Christine M. Williamson

Identification of an imprinting control region affecting the expression of all transcripts in the Gnas cluster

Genomic imprinting results in allele-specific silencing according to parental origin. Silencing is brought about by imprinting control regions (ICRs) that are differentially marked in gametogenesis. The group of imprinted transcripts in the mouse Gnas cluster (Nesp, Nespas, Gnasxl, Exon 1A and Gnas) provides a model for analyzing the mechanisms of imprint regulation. We previously identified an ICR that specifically regulates the tissue-specific imprinted expression of the Gnas gene. Here we identify a second ICR at the Gnas cluster. We show that a paternally derived targeted deletion of the germline differentially methylated region (DMR) associated with the antisense Nespas transcript unexpectedly affects both the expression of all transcripts in the cluster and methylation of two DMRs. Our results establish that the Nespas DMR is the principal ICR at the Gnas cluster and functions bidirectionally as a switch for modulating expression of the antagonistically acting genes Gnasxl and Gnas. Uniquely, the Nespas DMR acts on the downstream ICR at exon 1A to regulate tissue-specific imprinting of the Gnas gene.

A cis-acting control region is required exclusively for the tissue-specific imprinting of Gnas

Genomic imprinting brings about allele-specific silencing according to parental origin. Silencing is controlled by cis-acting regulatory regions that are differentially marked during gametogenesis and can act over hundreds of kilobases to silence many genes. Two candidate imprinting control regions (ICRs) have been identified at the compact imprinted Gnas cluster on distal mouse chromosome 2, one at exon 1A upstream of Gnas itself and one covering the promoters for Gnasxl and the antisense Nespas (ref. 8). This imprinted cluster is complex, containing biallelic, maternally and paternally expressed transcripts that share exons. Gnas itself is mainly biallelically expressed but is weakly paternally repressed in specific tissues. Here we show that a paternally derived targeted deletion of the germline differentially methylated region at exon 1A abolishes tissue-specific imprinting of Gnas. This rescues the abnormal phenotype of mice with a maternally derived Gnas mutation. Imprinting of alternative transcripts, Nesp, Gnasxl and Nespas (ref. 13), in the cluster is unaffected. The results establish that the differentially methylated region at exon 1A contains an imprinting control element that specifically regulates Gnas and comprises a characterized ICR for a gene that is only weakly imprinted in a minority of tissues. There must be a second ICR regulating the alternative transcripts.

Epigenetic Properties and Identification of an Imprint Mark in the Nesp-Gnasxl Domain of the Mouse Gnas Imprinted Locus

ABSTRACT The Gnas locus in the mouse is imprinted with a complex arrangement of alternative transcripts defined by promoters with different patterns of monoallelic expression. The Gnas transcript is subject to tissue-specific imprinted expression, Nesp is expressed only from the maternal allele, and Gnasxl is expressed only from the paternal allele. The mechanisms controlling these expression patterns are not known. To identify potential imprinting regulatory regions, particularly for the reciprocally expressed Nesp and Gnasxl promoters, we examined epigenetic properties of the locus in gametes, embryonic stem cells, and fetal and adult tissues. The Nesp and Gnasxl promoter regions are contained in extensive CpG islands with methylation of the paternal allele at Nesp and the maternal allele at Gnasxl. Parental allele-specific DNase I-hypersensitive sites were found at these regions, which correlate with hypomethylation rather than actual expression status. A germ line methylation mark was identified covering the promoters for Gnasxl and the antisense transcript Nespas. Prominent DNase I-hypersensitive sites present on paternal alleles in embryonic stem cells are contained within this mark. This is the second gametic mark identified at Gnas and suggests that the Nesp and Gnasxl promoters are under separate control from the Gnas promoter. We propose models to account for the regulation of imprinting at the locus.

Uncoupling antisense-mediated silencing and DNA methylation in the imprinted Gnas cluster.

