text:
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.
Author Summary
Genomic imprinting is a process resulting in expression of genes according to parental origin. Some imprinted genes are expressed when paternally derived and others when maternally derived. Thus imprinted genes are monoallelically expressed and one copy has to be silenced. There is evidence that some long non-coding RNAs, acting in cis, have a role in silencing. We investigated the role of Nespas, a gene for a non-coding RNA that is only expressed from the paternally derived chromosome in the Gnas cluster and runs antisense to its sense counterpart, Nesp. Expression of Nespas is associated with silencing of Nesp and a repressive methylation mark on the Nesp DNA. We generated a Nespas mutant with reduced levels of activity and showed that it down-regulated its sense counterpart Nesp, in the absence of a DNA methylation mark, but in the presence of an altered chromatin mark. We conclude that Nespas can repress Nesp by a mechanism independent of DNA methylation, by modulating a chromatin mark.
Citation: Williamson CM, Ball ST, Dawson C, Mehta S, Beechey CV, Fray M, et al. (2011) Uncoupling Antisense-Mediated Silencing and DNA Methylation in the Imprinted Gnas Cluster. PLoS Genet 7(3): e1001347. doi:10.1371/journal.pgen.1001347
Editor: Jeannie T. Lee, Massachusetts General Hospital, Howard Hughes Medical Institute, United States of America
Received: July 20, 2010; Accepted: February 18, 2011; Published: March 24, 2011
Copyright: © 2011 Williamson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the UK Medical Research Council and the Biotechnology and Biological Sciences Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Over recent years it has emerged that most of the mammalian transcriptome is non-coding [1]. Several long non-coding transcripts have been implicated in epigenetic gene regulation and play essential, but incompletely understood, roles in epigenetic gene silencing in X-inactivation and genomic imprinting in mammals. For the latter, more than 100 imprinted genes are known in the mouse and most occur in clusters [2]. Parental specific gene silencing throughout the clusters is brought about by imprinting control regions (ICRs). These are regions that are differentially methylated in gametogenesis and are active when unmethylated. ICRs for three clusters, the Igf2r cluster, the Kcnq1 cluster and the Gnas cluster, contain promoters for macroRNAs that are exclusively expressed from the paternally derived chromosome and run antisense to a protein coding gene that is repressed by the active unmethylated ICR (for reviews, see [3], [4]). Two of these macroRNA genes, Airn in the Igf2r cluster and Kcnq1ot1 in the Kcnq1 cluster, have been shown to be key elements in parental specific silencing of all protein coding genes in their respective clusters [5]-[7], although their mode of action is incompletely understood. However both are known to be associated with the acquisition of repressive histone marks and DNA methylation marks of some genes. It is not yet known if the third gene for a macroRNA, Nespas in the Gnas cluster has a functional role (Figure 1). It is likely that several other imprinted gene clusters may share this regulatory principle, but their ICRs have not been defined functionally.
Transcripts expressed from the maternally inherited allele are shown above the line, those from the paternally inherited allele are below the line and the direction of transcription is shown by arrow heads. Unspliced Nespas is not shown. The black filled boxes represent first exons of the protein-coding transcripts and the hashed filled boxes are the first exons of the non-coding transcripts. The first exons of Nesp, Gnasxl, Exon1A and Gnas all splice into exon 2 of Gnas. In a subset of tissues, Gnas itself is tissue-specifically imprinted; the dotted line shows the limited expression of Gnas from the paternal allele. The position of the differentially methylated regions (DMRs) are shown by rows of filled circles on the methylated allele; ICR, shows the position of the Imprinting Control Region [8]. Adapted from Peters and Williamson [49].
