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S M Tilghman

Publications and source records attributed to S M Tilghman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Igf2 imprinting does not require its own DNA methylation or H19 RNA.

Three models have been proposed to explain the imprinting of the mouse Igf2 gene on the maternal chromosome. We ruled out the importance of DNA methylation at Igf2 by showing that silencing of Igf2 accompanying the loss of DNA methylation could be overcome by a mutation at the neighboring H19 gene that activates Igf2. By replacing the H19 structural gene with a protein-coding gene, we have ruled out a role for H19 RNA in the imprinting of Igf2. This replacement resulted in sporadic activation of the H19 promoter on the paternal chromosome without affecting the level of expression of Igf2, a finding that is inconsistent with strict promoter competition between the genes. We conclude that a transcriptional model involving access to a common set of enhancers shared between Igf2 and H19 is the most likely explanation for Igf2 imprinting.

Animals↗
PubMed1998

Location of enhancers is essential for the imprinting of H19 and Igf2 genes.

Genomic imprinting is the process in mammals by which gamete-specific epigenetic modifications establish the differential expression of the two alleles of a gene. The tightly linked H19 and Igf2 genes are expressed in tissues of endodermal and mesodermal origin, with H19 expressed from the maternal chromosome and Igf2 expressed from the paternal chromosome. A model has been proposed to explain the reciprocal imprinting of these genes; in this model, expression of the genes is governed by competition between their promoters for a common set of enhancers. An extra set of enhancers might be predicted to relieve the competition, thereby eliminating imprinting. Here we tested this prediction by generating mice with a duplication of the endoderm-specific enhancers. The normally silent Igf2 gene on the maternal chromosome was expressed in liver, consistent with relief from competition. We then generated a maternal chromosome containing a single set of enhancers located equidistant from 1gf2 and H19; the direction of the imprint was reversed. Thus, the location of the enhancers determines the outcome of competition in liver, and the strength of the H19 promoter is not sufficient to silence Igf2.

Alleles↗
PubMed1998

Genomic imprinting is disrupted in interspecific Peromyscus hybrids.

Genomic imprinting, the unequal expression of gene alleles on the basis of parent of origin, is a major exception to mendelian laws of inheritance. By maintaining one allele of a gene in a silent state, imprinted genes discard the advantages of diploidy, and for this reason the rationale for the evolution of imprinting has been debated. One explanation is the parent-offspring conflict model, which proposes that imprinting arose in polyandrous mammals as the result of a parental conflict over the allocation of maternal resources to embryos. This theory predicts that there should be no selection for imprinting in a monogamous species. Crosses between the monogamous rodent species Peromyscus polionotus and the polyandrous Peromyscus maniculatus yield progeny with parent-of-origin growth defects that could be explained if imprinting was absent in the monogamous species. We find, however, that imprinting is maintained in P. polionotus, but there is widespread disruption of imprinting in the hybrids. We suggest that the signals governing genomic imprinting are rapidly evolving and that disruptions in the process may contribute to mammalian speciation.

Animals↗
PubMed1998

Chromatin conformation of the H19 epigenetic mark.

Genomic imprinting in mammals is an epigenetic process that results in differential expression of the two parental alleles. The tightly linked murine H19 and Igf2 genes are reciprocally imprinted: H19 is expressed from the maternal chromosome while Igf2 is expressed from the paternal chromosome. A single regulatory region in the 5' flank of the H19 gene has been implicated in silencing both genes. On the paternal chromosome, this region is heavily methylated at CpG residues, leading to repression of the H19 gene. The mechanism by which the same region in an unmethylated state on the maternal chromosome silences Igf2 is less well understood. We have probed the chromatin structure of the region by assessing its sensitivity to nuclease digestion. Two regions of nuclease hypersensitivity that are specific to the maternal chromosome were identified. These coincide with the region that is most heavily methylated on the paternal chromosome. As is the case with paternal methylation, hypersensitivity is present in all tissues surveyed, irrespective of H19 expression. We suggest that the chromatin structure of the maternal 5' flank of the H19 gene may represent an epigenetic mark involved in the silencing of Igf2.