There is increasing evidence that non-coding macroRNAs are major elements for silencing imprinted genes, but their mechanism of action is poorly understood. Within the imprinted Gnas cluster on mouse chromosome 2, Nespas is a paternally expressed macroRNA that arises from an imprinting control region and runs antisense to Nesp, a paternally repressed protein coding transcript. Here we report a knock-in mouse allele that behaves as a Nespas hypomorph. The hypomorph mediates down-regulation of Nesp in cis through chromatin modification at the Nesp promoter but in the absence of somatic DNA methylation. Notably there is reduced demethylation of H3K4me3, sufficient for down-regulation of Nesp, but insufficient for DNA methylation; in addition, there is depletion of the H3K36me3 mark permissive for DNA methylation. We propose an order of events for the regulation of a somatic imprint on the wild-type allele whereby Nespas modulates demethylation of H3K4me3 resulting in repression of Nesp followed by DNA methylation. This study demonstrates that a non-coding antisense transcript or its transcription is associated with silencing an overlapping protein-coding gene by a mechanism independent of DNA methylation. These results have broad implications for understanding the hierarchy of events in epigenetic silencing by macroRNAs.

Control of imprinting at the Gnas cluster.

Genomic imprinting is a form of epigenetic regulation in mammals whereby a small subset of genes is silenced according to parental origin. Early work had indicated regions of the genome that were likely to contain imprinted genes. Distal mouse chromosome 2 is one such region and is associated with devastating but ostensibly opposite phenotypes when exclusively maternally or paternally derived. Misexpression of proteins encoded at the Gnas complex, which is located within the region, can largely account for the imprinting phenotypes. Gnas is a complex locus with extraordinary transcriptional and regulatory complexity. It gives rise to alternatively spliced isoforms that show maternal-, paternal- and biallelic expression as well as a noncoding antisense transcript. The objective of our work at Harwell is to unravel mechanisms controlling the expression of these transcripts. We have performed targeted deletion analysis to test candidate regulatory regions within the Gnas complex and, unlike other imprinted domains, two major control regions have been identified. One controls the imprinted expression of a single transcript and is subsidiary to and must interact with, a principal control region that affects the expression of all transcripts. This principal region contains the promoter for the antisense transcript, expression of which may have a major role in controlling imprinting at the Gnas cluster.

Alternative non-coding splice variants of Nespas, an imprinted gene antisense to Nesp in the Gnas imprinting cluster.

Abstract. The Gnas locus on mouse Chr 2 represents a unique cluster of overlapping imprinted genes. Three of these in the order Nesp–Gnasxl–Gnas are transcribed in the sense direction with Nesp having maternal-specific expression, Gnasxl having paternal expression, and Gnas as being biallelically expressed in most tissues. A fourth imprinted gene, Nespas, is paternally expressed, lies antisense to Nesp, and expresses an unspliced transcript. Large unspliced antisense transcripts are emerging as a feature of imprinted gene clusters, and such non-coding RNAs may have a cis-regulatory function. Here we show that, in addition to an unspliced form of Nepas, we can detect five alternatively spliced forms of Nespas up to 1.4 kb in length that are non-coding. The splice variants are paternally expressed; they start approximately 2 kb upstream of Gnasxl in a region of maternal methylation and end 2.5 kb beyond the ATG of Nesp. These variants do not correspond to exons of the human antisense transcript although they start in the same region; the Nespas transcript, like its human counterpart, is spliced in various alternative patterns. The identification of a set of small spliced imprinted transcripts in the human and now in the mouse suggests that these antisense transcripts are functionally important.

