Novel conserved elements upstream of the H19 gene are transcribed and act as mesodermal enhancers

The reciprocally imprinted H19 and Igf2 genes form a co-ordinately regulated 130 kb unit in the mouse controlled by widely dispersed enhancers, epigenetically modified silencers and an imprinting control region (ICR). Comparative human and mouse genomic sequencing between H19 and Igf2 revealed two novel regions of strong homology upstream of the ICR termed H19 upstream conserved regions (HUCs). Mouse HUC1 and HUC2 act as potent enhancers capable of driving expression of an H19 reporter gene in a range of mesodermal tissues. Intriguingly, the HUC sequences are also transcribed bi-allelically in mouse and human, but their expression pattern in neural and endodermal tissues in day 13.5 embryos is distinct from their enhancer function. The location of the HUC mesodermal enhancers upstream of the ICR and H19, and their capacity for interaction with both H19 and Igf2 requires critical re-evaluation of the cis-regulation of imprinted gene expression of H19 and Igf2 in a range of mesodermal tissues. We propose that these novel sequences interact with the ICR at H19 and the epigenetically regulated silencer at differentially methylated region 1 (DMR1) of Igf2.     

The 5' flank of mouse H19 in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication

BACKGROUND: During mouse prenatal development, the neighbouring insulin-like growth factor II (Igf2) and H19 loci are expressed monoallelically from the paternal and maternal alleles, respectively. Identical spatiotemporal expression patterns and enhancer deletion experiments show that the Igf2 and H19 genes share a common set of enhancers. Deletion of a differentially methylated region in the 5' flank of the H19 gene partially relieves the repression of the maternal Igf2 and paternal H19 alleles in the soma. The mechanisms underlying the function of the 5' flank of the H19 gene are, however, unknown. RESULTS: Chromatin analysis showed that the 5' flank of the mouse H19 gene contains maternal-specific, multiple nuclease hypersensitive sites that map to linker regions between positioned nucleosomes. These features could be recapitulated in an episomal-based H19 minigene, which was propagated in human somatic cells. Although the 5' flank of the H19 promoter has no intrinsic silencer activity under these conditions, it unidirectionally extinguished promoter-enhancer communications in a position-dependent manner, without directly affecting the enhancer function. CONCLUSIONS: The unmethylated 5' flank of the H19 gene adopts an unusual and maternal-specific chromatin conformation in somatic cells and regulates enhancer-promoter communications, thereby providing an explanation for its role in manifesting the repressed state of the maternally inherited Igf2 allele.     

Deletion of a silencer element disrupts H19 imprinting independently of a DNA methylation epigenetic switch

The H19 imprinted gene is silenced when paternally inherited and active only when inherited maternally. This is thought to involve a cis-acting control region upstream of H19 that is responsible for regulating a number of functions including DNA methylation, asynchronous replication of parental chromosomes and an insulator. Here we report on the function of a 1.2 kb upstream element in the mouse, which was previously shown to function as a bi-directional silencer in Drosophila. The cre-loxP-mediated targeted deletion of the 1.2 kb region had no effect on the maternal allele. However, there was loss of silencing of the paternal allele in many endodermal and other tissues. The pattern of expression was very similar to the expression pattern conferred by the enhancer elements downstream of H19. We could not detect an effect on the expression of the neighbouring imprinted Igf2 gene, suggesting that the proposed boundary element insulating this gene from the downstream enhancers was unaffected. Despite derepression of the paternal H19 allele, the deletion surprisingly did not affect the differential DNA methylation of the locus, which displayed an appropriate epigenetic switch in the parental germlines. Furthermore, the characteristic asynchronous pattern of DNA replication at H19 was also not disrupted by the deletion, suggesting that the sequences that mediate this were also intact. The silencer is therefore part of a complex cis-regulatory region upstream of the H19 gene and acts specifically to ensure the repression of the paternal allele, without a predominant effect on the epigenetic switch in the germline.     

An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues

The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.     

Epigenetic regulation of the Igf2/H19 gene cluster

Igf2 (insulin‐like growth factor 2) and H19 genes are imprinted in mammals; they are expressed unevenly from the two parental alleles. Igf2 is a growth factor expressed in most normal tissues, solely from the paternal allele. H19 gene is transcribed (but not translated to a protein) from the maternal allele. Igf2 protein is a growth factor particularly important during pregnancy, where it promotes both foetal and placental growth and also nutrient transfer from mother to offspring via the placenta. This article reviews epigenetic regulation of the Igf2/H19 gene‐cluster that leads to parent‐specific expression, with current models including parental allele‐specific DNA methylation and chromatin modifications, DNA‐binding of insulator proteins (CTCFs) and three‐dimensional partitioning of DNA in the nucleus. It is emphasized that key genomic features are conserved among mammals and have been functionally tested in mouse. ‘The enhancer competition model’, ‘the boundary model’ and ‘the chromatin‐loop model’ are three models based on differential methylation as the epigenetic mark responsible for the imprinted expression pattern. Pathways are discussed that can account for allelic methylation differences; there is a recent study that contradicts the previously accepted fact that biallelic expression is accompanied with loss of differential methylation pattern.     

