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PubMed2003

The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains.

The repression of the maternally inherited Igf2 allele has been proposed to depend on a methylation-sensitive chromatin insulator organized by the 11 zinc finger protein CTCF at the H19 imprinting control region (ICR). Here we document that point mutations of the nucleotides in physical contact with CTCF within the endogenous H19 ICR lead to loss of CTCF binding and Igf2 imprinting only when passaged through the female germline. This effect is accompanied by a significant loss of methylation protection of the maternally derived H19 ICR. Because CTCF interacts with other imprinting control regions, it emerges as a central factor responsible for interpreting and propagating gamete-derived epigenetic marks and for organizing epigenetically controlled expression domains.

Animals↗

PubMed2003

Skewing X chromosome choice by modulating sense transcription across the Xist locus.

The X-inactive-specific transcript (Xist) locus is a cis-acting switch that regulates X chromosome inactivation in mammals. Over recent years an important goal has been to understand how Xist is regulated at the initiation of X inactivation. Here we report the analysis of a series of targeted mutations at the 5' end of the Xist locus. A number of these mutations were found to cause preferential inactivation, to varying degrees, of the X chromosome bearing the targeted allele in XX heterozygotes. This phenotype is similar to that seen with mutations that ablate Tsix, an antisense RNA initiated 3' of Xist. Interestingly, each of the 5' mutations causing nonrandom X inactivation was found to exhibit ectopic sense transcription in embryonic stem (ES) cells. The level of ectopic transcription was seen to correlate with the degree of X inactivation skewing. Conversely, targeted mutations which did not affect randomness of X inactivation also did not exhibit ectopic sense transcription. These results indicate that X chromosome choice is determined by the balance of Xist sense and antisense transcription prior to the onset of random X inactivation.

Animals↗

PubMed2006

A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.

During early mammalian female development, one of the two X chromosomes becomes inactivated. Although X-chromosome coating by Xist RNA is essential for the initiation of X inactivation, little is known about how this signal is transformed into transcriptional silencing. Here we show that exclusion of RNA Polymerase II and transcription factors from the Xist RNA-coated X chromosome represents the earliest event following Xist RNA accumulation described so far in differentiating embryonic stem (ES) cells. Paradoxically, exclusion of the transcription machinery occurs before gene silencing is complete. However, examination of the three-dimensional organization of X-linked genes reveals that, when transcribed, they are always located at the periphery of, or outside, the Xist RNA domain, in contact with the transcription machinery. Upon silencing, genes shift to a more internal location, within the Xist RNA compartment devoid of transcription factors. Surprisingly, the appearance of this compartment is not dependent on the A-repeats of the Xist transcript, which are essential for gene silencing. However, the A-repeats are required for the relocation of genes into the Xist RNA silent domain. We propose that Xist RNA has multiple functions: A-repeat-independent creation of a transcriptionally silent nuclear compartment; and A-repeat-dependent induction of gene repression, which is associated with their translocation into this silent domain.

Animals↗

PubMed2006

Tsix-mediated epigenetic switch of a CTCF-flanked region of the Xist promoter determines the Xist transcription program.

Initiation of X inactivation depends on the coordinated expression of the sense/antisense pair Xist/Tsix. We show here that a precisely defined Xist promoter region flanked by CTCF is maintained by Tsix in a heterochromatic-like state in undifferentiated embryonic stem (ES) cells and shifts to a pseudoeuchromatic structure upon Tsix truncation. We further demonstrate that the epigenetic state of the Xist 5' region prior to differentiation predicts the efficiency of transcriptional machinery recruitment to the Xist promoter during differentiation. Our results provide mechanistic insights into the Tsix-mediated epigenetic regulation of Xist resulting in Xist promoter activation and initiation of X inactivation in differentiating ES cells.

Animals↗

PubMed2001

Sharp, an inducible cofactor that integrates nuclear receptor repression and activation.

A yeast two-hybrid screen using the conserved carboxyl terminus of the nuclear receptor corepressor SMRT as a bait led to the isolation of a novel human gene termed SHARP (SMRT/HDAC1 Associated Repressor Protein). SHARP is a potent transcriptional repressor whose repression domain (RD) interacts directly with SMRT and at least five members of the NuRD complex including HDAC1 and HDAC2. In addition, SHARP binds to the steroid receptor RNA coactivator SRA via an intrinsic RNA binding domain and suppresses SRA-potentiated steroid receptor transcription activity. Accordingly, SHARP has the capacity to modulate both liganded and nonliganded nuclear receptors. Surprisingly, the expression of SHARP is itself steroid inducible, suggesting a simple feedback mechanism for attenuation of the hormonal response.

Amino Acid Motifs↗

PubMed1995

Biallelic expression of imprinted genes in the mouse germ line: implications for erasure, establishment, and mechanisms of genomic imprinting.