The ICR for the Gnas cluster contains the Nespas promoter [8] and lies within an extensive differentially methylated region, the Nespas-Gnasxl DMR, that acquires methylation in the maternal germline [9]. This DMR also contains the promoter for a protein coding paternally expressed transcript Gnasxl. The Gnas cluster, unusually, has a second maternally methylated germline DMR, the Exon1A DMR [10] that specifically controls maternal expression of transcripts arising from the Gnas promoter [11], [12]. The ICR regulates the Exon1A DMR that in turn regulates the imprinted expression of Gnas. There is a third DMR in the Gnas cluster and this is a somatic DMR that becomes methylated on the paternal allele post-fertilisation [9], [10]. This DMR covers the furthest upstream promoter in the cluster, the Nesp promoter. Nesp is maternally expressed, protein coding and is transcribed for about 80 kb through the whole cluster including the Nespas-Gnasxl DMR and the second germline DMR at Exon1A. Recently it was shown that truncation of this long Nesp transcript upstream of the two germline DMRs disrupted the acquisition of methylation at both DMRs in the oocyte [13]. Thus transcription of the Nesp protein coding transcript in the female germline is required for the establishment of maternal methylation across the whole Nespas-Gnasxl DMR including the ICR as well as the Exon1A DMR.
The Nesp transcript is the sense counterpart of the paternally expressed antisense Nespas. The macroRNA Nespas that arises from a promoter within the ICR transcribes through, and is associated with methylation of the Nesp DMR [8], but the mechanisms for induction of the post-fertilisation methylation of the Nesp DMR on the paternal allele or, for that matter, any somatic DMR, have not been established. De novo establishment of methylation at maternal and some paternal germline DMRs requires the DNA methyltransferase DNMT3A and its non-enzymatic cofactor DNMT3L [14]-[16]. The process of methylation is mediated by histone modifications; it appears that DNMT3A is recruited to the DNA by complexing with DNMT3L [17]-[19] which interacts with histone H3 but only when lysine 4 is unmethylated [17]. Interaction of DNMT3A with chromatin is also inhibited by H3K4 methylation [20] but promoted by H3K36 trimethylation [21]. Whether de novo DNA methylation of somatic DMRs depends upon DNMT3A, or the second de novo enzyme DNMT3B, and co-operation with DNMT3L has not been established.
In order to investigate a role for Nespas, we made use of a knock-in allele that behaved as a Nespas hypomorph. We show here that when Nespas is present at a low level in the hypomorph the Nesp promoter is unmethylated but Nesp expression is considerably down-regulated and the Nesp promoter is only partially enriched for an activating histone mark, methylated H3K4. Thus, in the hypomorph, Nespas can mediate the down-regulation of Nesp through chromatin modification even in the absence of DNA methylation at Nesp. An insufficiency of methylated H3K4 could be a major factor in repressing Nesp expression. Furthermore, the results suggest that Nespas has a role in the demethylation of H3K4me3 as a prerequisite for DNA methylation of the Nesp promoter on the paternal chromosome. The results provide the first evidence that Nespas has a functional role in regulating imprinted gene expression in the Gnas cluster.
Gene targeting generates a Nespas hypomorph (Nesp-Tint2)
In studies to analyse the regulatory function of Nespas, we made use of a Nespas hypomorph (Nesp-Tint2; phenotypic analysis to be reported elsewhere). The hypomorph resulted from insertion of a polyadenylation cassette from the rabbit β-globin gene, into exon 1 of Nespas, in the reverse orientation (pA) so that truncation of Nespas should not occur (Figure 2A and 2B). Although it was not anticipated that this insertion would influence expression of Nespas, the mutant was shown to be a hypomorph in three ways. Firstly, we used RT-PCR and primers (Figure 2C) that would amplify Nespas but not Nesp. Weak expression of a Nespas splice variant was found in brain from newborn mice with a paternally derived Nesp-Tint2 allele (+/Nesp-Tint2, note that the maternal allele precedes the paternal allele in all genotypes shown here; Figure 2D). Mice carrying a paternal copy of the null Nespas allele, ΔNAS-DMR, in which the Nespas promoter and first exon are deleted [8], were included as a negative control (Figure 2D). Secondly, the hypomorph was verified by RNA blot analysis using a single stranded probe, shown in Figure 2C, that would detect spliced and unspliced Nespas transcripts. The Nespas transcripts, which are detected as a smear [22], were considerably reduced in 15.5 dpc embryos with the paternally inherited Nesp-Tint2 allele when compared to the wild-type level (Figure 2E). Thirdly, we checked that the primary transcripts of Nespas were reduced in +/Nesp-Tint2 newborn brain using a TaqMan real-time qPCR assay. The assay was designed upstream of the Nesp promoter in intron 4 of Nespas [23] and is shown schematically in Figure 2C. We showed that Nespas levels were reduced by 94% in heterozygotes, +/Nesp-Tint2, that have a paternally inherited copy of the mutation when compared to wild-type levels (P = 5.91×10−11; Figure 2F). The reduced level of Nespas was not due to gain of methylation on the paternal allele at the Nespas-Gnasxl DMR (Figure S1). Thus the position of the cassette close to the Nespas promoter may have affected promoter activity, resulting in a low level of Nespas.