Animals↗
PubMed1998

The absence of enhancer competition between Igf2 and H19 following transfer into differentiated cells.

H19 and Igf2 are reciprocally imprinted genes that lie 90 kb apart on mouse chromosome 7. The two genes are coexpressed during development, with the H19 gene expressed exclusively from the maternal chromosome and Igf2 from the paternal chromosome. It has been proposed that their reciprocal imprinting is governed by a competition between the genes for a common set of enhancers. The competition on the paternal chromosome is influenced by extensive allele-specific methylation of the H19 gene and its 5' flank, which acts to inhibit H19 transcription and thus indirectly leads to the activation of the Igf2 gene. In contrast, no allele-specific methylation has been detected on the maternal chromosome, and the basis for the preference for H19 transcription on that chromosome is unresolved. In this investigation, the mechanism controlling the silencing of the Igf2 gene on the maternal chromosome was explored by studying the transcriptional activity of a yeast artificial chromosome (YAC) containing Igf2 and H19 following transfer into differentiated tissue culture cells. Contrary to expectations, both H19 and Igf2 were expressed from a single integrated copy of the YAC. Furthermore, Igf2 expression appeared to be independent of the H19 locus, based on deletions of the H19 gene promoter and its enhancers. These results suggest that an active process is responsible for the transcriptional bias toward H19 on the maternal chromosome and that the hypomethylated state of this chromosome cannot be viewed as a "default" state. Moreover, the active process is not reproduced in a differentiated cell and may require passage through the female germ line.

Animals↗
PubMed1998

Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster.

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57(Kip2), as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57(Kip2) during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57(Kip2), and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57(Kip2) have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57(Kip2) is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).

Animals↗
PubMed1997

Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith-Wiedemann and Simpson-Golabi-Behmel syndromes.

In mice, the imprinted Igf2 gene (expressed from the paternal allele), which encodes a growth-promoting factor (IGF-II), is linked closely to the reciprocally imprinted H19 locus on chromosome 7. Also imprinted (expressed from the maternal allele) is the Igf2r gene on chromsome 17 encoding the type 2 IGF receptor that is involved in degradation of excess IGF-II. Double mutant embryos carrying a deletion around the H19 region and also a targeted Igf2r allele, both inherited maternally, have extremely high levels of IGF-II (7- and 11-fold higher than normal in tissues and serum, respectively) as a result of biallelic Igf2 expression (imprint relaxation by deletion of H19-associated sequence) in combination with lack of the IGF2R-mediated IGF-II turnover. This excess of IGF-II causes somatic overgrowth, visceromegaly, placentomegaly, omphalocele, and cardiac and adrenal defects, which are also features of the Beckwith-Wiedemann syndrome (BWS), a genetically complex human disorder associated with chromosomal abnormalities in the 11p15.5 region where the IGF2 gene resides. In addition, the double mutant mouse embryos exhibit skeletal defects and cleft palate, which are manifestations observed frequently in the Simpson-Golabi-Behmel syndrome, another overgrowth disorder overlapping phenotypically, but not genetically, with BWS.

Abnormalities, Multiple↗
PubMed1997

Molecular characterization of four induced alleles at the Ednrb locus.

The piebald locus on mouse chromosome 14 encodes the endothelin-B receptor (EDNRB), a G protein-coupled, seven-transmembrane domain protein, which is required for neural crest-derived melanocyte and enteric neuron development. A spontaneous null allele of Ednrb results in homozygous mice that are predominantly white and die as juveniles from megacolon. To identify the important domains for EDNRB function, four recessive juvenile lethal alleles created by either radiation or chemical mutagens (Ednrb27Pub, Ednrb17FrS, Ednrb1Chlc, and Ednrb3Chlo) were examined at the molecular level. Ednrb27Pub mice harbor a mutation at a critical proline residue in the fifth transmembrane domain of the EDNRB protein. A gross genomic alteration within the Ednrb gene in Ednrb3Chlo results in the production of aberrantly sized transcripts and no authentic Ednrb mRNA. Ednrb17FrS mice exhibited a decreased level of Ednrb mRNA, supporting previous observations that the degree of spotting in piebald mice is dependent on the amount of EDNRB expressed. Finally, no molecular defect was detected in Ednrb1Chlc mice, which produce normal levels of Ednrb mRNA in adult brain, suggesting that the mutation affects important regulatory elements that mediate the expression of the gene during development.