New Mutations at the Imprinted Gnas Cluster Show Gene Dosage Effects of Gsα in Postnatal Growth and Implicate XLαs in Bone and Fat Metabolism but Not in Suckling

ABSTRACT The imprinted Gnas cluster is involved in obesity, energy metabolism, feeding behavior, and viability. Relative contribution of paternally expressed proteins XLαs, XLN1, and ALEX or a double dose of maternally expressed Gsα to phenotype has not been established. In this study, we have generated two new mutants (Ex1A-T-CON and Ex1A-T) at the Gnas cluster. Paternal inheritance of Ex1A-T-CON leads to loss of imprinting of Gsα, resulting in preweaning growth retardation followed by catch-up growth. Paternal inheritance of Ex1A-T leads to loss of imprinting of Gsα and loss of expression of XLαs and XLN1. These mice have severe preweaning growth retardation and incomplete catch-up growth. They are fully viable probably because suckling is unimpaired, unlike mutants in which the expression of all the known paternally expressed Gnasxl proteins (XLαs, XLN1 and ALEX) is compromised. We suggest that loss of ALEX is most likely responsible for the suckling defects previously observed. In adults, paternal inheritance of Ex1A-T results in an increased metabolic rate and reductions in fat mass, leptin, and bone mineral density attributable to loss of XLαs. This is, to our knowledge, the first report describing a role for XLαs in bone metabolism. We propose that XLαs is involved in the regulation of bone and adipocyte metabolism.

Imprinting of distal mouse chromosome 2 is associated with phenotypic anomalies in utero.

Previous studies have shown that the distal region on mouse chromosome (Chr) 2 is subject to imprinting as mice with maternal duplication/paternal deficiency (MatDp.dist2) and the reciprocal (PatDp.dist2) for this region exhibit phenotypic anomalies at birth and die neonatally. We show here that imprinting effects are detectable in utero. Notably PatDp.dist2 embryos show an increase in wet weight compared with normal, which peaks at 16.5 d post coitum (dpc), and diminishes by birth, whereas the wet weight of placenta is slightly reduced in the latter half of gestation. Newborns have increased length of the long bones. By contrast, the wet weight of MatDp.dist2 embryos decreases during the second half of gestation. Measurements of dry weights of embryos at 16.5 dpc have indicated that there is no difference in either PatDp.dist2 or MatDp.dist2 compared with normal so that the wet weight differences are due to fluid retention in PatDp.dist2 but fluid loss in MatDp.dist2. In PatDp.dist2 embryos excess fluid is particularly prominent in the subcuticular skin layer, whereas by birth fluid is evident around the neck and tongue. At 16.5 dpc the PatDp.dist2 embryos are severely oedematous, as the average fluid content per unit dry weight per embryo was increased by 40%, whereas the MatDp.dist2 embryos are dehydrated as the average water content per unit dry weight per embryo was reduced by 6%. A preliminary conclusion is that there is neither growth enhancement in PatDp.dist2 nor growth retardation in MatDp.dist2 offspring.

The spatial and temporal expression pattern of Nesp and its antisense Nespas, in mid-gestation mouse embryos

We describe the spatiotemporal expression pattern of Nesp, and its antisense transcript, Nespas. We found non-complementary expression of these two oppositely imprinted transcripts during mouse embryogenesis, in a number of forming embryonic structures. Nesp expression was primarily seen in the somites and vasculature, whereas Nespas was mainly detected in the progress zone, mesenchyme and ectoderm of the limb, and the neural tube.

MouseBook: an integrated portal of mouse resources

The MouseBook (http://www.mousebook.org) databases and web portal provide access to information about mutant mouse lines held as live or cryopreserved stocks at MRC Harwell. The MouseBook portal integrates curated information from the MRC Harwell stock resource, and other Harwell databases, with information from external data resources to provide value-added information above and beyond what is available through other routes such as International Mouse Stain Resource (IMSR). MouseBook can be searched either using an intuitive Google style free text search or using the Mammalian Phenotype (MP) ontology tree structure. Text searches can be on gene, allele, strain identifier (e.g. MGI ID) or phenotype term and are assisted by automatic recognition of term types and autocompletion of gene and allele names covered by the database. Results are returned in a tabbed format providing categorized results identified from each of the catalogs in MouseBook. Individual result lines from each catalog include information on gene, allele, chromosomal location and phenotype, and provide a simple click-through link to further information as well as ordering the strain. The infrastructure underlying MouseBook has been designed to be extensible, allowing additional data sources to be added and enabling other sites to make their data directly available through MouseBook.