(A) Overview of the mouse Gnas locus showing enlargement of the Nespas region and the site of insertion of the rabbit β-globin polyadenylation cassette in Nespas exon 1. The targeting vector shows the location of a 1.2 kb fragment (pA) from the rabbit β-globin gene to truncate Nesp, and the selection genes were flanked by loxP sites (open triangles). The selection genes were deleted from the pA-neo targeted allele upon male germline transmission by Cre-recombinase mediated excision to generate Nesp-Tint2. The DMRs are shown by the presence of filled circles on the methylated allele. A, AvrII; N, NdeI; Sp, SpeI; Sw, SwaI; P, Nespas promoter; ICR, Imprinting Control Region, the extent of which is defined by the deletion in the ΔNAS-DMR allele [8]. (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (pA-neo) cells. The pA-neo targeted clones were identified by the presence of an 8.5 kb NdeI fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb AvrII fragment with the 5′ external probe. (C) Schematic of the Nesp-Nespas region showing overlapping transcription on the sense and antisense strands and the position of RT-PCR primers, a single stranded RNA probe and TaqMan assay for detecting the primary Nespas transcript. (D) RT-PCR analysis of Nespas in neonatal brain after paternal transmission of Nesp-Tint2 (+/Nesp-Tint2) using primers F6 and R2 (Figure 2C; Table S1). +/ΔNAS-DMR, mice with paternal deletion of the ICR [8]; + and −, presence and absence of reverse transcriptase, respectively; Hprt, amplification control. (E) RNA blot analysis showing expression of Nespas in poly(A)+ RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C. (F) Bar chart showing the relative level of Nespas expression in newborn brain by RT-qPCR using a TaqMan assay within intron 4 (Figure 2C). The reference gene was Gapdh. Error bars (RQmin/RQmax) were based on a 95% confidence level.
Paternal inheritance of Nesp-Tint2 is associated with hypomethylation of the paternal Nesp somatic DMR and partial de-repression of Nesp
Previous work had shown paternal inheritance of the null ΔNAS-DMR allele was associated with loss of methylation of the Nesp promoter [8]. Therefore we investigated whether a low level of Nespas from the paternal allele in +/Nesp-Tint2 was associated with a change in the methylation status of the Nesp somatic DMR. Southern analysis of newborn liver showed complete loss of methylation at the Nesp promoter and first exon on the paternal allele in +/Nesp-Tint2 (Figure 3A, Top Right). This result was confirmed by bisulphite analysis of brain DNA from newborn offspring arising from crosses of Nesp-Tint2 carrier males with SD2 females carrying the Gnas cluster region from Mus spretus. The presence of single nucleotide variants in the parents enabled the distinction of maternal and paternal Nesp alleles. Two wild-type (+SD2/+) and four mutant (+SD2/Nesp-Tint2) newborns were analysed and Figure 3A (Bottom Right) shows loss of methylation on the paternal allele of +/Nesp-Tint2 compared with that of a wild-type. Thus the normally methylated paternal Nesp allele was unmethylated in +/Nesp-Tint2, probably due to the low level of Nespas (as summarised in Figure 3A, Left).