Alleles↗
PubMed1997

Structure and expression of the mouse L23mrp gene downstream of the imprinted H19 gene: biallelic expression and lack of interaction with the H19 enhancers.

The human L23 (mitochondrial)-related protein gene, located 40 kb downstream of the imprinted H19 gene, is biallelically expressed. We have cloned and characterized its mouse homolog, L23mrp, which maps to the conserved syntenic region on mouse chromosome 7. The promoter of L23mrp is a CpG island that is transcribed ubiquitously, but at different levels, in different fetal tissues. Allele-specific expression analysis revealed that both parental alleles are equally active. Since the enhancers located between H19 and L23mrp had been shown to be involved in the imprinted expression of Ins-2, Igf-2, and H19, we asked whether they also influence L23mrp. Analysis of mice with a targeted deletion of the enhancers demonstrated that they were not disrupted in the expression of L23mrp. These findings indicate that L23mrp is functionally insulated from the Ins-2/Igf-2/H19 domain in terms of both imprinting and enhancer action.

Alleles↗
PubMed1997

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.

Alleles↗
PubMed1997

The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation.

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-Protein Signaling) and Dishevelled proteins. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Furthermore, ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in mammals and amphibians.

Amino Acid Sequence↗
PubMed1997

Two dominant mutations in the mouse fused gene are the result of transposon insertions.

The mouse Fused locus encodes a protein that has been implicated in the regulation of embryonic axis formation. The protein, which has been named Axin to distinguish it from the product of the unrelated Drosophila melanogaster gene fused, contains regions of similarity to the RGS (regulators of G-protein signaling) family of proteins as well as to dishevelled, a protein that acts downstream of Wingless in D. melanogaster. Loss-of-function mutations at Fused lead to lethality between days 8 and 10 of gestation. Three dominant mutations result in a kinked tail in heterozygotes. Two of the dominant mutations, Fused and Knobbly, result from insertion of intracisternal A particle retrotransposons into the gene. The insertion in Fused, within the sixth intron, creates a gene that produces wild-type transcripts as well as mutant transcripts that initiate at both the authentic promoter and the 3'-most long terminal repeat of the insertion. Knobbly, an insertion of the retrotransposon into exon 7, precludes the production of wild-type protein. Thus the Fused homozygote is viable whereas Knobbly is a recessive embryonic lethal. In both mutants the dominant kink-tailed phenotype is likely to result from the synthesis of similar amino-terminal fragments of Axin protein that would contain the RGS domain, but lack the dishevelled domain.

Animals↗
PubMed1997

Genomic imprinting in mammals.

A handful of autosomal genes in the mammalian genome are inherited in a silent state from one of the two parents, and in a fully active form from the other, thereby rendering the organism functionally hemizygous for imprinted genes. To date 19 imprinted genes have been identified; 5 are expressed from the maternal chromosome while the rest are expressed from the paternal chromosome. Allele-specific methylation of CpG residues, established in one of the germlines and maintained throughout embryogenesis, has been clearly implicated in the maintenance of imprinting in somatic cells. Although the function of imprinting remains a subject of some debate, the process is thought to have an important role in regulating the rate of fetal growth.

Animals↗
PubMed1996

The structural H19 gene is required for transgene imprinting.