Genomic imprinting in mammals determines parental-specific (monoallelic) expression of a relatively small number of genes during development. Imprinting must logically be imparted in the germ line, where inherited maternal and paternal imprinting is erased and new imprinting established according to the individual's sex. We have assessed the allele-specific expression of four imprinted genes, two of which exhibit maternal-specific (H19 and Igf2r) and two of which exhibit paternal-specific (Igf2 and Snrpn) monoallelic somatic expression, in the germ line of F1 hybrid mice utilizing quantitative RT-PCR single-nucleotide primer extension assays. The expression of each gene was biallelic in the female and male germ line from the time that migratory mitotic PGCs entered the embryonic genital ridge and throughout gametogenesis, except that H19 RNA was not detected late in gametogenesis. These findings demonstrate that inherited imprinting is erased, or not recognized, in germ cells by the time of genital ridge colonization; also that new imprinting may not be established until late in gametogenesis, or that it is incomplete or not recognized at this stage. Regardless of imprinting status, a generalized neutralization of imprinting is evident in the germ line, associated with the totipotent state of this unique cell lineage.

Alleles↗

PubMed1995

An enhancer deletion affects both H19 and Igf2 expression.

The distal end of mouse Chromosome 7 contains four tightly linked genes whose expression is dependent on their parental inheritance. Mash-2 and H19 are expressed exclusively from the maternal chromosome, whereas Insulin-2 (Ins-2) and Insulin-like growth factor 2 (Igf2) are paternally expressed. The identical expression during development of the 3'-most genes in the cluster, Igf2 and H19, led to the proposal that their imprinting was mechanistically linked through a common set of transcriptional regulatory elements. To test this hypothesis, a targeted deletion of two endoderm-specific enhancers that lie 3' of H19 was generated by homologous recombination in embryonic stem cells. Inheritance of the enhancer deletion through the maternal lineage led to a loss of H19 gene expression in cells of endodermal origin, including cells in the liver, gut, kidney, and lung. Paternal inheritance led to a very similar loss in the expression of Igf2 RNA in the same tissues. These results establish that H19 and Igf2 utilize the same endoderm enhancers, but on different parental chromosomes. Mice inheriting the enhancer deletion from fathers were 80% of normal size, reflecting a partial loss-of-function of Igf2. The reduction was uniformly observed in a number of internal organs, indicating that insulin-like growth factor II (IGFII), the product of Igf2, acts systemically in mice to affect prenatal growth. A modest decline in Ins-2 RNA was observed in the yolk sac. In contrast Mash-2, which is expressed in spongiotrophoblast cells of the placenta, was unaffected by the enhancer deletion.

Animals↗

PubMed1995

Loss of methylation activates Xist in somatic but not in embryonic cells.

The mouse Xist gene, which is expressed only from the inactive X chromosome, is thought to play a role in the initiation of X inactivation. The 5' end of this gene is fully methylated on the active X chromosome and completely demethylated on the inactive X chromosome, suggesting that DNA methylation may be involved in controlling allele-specific transcription of this gene. To directly investigate the importance of DNA methylation in the control of Xist expression, we have examined its methylation patterns and expression in ES cells and embryos that are deficient in DNA methyltransferase activity. We report here that demethylation of the Xist locus in male mutant embryos induces Xist expression, thus establishing a direct link between demethylation and expression of the Xist gene in the postgastrulation embryo. The transcriptional activity of Xist in undifferentiated ES cells, however, appears to be independent of its methylation status. These results suggest that methylation may only become essential for Xist repression after ES cells have differentiated or after the embryo has undergone gastrulation.

Animals↗

PubMed1995

Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms.

Genomic imprinting determines the monoallelic expression of a small number of genes during at least later stages of development. To obtain information necessary for the elucidation of imprinting mechanisms, we assessed the allele-specific expression and total expression level of four imprinted genes during early stages of development of normal F1 hybrid mice utilizing quantitative allele-specific reverse transcription-PCR (RT-PCR) single-nucleotide primer extension assays. The Igf2r and Snrpn genes were activated by the early 4-cell stage and exhibited biallelic and monoallelic expression, respectively, throughout preimplantation development. Thus, with respect to different imprinted genes, epigenetic systems determining monoallelic expression are not uniform in their time of establishment. Biallelic expression of Igf2r was observed in single blastomeres, discounting the possibility of random allelic inactivation at this stage. The closely linked H19 and Igf2 genes were activated after the blastocyst stage and often exhibited biallelic and monoallelic expression respectively in tissues of pregastrulation postimplantation-stage embryos, rather than reciprocal monoallelic modes as observed at later stages. This raises the possibility that imprinting of H19 is involved only in the maintenance and not in the initiation of monoallelic expression of Igf2. Monoallelic expression of Snrpn was observed in each blastomere at the 4-cell stage, demonstrating that the germ line, which exhibits biallelic expression of imprinted genes, must be derived from cells in which imprinting was once manifest.

