Long-range chromatin interactions at the mouse Igf2/H19 locus reveal a novel paternally expressed long non-coding RNA

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

H19 Antisense RNA Can Up-Regulate Igf2 Transcription by Activation of a Novel Promoter in Mouse Myoblasts

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

Comparison between the endogenous <i>Igf2</i> transcription levels and the steady state levels of the ectopic RNAs.

<p>In these graphs, we compared, for each transfected <i>H19</i> KO clones, <i>Igf2</i> transcription data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g005" target="_blank">Figure 5B</a> with the steady state <i>91H</i> and <i>H19</i> ectopic RNA levels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g004" target="_blank">Figure 4B</a>. (A) <i>Igf2</i> transcription <i>versus 91H</i> RNA levels. (B) <i>Igf2</i> transcription <i>versus H19</i> RNA levels. In untransfected H19 KO myoblasts (−/−), both <i>91H</i> and <i>H19</i> are not expressed (RNA levels  = 0) and <i>Igf2</i> transcription level is below the “empty plasmid” background (see Fig. 5A) which is inferior to 0.11. (C) <i>Igf2</i> transcription level <i>versus</i> the ratio of <i>91H/H19</i> RNA levels. Clones expressing large amount of ectopic <i>H19</i> RNA (clones 4, 11 and 12ND, black diamonds) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g004" target="_blank">Figure 4B</a>) were analysed separately from the others (ratio of <i>91H</i>/<i>H19</i> RNA levels >0.2; open diamonds). In clones expressing high <i>H19</i> RNA levels (black diamonds), the ectopic <i>H19</i> RNA level relative to the <i>91H</i> RNA level (which leads to a decrease of the <i>91H/H19</i> RNA ratio) is inversely proportional to <i>Igf2</i> transcription levels (R² = 0.8173).</p

Map of the mouse <i>H19/91H</i> region showing the PCR amplicons used in RT-qPCR experiments.

<p>The region corresponding to the sequence removed by the H19^Δ3 deletion in the <i>H19</i> KO myoblasts (see below) is indicated in blue. The <i>H19/91H</i> insert transfected into the <i>H19</i> KO myoblast cells is also shown in the figure (green lane). The insert is a 16 kb BamHI-BamHI fragment encompassing the <i>H19</i> endodermic enhancers and the whole <i>H19</i> gene. PCR amplicons used to quantify the ectopic RNAs are indicated (<i>H19</i> RNA, mI1-mI3, mJ and mC’). DNA methylation is indicated by black lollipops and RNAs are depicted in red. Positions of restriction sites and PCR amplicons used for real-time PCRs are indicated relative to the <i>H19</i> transcription start site. The mA and mB PCR amplicons have been used in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone.0037923-Berteaux1" target="_blank">[34]</a> and are indicated here solely for clarity of our PCR nomenclature. For primer sequences see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone.0037923.s008" target="_blank">Table S1</a>.</p

Nuclear Run-on experiments.

<p>(A) Autoradiographies of nuclear Run-on experiments on transfected and untransfected <i>H19</i> KO myoblasts. Nuclear Run-on experiments were performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone.0037923-Milligan1" target="_blank">[38]</a> and the α³²P UTP labelled transcripts were hybridized on filters to denatured plasmids containing the insert DNA of genes indicated on the figure. <i>91H</i>/<i>H19</i> transcription was assayed using an insert corresponding to the <i>H19</i> sequence and <i>Igf2</i> with a genomic 2.4 kb BamHI-BamHI DNA fragment encompassing the exon 4-exon 6 region. Such nuclear run-on experiments were performed on undifferentiated (ND) and differentiated (D) cells either on the whole hygromycin-resistant transfected <i>H19</i> KO myoblast cell population (“Whole”) and transfected clones. (B) The same filters as those used for the autoradiographies shown in A were used for PhosphorImager quantifications. The ectopic <i>91H/H19</i> transcription levels (open bars) were compared to the endogenous <i>Igf2</i> transcription levels (black bars). For each hybridized filter, the relative transcription levels were determined for each gene by normalizing to the <i>Gapdh</i> transcription level.</p

<i>Igf2</i> Pm mRNA levels in mouse tissues and <i>Igf2</i> promoter usage in transfected <i>H19</i>KO myoblasts.

<p>(A) Pm <i>Igf2</i> mRNA levels relative to total <i>Igf2</i> mRNA level (100%) in different mouse tissues. Total <i>Igf2</i> mRNA levels were determined by RT-qPCR using a PCR primer pair (Igf2exon6 PCR amplicon) located in the exon 6 common to all <i>Igf2</i> transcripts. (B) <i>Igf2</i> promoter usage in untransfected/transfected <i>H19</i> KO myoblasts. Relative mRNA levels (%) are calculated relative to the P3 <i>Igf2</i> transcript level in the control cells (+/−) (100%).</p

Characterisation of TSS of the endogenous mouse <i>91H</i> RNA.

<p>5′RACE experiment was performed on unpolyadenylated and capped RNA from 7 days-old mouse liver. (A) The RT Primer was designed in the mFb region (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037923#pone-0037923-g001" target="_blank">Figure 1</a>) and a band was successfully amplified by nested PCRs. The RT primer corresponds with the forward primer of PCRa and nested PCR reactions were performed using the GeneRacer DNA oligonucleotide as reverse primer. (B) Ethidium bromide staining of an agarose gel showing PCRs product obtained from amplifications indicated above (MW: Molecular Weight). Sequencing of PCRa and PCRc products showed that these bands correspond essentially to unspecific amplifications while PCRb correspond to the TSS of the <i>91H</i> RNA. (C) Electrophoregram of the sequenced 5′RACE product amplified from the capped RNA fraction (PCRb). This sequence identified a unique Cap site located in the endodermic enhancer 2 at position chr7:149,755,206 or chr7:149,755,207 on mouse July 2007/ mm9 Assembly. Due to the presence of a C residue at the end of the GeneRacer RNA oligonucleotide primer and/or the possibility that the last C residue may derive from the cap of the RNA, the exact position of the TSS remains ambiguous between two consecutive C residues found in the mouse genome sequence. (D) The sequence of the endodermic enhancer 2 is indicated in bold. The position of the TSS of the <i>91H</i> RNA is indicated (black arrow).</p