Parental genomic imprinting at the Igf2/H19 locus is controlled by a methylation-sensitive CTCF insulator that prevents the access of downstream enhancers to the Igf2 gene on the maternal chromosome. However, on the paternal chromosome, it remains unclear whether long-range interactions with the enhancers are restricted to the Igf2 promoters or whether they encompass the entire gene body. Here, using the quantitative chromosome conformation capture assay, we show that, in the mouse liver, the endodermal enhancers have low contact frequencies with the Igf2 promoters but display, on the paternal chromosome, strong interactions with the intragenic differentially methylated regions 1 and 2. Interestingly, we found that enhancers also interact with a so-far poorly characterized intergenic region of the locus that produces a novel imprinted long non-coding transcript that we named the paternally expressed Igf2/H19 intergenic transcript (PIHit) RNA. PIHit is expressed exclusively from the paternal chromosome, contains a novel discrete differentially methylated region in a highly conserved sequence and, surprisingly, does not require an intact ICR/H19 gene region for its imprinting. Altogether, our data reveal a novel imprinted domain in the Igf2/H19 locus and lead us to propose a model for chromatin folding of this locus on the paternal chromosome.

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions.

BackgroundDespite its critical role for mammalian gene regulation, the basic structural landscape of chromatin in living cells remains largely unknown within chromosomal territories below the megabase scale.ResultsHere, using the 3C-qPCR method, we investigate contact frequencies at high resolution within interphase chromatin at several mouse loci. We find that, at several gene-rich loci, contact frequencies undergo a periodical modulation (every 90 to 100 kb) that affects chromatin dynamics over large genomic distances (a few hundred kilobases). Interestingly, this modulation appears to be conserved in human cells, and bioinformatic analyses of locus-specific, long-range cis-interactions suggest that it may underlie the dynamics of a significant number of gene-rich domains in mammals, thus contributing to genome evolution. Finally, using an original model derived from polymer physics, we show that this modulation can be understood as a fundamental helix shape that chromatin tends to adopt in gene-rich domains when no significant locus-specific interaction takes place.ConclusionsAltogether, our work unveils a fundamental aspect of chromatin dynamics in mammals and contributes to a better understanding of genome organization within chromosomal territories.

When U1 and U2 small nuclear ribonucleoproteins (snRNPs) purified by a procedure which preserves their immunoprecipitability by autoimmune antibodies (Hinterberger et al., J. Biol. Chem. 258:2604-2613, were submitted to extensive digestion with micrococcal nuclease, we found that their degradation pattern was sharply dependent upon magnesium concentration, indicating that they undergo a profound structural modification. At low Mg2+ (s5 mM), both particles only exhibit a core-resistant structure previously identified as being common to all but U6 snRNAs (Liautard et al., J. Mol. Biol. 162:623-643, 1982). At high Mg2+ (.7 mM), U1 and U2 snRNPs behave differently from one another. In U1 snRNP, most U1 snRNA sequence is protected, except for the 10 5'-terminal nucleotides presumably involved in splicing and a short sequence between nucleotides 102 and 108. Another region spanning nucleotides 60 to 79 is only weakly protected. This structural modification was demonstrated to be reversible. In U2 snRNP, the U2 snRNA sequence remains exposed in its 5' part up to nucleotide 92, and the 3'-terminal hairpin located outside the core structure becomes protected.All but U3 small nuclear RNAs (snRNAs) assemble in the cell with specific proteins to form small nuclear ribonucleoproteins (snRNPs) which are precipitable by sera from patients with systemic lupus erythematosus or mixed connective tissue disease. These U snRNPs (for a review, see C. Brunel, J. Sri-Widada, and P. Jeanteur, Progress in Molecular and Subcellular Biology, in press) interact with heterogenous nuclear RNPs (containing the heterogenous population of premessengers), which are now recognized as the real splicing substrate (8). Recent reports have strengthened the initial suggestions by Lerner et al. (13) and Rogers and Wall (20) that U1 snRNP is involved in splicing. Among these are the crucial demonstration that an in vitro splicing system is inhibited by monoclonal anti-Sm, patient anti-Sm, or anti-RNP antibodies but not by scleroderma-polymyositis overlap syndrome serum-precipitating U2 snRNP (18) and the biochemical evidence that U1 snRNP binds to an in vitro transcript containing splice sites (16) or that U1 snRNP stimulates in vitro splicing of adenoviral sequences (8).In the course of isolating U snRNPs from HeLa cells, we first purified a mixture containing Ul, Ul-, U4, U5, and U6 particles (5) and then purified the individual U1 snRNP (21). These particles have a common and simple protein complement which constitutes only the low-molecular-weight polypeptides (9 to 14 kilodaltons) among those immunoprecipitated by anti-RNP or anti-Sm antibodies. As such, they retained antigenicity with respect to anti-Sm but not anti-RNP (1) and were therefore designated as core snRNPs. Although certainly far from being functional, these core particles allowed us to identify the binding site for these common core proteins as being a single-stranded region containing the sequence A(U)nG, with n -3, bordered by two double-stranded stems (14). This site corresponds t...

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