Alleles↗

PubMed2000

Comparative genomic sequencing identifies novel tissue-specific enhancers and sequence elements for methylation-sensitive factors implicated in Igf2/H19 imprinting.

A differentially methylated region (DMR) and endoderm-specific enhancers, located upstream and downstream of the mouse H19 gene, respectively, are known to be essential for the reciprocal imprinting of Igf2 and H19. To explain the same imprinting patterns in non-endodermal tissues, additional enhancers have been hypothesized. We determined and compared the sequences of human and mouse H19 over 40 kb and identified 10 evolutionarily conserved downstream segments, 2 of which were coincident with the known enhancers. Reporter assays in transgenic mice showed that 5 of the other 8 segments functioned as enhancers in specific mesodermal and/or ectodermal tissues. We also identified a conserved 39-bp element that appeared repeatedly within the DMR and formed complexes with specific nuclear factors. Binding of one of the factors was inhibited when the target sequence contained methylated CpGs. These complexes may contribute to the presumed boundary function of the unmethylated DMR, which is proposed to insulate maternal Igf2 from the enhancers. Our results demonstrate that comparative genomic sequencing is highly efficient in identifying regulatory elements.

5' Untranslated Regions↗

PubMed2002

Domain regulation of imprinting cluster in Kip2/Lit1 subdomain on mouse chromosome 7F4/F5: large-scale DNA methylation analysis reveals that DMR-Lit1 is a putative imprinting control region.

Mouse chromosome 7F4/F5, where the imprinting domain is located, is syntenic to human 11p15.5, the locus for Beckwith-Wiedemann syndrome. The domain is thought to consist of the two subdomains Kip2 (p57(kip2))/Lit1 and Igf2/H19. Because DNA methylation is believed to be a key factor in genomic imprinting, we performed large-scale DNA methylation analysis to identify the cis-element crucial for the regulation of the Kip2/Lit1 subdomain. Ten CpG islands (CGIs) were found, and these were located at the promoter sites, upstream of genes, and within intergenic regions. Bisulphite sequencing revealed that CGIs 4, 5, 8, and 10 were differentially methylated regions (DMRs). CGIs 4, 5, and 10 were methylated paternally in somatic tissues but not in germ cells. CGI8 was methylated in oocyte and maternally in somatic tissues during development. Parental-specific DNase I hypersensitive sites (HSSs) were found near CGI8. These data indicate that CGI8, called DMR-Lit1, is not only the region for gametic methylation but might also be the imprinting control region (ICR) of the subdomain.

Alleles↗

PubMed2000

Novel imprinted DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic those implicated in IGF2/H19 regulation.

The evolution of genomic imprinting in mammals occurred more than 100 million years ago, and resulted in the formation of genes that are functionally haploid because of parent-of-origin-dependent expression. Despite ample evidence from studies in a number of species suggesting the presence of imprinted genes on human chromosome 14, their identity has remained elusive. Here we report the identification of two reciprocally imprinted genes, GTL2 and DLK1, which together define a novel imprinting cluster on human chromosome 14q32. The maternally expressed GTL2 (gene trap locus 2) gene encodes for a nontranslated RNA. DLK1 (delta, Drosophila, homolog-like 1) is a paternally expressed gene that encodes for a transmembrane protein containing six epidermal growth factor (EGF) repeat motifs closely related to those present in the delta/notch/serrate family of signaling molecules. The paternal expression, chromosomal localization, and biological function of DLK1 also make it a likely candidate gene for the callipyge phenotype in sheep. Many of the predicted structural and regulatory features of the DLK1/GTL2 domain are highly analogous to those implicated in IGF2/H19 imprint regulation, including two hemimethylated consensus binding sites for the vertebrate enhancer blocking protein, CTCF. These results provide evidence that a common mechanism and domain organization may be used for juxtapositioned, reciprocally imprinted genes.

Amino Acid Motifs↗

PubMed2001

Characterization of the genomic Xist locus in rodents reveals conservation of overall gene structure and tandem repeats but rapid evolution of unique sequence.

The Xist locus plays a central role in the regulation of X chromosome inactivation in mammals, although its exact mode of action remains to be elucidated. Evolutionary studies are important in identifying conserved genomic regions and defining their possible function. Here we report cloning, sequence analysis, and detailed characterization of the Xist gene from four closely related species of common vole (field mouse), Microtus arvalis. Our analysis reveals that there is overall conservation of Xist gene structure both between different vole species and relative to mouse and human Xist/XIST. Within transcribed sequence, there is significant conservation over five short regions of unique sequence and also over Xist-specific tandem repeats. The majority of unique sequences, however, are evolving at an unexpectedly high rate. This is also evident from analysis of flanking sequences, which reveals a very high rate of rearrangement and invasion of dispersed repeats. We discuss these results in the context of Xist gene function and evolution.

3' Untranslated Regions↗

PubMed1994