An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

A differentially methylated imprinting control region within the Kcnq1 locus harbors a methylation-sensitive chromatin insulator

The mechanisms underlying the phenomenon of genomic imprinting remain poorly understood. In one instance, a differentially methylated imprinting control region (ICR) at the H19 locus has been shown to involve a methylation-sensitive chromatin insulator function that apparently partitions the neighboring Igf2 and H19 genes in different expression domains in a parent of origin-dependent manner. It is not known, however, if this mechanism is unique to the Igf2/H19 locus or if insulator function is a common feature in the regulation of imprinted genes. To address this question, we have studied an ICR in the Kcnq1 locus that regulates long range repression on the paternally derived p57Kip2 and Kcnq1 alleles in an imprinting domain that includes Igf2 and H19. We show that this ICR appears to possess a unidirectional chromatin insulator function in somatic cells of both mesodermal and endodermal origins. Moreover, we document that CpG methylation regulates this insulator function suggesting that a methylation-sensitive chromatin insulator is a common theme in the phenomenon of genomic imprinting.     

Multiple nucleosome positioning sites regulate the CTCF-mediated insulator function of the H19 imprinting control region

The 5' region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.     

Dynamic readjustment of parental methylation patterns of the 5'-flank of the mouse H19 gene during in vitro organogenesis

Gametic marks are stably propagated in order to manifest parent of origin-specific expression patterns of imprinted genes in the developing conceptus. Although the character of the imprint has not yet been fully elucidated, there is compelling evidence that it involves a methylation mark. This is exemplified by a region upstream of the H19 gene, which is not only methylated in a parent of origin-specific manner, but also regulates the silencing of the maternal Igf2 and paternal H19 alleles, respectively. We show here that the parental-specific methylation patterns within the differentially methylated domain (DMD) are perturbed in the soma during in vitro organogenesis. Under these conditions, the paternal DMD allele becomes partially demethylated, whereas the maternal DMD allele gains methylation. Despite these effects, there were no changes in allelic Igf2 or H19 expression patterns in the embryo. Finally, we show that although TSA derepresses the paternal H19 allele in ectoplacental cone when in vitro developed, there is no discernible effect on the methylation status of the paternally inherited 5'-flank in comparison to control samples. Collectively, this data demonstrates that the parental mark is sensitive to a subset of environmental cues and that a certain degree of plasticity of the gametic mark is tolerated without affecting the manifestation of the imprinted state.     

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.     

Network properties of human disease genes with pleiotropic effects

Background  

The ability of a gene to cause a disease is known to be associated with the topological position of its protein product in the molecular interaction network. Pleiotropy, in human genetic diseases, refers to the ability of different mutations within the same gene to cause different pathological effects. Here, we hypothesized that the ability of human disease genes to cause pleiotropic effects would be associated with their network properties.     

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

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