The H19 gene: regulation and function of a non-coding RNA.

The H19 gene encodes a 2.3-kb non-coding mRNA which is strongly expressed during embryogenesis. This gene belongs to an imprinted cluster, conserved on mouse chromosome 7 and human chromosome 11p15. H19 is maternally expressed and the neighbouring Igf2 gene is transcribed from the paternal allele. These two genes are co-expressed in endoderm- and mesoderm-derived tissues during embryonic development, which suggests a common mechanism of regulation. The regulatory elements (imprinted control region, CTCF insulation, different enhancer sequences, promoters of the two genes, matrix attachment regions) confer a differential chromatin architecture to the two parental alleles leading to reciprocal expression. The role of the H19 gene is unclear but different aspects have been proposed. H19 influences growth by way of a cis control on Igf2 expression. Although H19(-/-) mice are viable, a role for this gene during development has been suggested by viable H19(-/-) parthenogenetic mice. Finally it has been described as a putative tumour suppressor gene. H19 has been studied by numerous laboratories over the last fifteen years, nevertheless the function of this non-coding RNA remains to be elucidated.

The 3' portion of the mouse H19 Imprinting-Control Region is required for proper tissue-specific expression of the Igf2 gene.

Genomic imprinting at the H19/Igf2 locus is governed by a cis-acting Imprinting-Control Region (ICR), located 2 kb upstream of the H19 gene. This region possesses an insulator function which is activated on the unmethylated maternal allele through the binding of the CTCF factor. It has been previously reported that paternal transmission of the H19(SilK) deletion, which removes the 3' portion of H19 ICR, leads to the loss of H19 imprinting. Here we show that, in the liver, this reactivation of the paternal H19 gene is concomitant to a dramatic decrease in Igf2 mRNA levels. This deletion alters higher-order chromatin architecture, Igf2 promoter usage and tissue-specific expression. Therefore, when methylated, the 3' portion of the H19 ICR is a bi-functional regulatory element involved not only in H19 imprinting but also in 'formatting' the higher-order chromatin structure for proper tissue-specific expression of both H19 and Igf2 genes.

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.

Appropriate expression of the mouse H19 gene utilises three or more distinct enhancer regions spread over more than 130 kb.

H19 and Igf2 are closely linked, reciprocally imprinted genes which lie on distal chromosome 7 in the mouse. Data suggests that common elements are used for expression and imprinting of both genes, and simple models have been proposed based on the presence of a single set of enhancers located downstream of H19. In this study we have investigated the H19 expression pattern from a 130 kb YAC transgene, which imprints H19 appropriately at ectopic loci. However, we show that while enhancers for expression in many cell types are present on the YAC, those for expression in mesodermal components of the heart, kidney, lung and thymus are located at a greater distance. Based on the available evidence, we conclude that regulation of H19 is complex, requiring contribution from at least three different sets of cell-type specific enhancers. Thus, the mechanism of reciprocal imprinting of H19 and Igf2 utilises different regulatory elements in different cell types during mouse development.

Loss of the maternal H19 gene induces changes in Igf2 methylation in both cis and trans.

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (Igf2) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of Igf2, and the H19 gene (lying 90 kb downstream of Igf2 gene), which upon inactivation leads to relaxation of the Igf2 imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of Igf2 imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the Igf2 gene in two strains of mouse with distinct deletions of the H19 gene. The results show that maternal transmission of H19 gene deletions leads the maternal allele of Igf2 to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the H19 gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the Igf2 gene is affected in trans by deletions of the active maternal allele of the H19 gene. Selection during development for the appropriate expression of Igf2, dosage-dependent factors that bind to the Igf2 gene, or methylation transfer between the parental alleles could be involved in this trans effect.

Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element.

The distal region of mouse chromosome 7 contains a cluster of imprinted genes that includes H19 and Igf2 (insulin-like growth factor 2). H19 is expressed as an untranslated RNA found at high levels in endodermal and mesodermal embryonic tissues. This gene is imprinted and exclusively expressed from the allele of maternal origin. The Igf2 gene shows a similar pattern of expression but is expressed from the paternal allele. We have generated a targeted deletion of the H19 transcription unit by insertion of a neo replacement cassette. The homozygous mutant animals are viable and fertile and display an overgrowth phenotype of 8% compared with wild-type littermates. This is associated with the disruption of Igf2 imprinting and the consequent biallelic expression of this gene. A striking feature of the recombinant H19 allele is the occurrence of a parental imprint set on the neo replacement cassette. Therefore imprinting of the H19 locus is independent of the H19 gene itself. Taken together with the results of a larger H19 mutation described previously, this indicates that an imprinting control element is located within the region 10 kb upstream of H19.

Inactivation of an X-linked transgene in murine extraembryonic and adult tissues.