(A) Loss of methylation at the Nesp DMR in +/Nesp-Tint2. (A Left) Schematic summary of the transcriptional status of Nespas and the methylation status of Nespas and Nesp on the paternal allele of wild-type and +/Nesp-Tint2. Dashed line indicates low level of transcript. Row of filled circles, methylated allele; row of open circles, unmethylated allele. (A Top Right) Southern analysis showing promoter methylation at the Nesp DMR is lost in +/Nesp-Tint2. Genomic DNA from newborn liver was digested with EcoRI (-), EcoRI and HpaII (H), or EcoRI and MspI (M). (A Bottom Right) Bisulphite profile of the paternal allele of the Nesp DMR in newborn brain from +SD2/+ and +SD2/Nesp-Tint2. Sequence variants allowed the maternal and paternal alleles to be discriminated; see Table S1 for primers. Each row of circles represented a clone derived from the paternal allele and each circle corresponded to a separate CpG (filled circles, methylated CpGs; open circles, unmethylated CpGs). Each block of circles represented the data from an individual mouse. (B) Low level of truncated Nesp from the paternal allele in +/Nesp-Tint2. (B Top Left) RNA blot analysis showing expression of Nesp in poly(A)+ RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C. The full length transcript was expressed from the maternal allele. (B Bottom Left)The smaller truncated transcript was expressed from the targeted paternal allele as summarised in the schematic. (B Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR, measured using a TaqMan assay that detects exon 1 spliced onto 2. The reference gene was Gapdh. Error bars (RQmin/RQmax) were based on a 95% confidence level.
As the paternally derived Nesp DMR was unmethylated in +/Nesp-Tint2, we expected that Nesp would be expressed from the mutant paternal allele. In addition, as Nesp and Nespas overlap (Figure 1), insertion of the polyadenylation cassette in exon 1 of Nespas on the antisense strand is also an insertion into intron 2 of Nesp on the sense strand and might truncate Nesp. Sequence analysis of RT-PCR products derived from using a Nesp exon 2-specific forward primer and a reverse primer specific to the polyadenylation cassette and therefore specific to the mutant paternal allele revealed a number of splice variants (Figure S2) whereby Nesp splices into the inserted β-globin sequence. Thus Nesp was expressed from the paternal allele and was likely to be truncated. The sizes of the Nesp transcripts in +/Nesp-Tint2 embryos were analysed by northern blotting (Figure 3B). Two major transcripts were detected: full length Nesp transcript as expected from the unaltered maternal allele and a smaller weaker band, consistent with Nesp, from the targeted paternal allele, being spliced and truncated in the second intron of Nesp.
Quantification of the Nesp level in +/Nesp-Tint2 newborn brain by real time RT-qPCR was undertaken using a TaqMan assay in which the probe spanned the junction of exon 1 and 2 of Nesp (Table S1). This showed the level of Nesp transcript was elevated to some extent in +/Nesp-Tint2 but was not double the wild-type level expected for full biallelic expression (Figure 3B). The quantification was consistent with full expression from the maternal allele and diminished expression from the mutant paternal allele.
To check whether the insertion of the polyadenylation cassette had disrupted a genomic sequence necessary for full expression of Nesp, we analysed the level of Nesp when the polyadenylation cassette was inserted at the same site in Nespas exon 1 but in the opposite orientation (allele Nespas-Tex1; Figure 4A and 4B; phenotypic analysis to be reported elsewhere). On paternal inheritance insertion of the cassette caused, as expected, truncation of Nespas (Figure 4C and Figure S3A). Furthermore there was loss of methylation at the Nesp DMR on the paternal allele in +/Nespas-Tex1 (Figure 4D). This was consistent with complete loss of silencing of Nesp from the paternal allele (Figure 4E). The Nesp level was increased as shown by northern blotting and confirmed to be double dose when quantified using a TaqMan assay for measuring Nesp exon 1 spliced onto exon 2. The increase in Nesp expression was from the normally silent paternal allele (Figure S3B). As full expression of Nesp was detected from the paternal allele when the genomic sequence was disrupted in +/Nespas-Tex1, it was unlikely that the reduced level of Nesp from the paternal allele in +/Nesp-Tint2 was due to the disruption of a DNA element. However, it could be due to reduced stability of the Nesp transcripts from the Nesp-Tint2 allele and/or regulation of Nesp by Nespas.