The product of the H19 gene is an untranslated RNA that is expressed exclusively from the maternal chromosome during mammalian development. The H19 gene and its 5'-flanking sequence are required for the genomic imprinting of two paternally expressed genes, Ins-2 (encodes insulin-2) and Igf-2 (encodes insulin-like growth factor-2), that lie 90 and 115 kb 5' to the H19 gene, respectively. In this report, the role of the H19 gene in its own imprinting is investigated by introducing a Mus spretus H19 gene into heterologous locations in the mouse genome. Multiple copies of the transgene were sufficient for its paternal silencing and DNA methylation. Replacing the H19 structural gene with a luciferase reporter gene resulted in loss of imprinting of the transgene. That is, high expression and low levels of DNA methylation were observed upon both paternal and maternal inheritance. The removal of 701 bp at the 5' end of the structural gene resulted in a similar loss of paternal-specific DNA methylation, arguing that those sequences are required for both the establishment and maintenance of the sperm-specific gametic mark. The M. spretus H19 transgene could not rescue the loss of Igf-2 imprinting in trans in H19 deletion mice, implying a cis requirement for the H19 gene. In contrast to a previous report in which overexpression of a marked H19 gene was a prenatal lethal, expression of the M. spretus transgene had no deleterious effect, leading to the conclusion that the 20-base insertion in the marked gene created a neomorphic mutation.

Animals↗
PubMed1996

Linkage between vitamin D-binding protein and alpha-fetoprotein in the mouse.

The albumin gene family consists of four evolutionarily related genes that code for serum transport proteins. In rodents, the genes for albumin, alpha-fetoprotein, and alpha ALB are physically linked within 100 kilobases of DNA. The fourth gene, Gc, encoding vitamin D-binding protein or group-specific component, maps to the same chromosome as the other family members, but linkage has not been established. This report describes the genetic and physical mapping of Gc in mouse and establishes that, although Gc is genetically linked to the other genes, its physical distance from them extends beyond the resolution range of yeast artificial chromosome cloning and pulsed-field gel electrophoresis.

Animals↗
PubMed1996

Complementation mapping of skeletal and central nervous system abnormalities in mice of the piebald deletion complex.

The s15DttMb, s36Pub, s1Acrg and s24Pub piebald deletion alleles belong to a set of overlapping deficiencies on the distal portion of chromosome 14. Molecular analysis was used to define the extent of the deletions. Mice homozygous for the smallest deletion, s15DttMb, die shortly after delivery and display alterations in the central nervous system, including hydrocephalus and a dorsally restricted malformation of the spinal cord. These mice also display homeotic transformations of vertebrae in the midthoracic and lumbar regions. Homozygous s27Pub mice contain a point mutation in the piebald gene, survive to weaning, and display no central nervous system or skeletal defects, arguing that the s15DttMb phenotype results from the loss of genes in addition to piebald. A larger deletion, s36Pub, exhibits additional cartilage malformations and defects in the anterior axial and cranial skeleton. The skeletal defects in both s15DttMb and s36Pub mice resemble transformations associated with the targeted disruption of Hox genes and genes encoding the retinoic acid receptors, which play a role in the specification of segmental identity along the anteroposterior axis. Complementation analysis of the s15DttMb and s36Pub phenotypes, using two additional deletions, localized the gene(s) associated with each phenotype to a defined chromosomal region.

Abnormalities, Multiple↗
PubMed1996

Genomic imprinting in mice: its function and mechanism.

Genomic imprinting is an epigenetic phenomenon by which the two parental alleles of a gene are differentially expressed. Although the function of genomic imprinting is not clear, it has been proposed that it evolved in mammals to regulate intrauterine growth. This proposal is consistent with experiments that were designed to reveal the mechanism and impact of genomic imprinting in a region of mouse chromosome 7 that contains four imprinted genes: Mash-2 (a transcription factor) and H19 (a noncoding RNA) are maternally expressed, whereas Insulin-2 (Ins-2) and Insulin-like growth factor 2 (Igf-2) are paternally expressed. Two targeted disruptions at the locus were generated in mice; these support the hypothesis that the function of the H19 gene is to set up the imprinting of both Igf-2 and Ins-2. H19 transcription on the maternal chromosome precludes transcription of the other two genes by a mechanism that involves competition for a common set of enhancers. On the paternal chromosome the H19 gene is silenced by DNA methylation, thus permitting the use of enhancers by the other genes.

Animals↗