generally silent allele B of IGF2 was only expressed in H19-negative cells, whereas the majority of the tumor cells expressed only the normally active allele A in H19-positive cells. If H19 expression modifies the translatability of IGF2 mRNAs, we would predict that the over-all sedimentation properties of transcripts derived from alleles A and B would differ in a sucrose gradient analysis.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   The rationale for examining the effect of H19 expression on the association of IGF2 RNA with polysomes in the Wilms' tumor.

View larger version (64K): [in this window] [in a new window]   Fig. 5.   Parent of origin-dependent sedimentation properties of Wilms' tumor-derived IGF2 mRNAs in sucrose gradients. A, Northern blot analysis of IGF2 mRNA species extracted from every second fraction. B, selected fractions of the sucrose gradient of panel A were subjected to RT-PCR analysis to examine allelic origin of IGF2 transcripts. Panels A and B denote the ApaI noncutting and cutting alleles, respectively.  and + denote minus and plus reverse transcriptase, respectively.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Analysis of allelic expression pattern of IGF2 in the normal kidney tissue and Wilm's tumor of the same patient. The analysis was carried out by ApaI digestion of genomic PCR products (DNA) and RT-PCR products (RNA). The molecular weight (MW) marker lane displays Sau3A I/TaqI-restricted pUC 19 DNA (Stratagene). The extra band, migrating as a 140-base pair fragment, represents a PCR artifact.

	DISCUSSION

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A function for H19 in trans has been disputed because a previous study claims that the H19 transcript is not associated with polysomes and does not produce a protein in the mouse (2). In addition, the lack of a reported increased incidence of cancer in H19-deficient mice (4) would appear to question a link to the suggested tumor suppressor function of human H19 in trans. On the other hand, the H19 knock out data does not rule out that an H19 trans-function is redundant and/or that a removal of an H19 function in trans contributes to the overgrowth phenotype of H19-deficient conceptuses. In addition, it is possible that human H19 has acquired a trans-function that is absent or has been lost in the mouse during evolution. Moreover, a recent transgenic study has strongly argued against the possibility that the mouse H19 gene represses the transcription of Igf2 in cis, by means of its own expression (24). We have, therefore, reexamined a putative role for the H19 transcript in humans.

First, we document that in contrast to previous reports, H19 mRNA co-sediments with polysomes in a variety of cell types, in both mouse and man. In cell cultures, this sedimentation is sensitive to pactamycin, which is an inhibitor of initiation of mRNA translation. This observation would seem to support a previous observation that the human H19 transcript is translatable in vitro and has an open reading frame that could formally encode a protein of 26 kDa (25). Second, by both direct and indirect lines of evidence, we were able to document that the cytoplasmic levels of H19 transcripts inversely correlated with cytoplasmic levels of IGF2 in a Wilms' tumor. Because the parental H19 alleles were identical with respect to a number of polymorphic markers, we have been unable to trace the parental origin of the expressed allele. On the other hand, because absence of H19 transcripts correlates both with increased levels of cytoplasmic transcripts derived from one allele and with increased translatability of transcripts derived from the other allele, we argue that one or both of these effects can be attributed to an H19 function in trans. Collectively, the data support the notion that H19 modifies IGF2 mRNA cytoplasmic levels and, potentially, polysome association in trans.

The observations of this report provide yet another glimpse into the complex regulation of the function of IGF2. This involves multiple steps of controls from gene dosage, differential promoter usage, splicing patterns, translational control(s) to postsecretory attenuation of IGF2 function by IGF-binding proteins (26). Whereas some of the cytoplasmic and extra-cellular levels of control appear to be uncoordinated with IGF2 expression levels, the expression of H19, and hence the antagonistic function of H19 in trans, is expected to be coordinated (27). This allows us to formulate a model in which H19 serves to prevent overshoot of IGF2 expression in trans. In this model, an increase in H19 expression would accompany an increase in IGF2 expression because their expression patterns are coordinated (10, 27) (Fig. 7). This coordination would be of particular importance in cases where the high levels of IGF2 expression could be expected to saturate the uncoordinated types of negative cytoplasmic and/or extracellular controls, such as IGF-binding proteins. In practice, this would mean that the higher the levels of IGF2 expression, the more important the H19 regulatory pathway would become. According to this model, a loss of the H19 function in trans would be expected to be a key event in cells expressing high levels of IGF2 mRNAs, resulting in a significant increase of free IGF-II ligand. Our model would highlight the consequences of losing the H19 function in trans when IGF2 is overexpressed. In a parallel study, we have been able to show that this loss of H19 expression is an early event which may predispose for Wilms' tumors (14). Another interesting case is the previous documentation that IGF2 could not be genetically linked with a familial form of BWS (28), despite the close link between BWS and IGF2 (29, 30). The possibility that H19 can be the direct culprit in this cancer-predisposing disease, at least in some instances, and that the role of IGF2 may be more indirect would appear to be compatible with the proposed model.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   A model of the postulated H19-specific control loop. Whereas the H19-specific control would be coordinated with IGF2 activity, other levels of controls, represented by IGF-binding proteins for example, may be uncoordinated. In the normal context with cells displaying low levels of IGF2 mRNAs, the noncoordinated controls would be expected to successfully prevent production of high levels of IGF-II peptide. The coordinated control, represented by H19 transcripts, will be of minor importance because the levels and pattern of H19 expression closely follows those of IGF2 (27). By the same token, increased activity of the IGF2 gene will be followed by increased activity of the H19 gene. In this scenario, the coordinated H19 control would gain importance in direct proportion to the inability of the uncoordinated controls to deal with abnormal levels of IGF2 activity. If the H19 function is abnormally silenced in a situation with high levels of IGF2 transcripts, as has been documented in numerous contexts (31), this report suggests that production of the IGF-II peptide will increase abnormally to contribute to overgrowth syndrome syndromes, such as the Beckwith-Wiedemann syndrome, and neoplasia (outlined in the right-most panel). This model may be valid only in the absence of prominent translational suppression of IGF2 mRNAs.

Our observations are consistent with the possibility that the human H19 gene modifies the expression of IGF2 in trans. These data may have an impact in our understanding of human diseases, such as BWS, and allows us to further penetrate the nature of disease-associated (epi)genetic abnormalities.