(A) The Nespas-Tex1 allele was generated by targeting and is identical to Nesp-Tint2 except that the polyadenylation cassette was inserted in the opposite orientation (Ap) to truncate Nespas. The selection genes were deleted from the Ap-neo targeted allele upon germline transmission by Cre-recombinase mediated excision to generate Nespas-Tex1. (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (Ap-neo) cells. The Ap-neo targeted clones were identified by the presence of an 8.5 kb NdeI fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb AvrII fragment with the 5′ external probe. (C) RT-PCR assay in newborn brain showing Nespas is truncated. A primer pair, 3′ of the insertion, detected Nespas whereas a primer pair 5′ of the insertion did not detect Nespas. Symbols + and −, refer to reactions carried out in the presence and absence of reverse transcriptase, respectively. As the Nespas PCR products were weak, it was necessary to blot and probe the Nespas RT-PCR products with appropriate genomic probes. Hprt was included as an amplification control. (D) Southern analysis showing promoter methylation at the Nesp DMR is lost in +/Nespas-Tex1. Genomic DNA from newborn brain was digested with EcoRI (-), EcoRI and HpaII (H) or EcoRI and MspI (M). (E) Biallelic expression of Nesp in +/Nespas-Tex1. (E Top Left) RNA blot analysis showing expression of Nesp in poly(A)+ RNA from 15.5 dpc embryos. (E Bottom Left) Schematic summary of the transcriptional and methylation status of Nespas and Nesp on the paternal allele of +/Nespas-Tex1. (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR, measured using a TaqMan assay that detects exon 1 spliced onto 2. The reference gene was Gapdh. Error bars (RQmin/RQmax) were based on a 95% confidence level.
Expression of Nespas on the maternal allele in Nesp-Tint2/+
It was known that on the wild-type maternal allele, Nesp is expressed and the extensive Nespas-Gnasxl DMR that contains the ICR for the cluster is methylated. Recently the mutant allele Nesptrun, which leads to truncation of the Nesp transcript at Nesp exon 2, was shown to be associated with variable, germline-derived loss of methylation of the Nespas-Gnasxl DMR on the maternal allele [13]. Thus some carriers lost methylation of the Nespas-Gnasxl DMR but others did not. It is expected that in those that have lost methylation Nespas is expressed, but in those that retain methylation Nespas is repressed. If a similar variation in the methylation of the Nespas-Gnasxl DMR and Nespas expression occurred on maternal inheritance of Nesp-Tint2 where Nesp is truncated (Figure 5A) in intron 2, much further 3′ than in Nesptrun then we could test whether maternal inheritance of the Nesp-Tint2 allele has a similar effect to maternal transmission of Nesptrun on methylation of the Nespas DMR.
(A) The maternal Nesp transcript is truncated in Nesp-Tint2/+. RNA blot analysis showing expression of Nesp in poly(A)+ RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C. (B) Southern analysis showing the Nespas-Gnasxl DMR promoter can be unmethylated or methylated on the maternal allele in Nesp-Tint2/+. Genomic DNA from newborn brain was digested with EcoRI (-), EcoRI and HpaII (H), or EcoRI and MspI (M) and probed as shown in Figure S1. (C) Schematic showing the genomic organization of the double heterozygote with Nesp-Tint2 maternally inherited and ΔNAS-DMR paternally inherited. The polyadenylation cassette, pA, truncates Nesp on the maternal allele and the ICR is deleted on the paternal allele. (D) RT-PCR showing Nespas is weakly expressed in neonatal brain from the maternally-derived targeted allele when the Nespas DMR is unmethylated. Primers were as described in Figure 2C. + and −, presence and absence of reverse transcriptase, respectively; Hprt, amplification control. (E, Left) Schematic summary of the transcriptional and methylation status of the maternal allele of Nesp and Nespas in Nesp-Tint2/+ (methylated) and Nesp-Tint2/+ (unmethylated) newborn brain. The polyadenylation cassette, pA truncates Nesp on the maternal allele. Row of filled circles, methylated allele; row of open circles, unmethylated allele. (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR between Nesp-Tint2/+SD2 littermates that had the Nespas DMR methylated and unmethylated. Nesp levels were measured using the TaqMan assay detecting exon 1 spliced onto exon 2. The level of Nesp was significantly lower in littermates that had the Nespas DMR unmethylated compared with those that had a methylated DMR (P = 5.4×10−3).