Transgenes located on the X chromosome have been used to study the mechanisms involved in X-chromosome inactivation. Analysis of the transgenic mouse strain M-TKneo1 carrying a neomycin resistance gene inserted in the X chromosome showed that, in adult somatic tissues, this transgene is subject to X-inactivation and to de novo methylation as other endogenous X-linked genes. During mouse embryogenesis, X-linked genes show a preferential paternal inactivation in extraembryonic tissues, whereas these genes are subject to random inactivation in embryonic tissues. It has been suggested that, in the mouse, the extraembryonic tissues carry a parental imprint at the time of inactivation. The study of the neo transgene expression in extraembryonic endoderm has shown not only that neo is inactivated but also that, at the RNA level, paternal inactivation of the transgene seems essentially complete. The differences between our results and previously obtained results with a mouse alpha-fetoprotein transgene, which was only inactivated in neonatal tissues but not in extraembryonic tissues, are discussed.

Characterization of a murine gene expressed from the inactive X chromosome.

In mammals, equal dosage of gene products encoded by the X chromosome in male and female cells is achieved by X inactivation. Although X-chromosome inactivation represents the most extensive example known of long range cis gene regulation, the mechanism by which thousands of genes on only one of a pair of identical chromosomes are turned off is poorly understood. We have recently identified a human gene (XIST) exclusively expressed from the inactive X chromosome. Here we report the isolation and characterization of its murine homologue (Xist) which localizes to the mouse X inactivation centre region and is the first murine gene found to be expressed from the inactive X chromosome. Nucleotide sequence analysis indicates that Xist may be associated with a protein product. The similar map positions and expression patterns for Xist in mouse and man suggest that this gene may have a role in X inactivation.

Genetic and molecular evidence of an X-chromosome deletion spanning the tabby (Ta) and testicular feminization (Tfm) loci in the mouse.

A new radiation-induced mutation in the mouse, tabby-25H (Ta25H), has proved to be a deletion which spans both the tabby and testicular feminization (Tfm) loci on the X chromosome. The Ta phenotype closely resembles that of the original TaFa mutation in both the heterozygous and hemizygous conditions but Ta25H/Y animals additionally show the Tfm/Y phenotype, being externally female but possessing abdominally located testes. There is a shortage of both Ta25H/+ and Ta25H/Y classes relative to their normal sibs among the progeny of Ta25H/+ females at weaning age and this was indicated to be due to prenatal or neonatal losses. Exencephaly was observed in some members of both classes prior to birth. Both Ta25H classes tend to be runted at weaning but, remarkably, Ta25H/+ females often show a range of abnormalities not evident in Ta25H/Y animals. When probes for the Zfx, Ccg-1, Phk, and DXPas19 loci, which lie close to Ta, were hybridised to DNAs from Ta25H hemizygotes, the profiles of the X-linked bands were similar to those of control DNAs, suggesting these loci lie outside the deletion. However, a clear absence of an X-linked band was found with human androgen receptor probes, indicating that the Tfm locus is indeed missing. The deletion, therefore, extends a minimum of 1.5 cM and, with its proximal and distal boundaries partially defined, it could be as large as 4 cM. As Ta25H/+ females show the striped X-inactivation coat pattern, the putative X-inactivation centre, Xce, which lies close to Ta, cannot be located within the region deleted. The greasy (Gs) locus similarly appears to lie outside the deletion.

Conservation and reorganization of loci on the mammalian X chromosome: a molecular framework for the identification of homologous subchromosomal regions in man and mouse.

By means of cross-reacting molecular probes, some 18 loci specific for the X chromosome of both man and mouse have been localized on the mouse X chromosome using an interspecific mouse cross involving the inbred SPE/Pas strain derived from Mus spretus. Comparison of the localizations of these loci on the mouse X with their positions on the human X chromosome suggests that intrachromosomal rearrangements involving at least five X chromosome breakage events must have occurred during the period of evolutionary divergence separating primates from rodents. Within the five blocks of chromosomal material so defined, there is for the moment little or no evidence that either chromosomal inversion events or extensive rearrangements have occurred. These data confirm the remarkable evolutionary conservation of the X chromosome apparent in mammalian species, compared to autosomal synteny groups in which both inter- and intrachromosomal rearrangement events appear to have occurred frequently. The breakage events described here for the X chromosome should therefore provide a minimal estimate for the frequency of chromosomal rearrangement events, such as breakage and inversion, which have affected autosomal synteny groups during the evolutionary period separating man from mouse. The definition of the number of chromosome breakage events by which the X chromosomes of these species differ, together with their localization, provides a framework for the use of interspecies mouse crosses for further detailed mapping of particular subchromosomal regions of the human X chromosome and for defining loci in the mouse homologous to those implicated in human congenital diseases.

Specific tissue targeting of polyoma virus oncogenicity in athymic nude mice.