Novel cis-regulatory function in ICR-mediated imprinted repression of H19

Expression of coregulated imprinted genes, H19 and Igf2, is monoallelic and parent-of-origin-dependent. Like most imprinted genes, H19 and Igf2 are regulated by a differentially methylated imprinting control region (ICR). CTCF binding sites and DNA methylation at the ICR have previously been identified as key cis-acting elements required for proper H19/Igf2 imprinting. Here, we use mouse models to elucidate further the mechanism of ICR-mediated gene regulation. We specifically address the question of whether sequences outside of CTCF sites at the ICR are required for paternal H19 repression. To this end, we generated two types of mutant ICRs in the mouse: (i) deletion of intervening sequence between CTCF sites (H19^ICR?IVS), which changes size and CpG content at the ICR; and (ii) CpG depletion outside of CTCF sites (H19^ICR-8nrCG), which only changes CpG content at the ICR. Individually, both mutant alleles (H19^ICR?IVS and H19^ICR-8nrCG) show loss of imprinted repression of paternal H19. Interestingly, this loss of repression does not coincide with a detectable change in methylation at the H19 ICR or promoter. Thus, neither intact CTCF sites nor hypermethylation at the ICR is sufficient for maintaining the fully repressed state of the paternal H19 allele. Our findings demonstrate, for the first time in vivo, that sequence outside of CTCF sites at the ICR is required in cis for ICR-mediated imprinted repression at the H19/Igf2 locus. In addition, these results strongly implicate a novel role of ICR size and CpG density in paternal H19 repression.     

Several copies of insulator from MDG4 can determine the interaction between positively and negatively acting regulatory elements and promoter of the miniwhite gene of Drosophila melanogaster

Fine regulation of complex gene loci in higher eukaryotes is realized through the interaction of promoters with enhancers and repressors, which can be located long distance from the promoter regulated. A question arises, what mechanisms determine proper contacts between the regulatory elements over large distances in the genome. It is suggested that the important role in this process is played by a special class of regulatory elements, insulators, which block the interaction of enhancer and promoter, if they are positioned between them. Furthermore, enhancers do not directly inactivate the activities of enhancer and promoter. Nevertheless, an enhancer, isolated from one of the promoters by an insulator, can activate another, not isolated promoter. The best studied insulator of Drosophila melanogaster was found in the 5′ regulatory region of retrotransposon MDG4. It consists of 12 binding sites for the Su(Hw) protein, which is critical for the activity of this insulator. It was demonstrated that Su(Hw) insulator could protect the gene expression from the negative influence of heterochromatin and from repression, induced by the Polycomb group proteins (Pc proteins). In the present study, it was demonstrated that in transgenic lines, two or three copies of the Su(Hw) insulator could determine the interaction of the miniwhite enhancer and Pc dependant silencer with the miniwhite promoter. Thus, it was first demonstrated that insulators could participate in the regulation of the contacts between promoter and functionally opposite elements, responsible for either gene activation, or repression. Original Russian Text ? M.V. Kostyuchenko, E.E. Savitskaya, M.N. Krivega, P.G. Georgiev, 2008, published in Genetika, 2008, Vol. 44, No. 12, pp. 1693–1697.     

A complex deoxyribonucleic acid looping configuration associated with the silencing of the maternal Igf2 allele

Alternate interactions between the H19 imprinting control region (ICR) and one of the two Igf2 differentially methylated regions has been proposed as a model regulating the reciprocal imprinting of Igf2 and H19. To study the conformation of this imprint switch, we performed a systematic structural analysis across the 140 kb of the mouse Igf2-H19 region, which includes enhancers located both between the two genes as well as downstream of H19, by using a scanning chromosome conformation capture (3C) technique. Our results suggest that on the active paternal Igf2 allele, the various enhancers have direct access to the Igf2 promoters, whereas the imprinted silent maternal Igf2 allele assumes a complex three-dimensional knotted loop that keeps the enhancers away from the Igf2 promoters and allows them to interact with the H19 promoter. This complex DNA looping of the maternal allele is formed by interactions involving differentially methylated region 1, the ICR, and enhancers. Binding of CTC-binding factor to the maternal, unmethylated ICR in conjunction with the presence of multicomplex components including interchromosomal interactions, create a barrier blocking the access of all enhancers to Igf2, thereby silencing the maternal Igf2. This silencing configuration exists in newborn liver, mouse embryonic fibroblast, and embryonic stem cells and persists during mitosis, conferring a mechanism for epigenetic memory.     

A skeletal muscle-specific mouse Igf2 repressor lies 40 kb downstream of the gene

Igf2 and H19 are closely linked and reciprocally expressed genes on distal chromosome 7 in the mouse. We have previously shown that a 130 kb YAC transgene contains multiple tissue-specific enhancers for expression of both genes during embryogenesis. The YAC also contains all the crucial elements responsible for initiating and maintaining appropriate parent-of-origin-specific expression of these genes at ectopic sites, with expression of Igf2 after paternal inheritance and of H19 after maternal inheritance. Located centrally between Igf2 and H19 are two prominent DNaseI hypersensitive sites, and two stretches of sequence that are conserved between mouse and human. In this study, we have deleted, from the transgene, a one kb part of the intergenic region that contains the hypersensitive sites and one of the homologous stretches. We demonstrate that this deletion results in loss of maternal Igf2 repression in skeletal muscle cells, most strikingly in the tongue, late in embryogenesis. We propose that the intergenic region functions as a tissue-specific repressor element, forming an integral part of the complex regulatory mechanism that controls monoallelic gene expression in this domain.     

Functional interaction between an insulator and a Pc-dependent silencer by the example of the Mcp boundary of the <Emphasis Type="Italic">Drosophila melanogaster Abd-B</Emphasis> gene

Insulators are cis-regulatory elements that prevent improper gene activation and heterochromatin spreading. As shown previously, the boundary element, Mcp, from the regulatory region of the Drosophila melanogaster Abd-B gene, contains insulator. Here, we studied the boundary function of the Mcp insulator and showed that this function is provided by two modules. One is responsible for long-distance interactions and the capability of blocking enhancers. The other is essential for blocking Pc-dependent repression. It was observed for the first time that an insulator increased the repressor activity of a neighbor silencer.