Guy Cathala

Quantitative analysis of chromosome conformation capture assays (3C-qPCR)

Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7–9 days.

A Novel H19 Antisense RNA Overexpressed in Breast Cancer Contributes to Paternal IGF2 Expression

ABSTRACT The H19/IGFf2 locus belongs to a large imprinted domain located on human chromosome 11p15.5 (homologue to mouse distal chromosome 7). The H19 gene is expressed from the maternal allele, while IGF2 is paternally expressed. Natural antisense transcripts and intergenic transcription have been involved in many aspects of eukaryotic gene expression, including genomic imprinting and RNA interference. However, apart from the identification of some IGF2 antisense transcripts, few data are available on that topic at the H19/IGF2 locus. We identify here a novel transcriptional activity at both the human and the mouse H19/IGF2 imprinted loci. This activity occurs antisense to the H19 gene and has the potential to produce a single 120-kb transcript that we called the 91H RNA. This nuclear and short-lived RNA is not imprinted in mouse but is expressed predominantly from the maternal allele in both mice and humans within the H19 gene region. Moreover, the transcript is stabilized in breast cancer cells and overexpressed in human breast tumors. Finally, knockdown experiments showed that, in humans, 91H, rather than affecting H19 expression, regulates IGF2 expression in trans.

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting.

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.

Activation and inhibition processes of alkaline phosphatase from bovine brain by metal ions (Mg2+ and Zn2)

Abstract 1. 1 Alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC3.1.3.1) from bovine brain is strongly activated by Mg2+ (5–4-fold). In this respect the enzymes behaves like that of kidney or bone marrow but is different from the intestinal and placental enzymes which are much less activated. 2. 2 The rate of the activation process is pH-dependent. At pH 10.0, the pH optimum of p nitrophenyl phosphate hydrolysis, the activation process is rapid. At lower pH values, it is slow and can be described by an exponential relationship vs time. The results suggest that the activation by Mg2+ proceeds through a binding of the metal with free enzyme which might be followed by a conformational change, rather than by an action on the substrate. 3. 3 Other metal ions can also bind to the brain enzyme. Mn2+, Co2+, Ni2+ with an activating effect, Zn2+ with an inhibitory effect. They all bind to the enzyme at the same site and also induce the supposed conformational change. However, Zn2+ is able to produce interactions, perhaps with Pi which is one of the products of hydrolysis, which might explain its inhibitory effect.

Genomic imprinting controls matrix attachment regions in the Igf2 gene.

ABSTRACT Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.

H19 gene expression is up-regulated exclusively by stabilization of the RNA during muscle cell differentiation

H19 is a paternally imprinted gene whose expression produces a 2.4 kb RNA in most tissues during development and in mammalian myoblastic cell lines upon differentiation. Deletion of the active maternal allele of H19 and its flanking regions in the mouse leads to biallelic methylation and loss of imprinting of the neighbouring Igf2 gene. The function of H19 RNA remains unknown and, although polysome-associated, the absence of a conserved open reading frame suggests that it does not encode a protein product. We describe a novel post-transcriptional regulation of H19 gene expression which, in spite of this lack of coding capacity, is dependent on translational activity. We show that stabilization of the RNA is solely responsible for its accumulation during in vitro muscle cell differentiation. This conclusion is based on the finding that inhibition of protein synthesis results in a dramatic destabilization of H19 RNA in proliferating mouse C2C12 myoblastic cells but not in differentiated cells, and on run-on experiments which showed that the rate of transcription of H19 RNA remains constant during muscle cell differentiation. This mechanism could also be involved in H19 gene expression during mouse development in addition to its transcriptional activation which we have shown to occur.

A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias

Allele-specific epigenetic modifications are crucial for several important biological functions, including genomic imprinting and X-inactivation in mammals. Consequently, an ever increasing number of investigations requires accurate quantification of the relative abundance of parental alleles of a specific sequence in a DNA sample. Here, combining the use of polymorphic restriction sites with real-time polymerase chain reaction (PCR) amplification, we describe a simple and quantitative assay to measure allele ratios. The efficiency of the assay was assessed on genomic DNA for several polymorphic restriction sites located in the mouse Igf2/H19 imprinted locus. The assay was also successfully applied to quantify allele ratio in cDNA samples. In addition, we provide an experimental procedure for detection and correction of potential PCR amplification bias which significantly extends the range of application of the assay.

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.

Turnover of primary transcripts is a major step in the regulation of mouse H19 gene expression

In the gene expression pathway, RNA biogenesis is a central multi‐step process where both message fidelity and steady‐state levels of the mature RNA have to be ascertained. An emerging question is whether RNA levels could be regulated at the precursor stage. Until recently, because it was technically very difficult to determine the level of a pre‐mRNA, discrimination between changes in transcriptional activity and in pre‐mRNA metabolism was extremely difficult. H19 RNA, the untranslated product of an imprinted gene, undergoes post‐transcriptional regulation. Here, using a quantitative real‐time RT–PCR approach, we accurately quantify its precursor RNA levels and compare these with the transcriptional activity of the gene, assessed by run‐on assays. We find that the levels of H19 precursor RNA are regulated during physiological processes and this regulation appears to be related to RNA polymerase II transcription termination. Our results provide direct evidence that turnover of polymerase II primary transcripts can regulate gene expression in mammals.