Inbred athymic nu/nu mice (BALB/c and C57BL/6) were injected subcutaneously with polyoma virus A2 strain or with polyoma mutants which are able to infect undifferentiated embryonal carcinoma cells and harbor mutations in their enhancer sequences. Mammary adenocarcinomas were induced exclusively in females in which they represent the majority of the tumors. Both males and females developed sarcomas, mostly osteosarcomas, with a similar low frequency. No other type of neoplasm was observed. Mutations affecting the enhancers do not have any effect on the histotype of the tumors. Multiple copies of intact or defective free viral DNA were detected in all tumors. Such a sex-linked specific tissue targeting suggests a hormonal control of tumor initiation and/or promotion. From a pathological point of view, polyoma-induced adenocarcinomas are very similar to human early breast cancers. Tumor induction in nude mice by polyoma virus therefore represents a unique experimental model which differs from the more extensively used newborn immunocompetent mice.

Localization of the region homologous to the Duchenne muscular dystrophy locus on the mouse X chromosome.

Recent progress has resulted in part of the gene mutated in Duchenne and the milder Becker muscular dystrophies being cloned and has suggested that the gene itself extends over 1,000 to 2,000 kilobases (kb). To study how mutations in this gene affect muscle development and integrity, it would be of interest to have available a mouse model of the human disease. The mouse mdx mutation affects muscle and confers a mild dystrophic syndrome, but it is not clear whether this mutation is equivalent to Duchenne/Becker muscular dystrophy in man. Here we describe the use of two sequences from the human Duchenne muscular dystrophy (DMD) gene that cross-hybridize to mouse X-linked sequences to localize the gene homologous to DMD in the mouse. Both sequences map to the region of 10 centimorgan lying between the Tabby (Ta) and St14-1 (DxPas8) loci, close to the phosphorylase b kinase locus (Phk). By analogy with the human X-chromosome, we conclude that the region in the mouse around the G6pd and St14-1 loci may contain two genes corresponding to distinct human myopathies: Emery Dreifuss muscular dystrophy which is known to be closely linked to St14-1 in man and the DMD homologue described here.

T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). Our results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

Regulation of polyoma virus transcription in murine embryonal carcinoma cells.

Undifferentiated murine embryonal carcinoma (EC) cells are resistant to infection with wild-type polyoma virus. The block appears to be located at the transcriptional level. Polyoma host range mutants capable of expressing early and late functions in EC cells have been isolated. The modifications responsible for the phenotype of these mutants are localized in the noncoding region of polyoma DNA genome, containing regulatory sequences for replication and transcription. We compared the 5' termini of early and late mRNAs of wild-type polyoma and mutant viruses in EC cells and in permissive cells. Our results show that wild-type mRNA is normally spliced in EC cells but present at a very low level. The sequence modifications of the mutant viruses lead to a 100-fold increase in the production of mRNA in these cells, but the major 5' termini of early and late mRNAs are identical to those in wild-type-infected 3T6 cells.

Temperature dependent expression of polyoma virus in murine embryonal carcinoma cells.

At 37 degrees C, undifferentiated murine teratocarcinoma cells (PCC4) are resistant to infection with SV40 and Polyoma virus. When infection is carried out at 31 degrees C, these cells become fully susceptible to a variety of polyoma virus strains, including wt, ts-a, and hr-t; they also display an increased susceptibility to polyoma virus mutants (PyE.C.) which have been selected for their ability to develop in PCC4 cells at 37 degrees C (Vasseur et al., '80). However, expression of SV40 is still restricted at 31 degrees C and no T antigen can be detected. PCC4 cells grown at 31 degrees C express the characteristic embryonal surface antigen(s), but no H2 antigen, and do not produce plasminogen activator. PyE.C. mutants and other polyoma virus strains cannot develop at 37 degrees C in nullipotent F9 embryonal carcinoma cells and restriction is not abolished at 31 degrees C. The results indicate that: i) Resistance of PCC4 cells to polyoma virus and to SV40 are not mediated by the same process; ii) loss of restriction of polyoma in PCC4 cells does not require cell differentiation; iii) F9 and PCC4 cells control polyoma virus expression through different mechanisms.

Transcription of polyoma DNA by Escherichia coli RNA polymerase: influence of ionic strength on promoter selection.

The influence of ionic strength on transcription of polyoma DNA by Escherichia coli RNA polymerase was investigated. At 0.15 M KCl, transcription was highly symmetrical and, due to the lack of reinitiation, a limited extent of RNA synthesis was observed. When the concentration of KCl was raised to 0.45 M, the affinity of the enzyme for its template, as well as its apparent affinity for ribonucleoside triphosphates, was reduced. Under optimal conditions, the rate and extent of RNA synthesis at 0.45 M KCl were greater than at 0.15 M KCl, and transcription was mostly asymmetric. Binding and initiation sites at both ionic strengths were identified; at 0.15 M KCl, transcription was initiated from two major sites, located at 0.99 and 0.06 map unit, whereas at 0.45 M KCl, a unique initiation site, at 0.99 map unit, was selected by RNA polymerase.