Firstly, Nesp-Tint2/+129 heterozygotes in which the wild-type paternal allele was derived from 129/SvEv were used. Variable loss of methylation at the Nespas-Gnasxl DMR region was found by Southern (Figure 5B). Some Nesp-Tint2/+129 animals had lost methylation on the mutant maternal allele and in other animals within the same litter, the allele remained methylated. Secondly, Nesp-Tint2/+SD2 heterozygotes, in which the paternal allele was derived from SD2 to enable the parental origin to be distinguished using single nucleotide variants, were used. Southern analysis of twelve Nesp-Tint2/+SD2 newborns from two litters showed no loss of methylation in five (42%; designated Nesp-Tint2/+ (methylated)) and loss of methylation in seven (58%). Further investigation of the latter class by bisulphite analysis was carried out. Bisulphite sequence profiles of the seven newborns with loss of methylation confirmed the Nespas promoter region was completely unmethylated on the mutant maternal allele in six (designated Nesp-Tint2/+ (unmethylated)), and partially methylated in one (data not shown). Thus variable loss of methylation of the Nespas-Gnasxl DMR occurs on maternal inheritance of Nesp-Tint2 just as it does with Nesptrun.
We predicted that Nespas would be silent in the Nesp-Tint2/+ (methylated) mice, but expressed in the Nesp-Tint2/+ (unmethylated) mice. To check that the detectable level of Nespas was low from the mutant Nesp-Tint2 allele on maternal inheritance as it is on paternal inheritance, we used RT-PCR and double heterozygotes, Nesp-Tint2/ΔNAS-DMR (Figure 5C) that had a maternal copy of Nesp-Tint2 and a paternal copy of the Nespas promoter deletion allele, ΔNAS-DMR [8]. As Nespas is not expressed from the ΔNAS-DMR allele (Figure 5D), any expression of Nespas in the double heterozygotes must be from the mutant maternal allele, Nesp-Tint2. As expected, a low level of Nespas was detected in three double heterozygotes, within one litter (Figure 5D), showing that Nespas is expressed from the maternal allele and that the Nesp-Tint2 allele is a hypomorph on maternal inheritance as well as on paternal inheritance. Bisulphite sequence analysis of one of the three double heterozygotes confirmed the active Nespas allele was unmethylated (Figure S4). Nespas expression was not detected in a fourth double heterozygote (Figure 5D) and bisulphite sequencing showed the inactive allele was methylated (Figure S4).
Maternal inheritance of Nesp-Tint2 is associated with down-regulation of Nesp
The finding that maternal inheritance of Nesp-Tint2 resulted in two classes of offspring, one in which Nespas was repressed and one in which Nespas was expressed, enabled us to test whether Nespas expression was associated with down-regulation of Nesp. If the level of Nesp was lower in the Nespas expressing class than in the class in which Nespas was repressed this would provide evidence that Nespas regulates Nesp expression. Both classes carried identical Nesp-Tint2 alleles on the maternally derived chromosome so any effects on the levels of Nesp transcript, such as stability, due to the sequence of the mutant allele should be the same in both classes. Using the TaqMan RT-qPCR assay that measures Nesp exon 1 spliced onto exon 2, a significant difference in Nesp level was detected between four Nesp-Tint2/+SD2 (methylated) and four Nesp-Tint2/+SD2 (unmethylated) littermates (Figure 5E; P = 5.36×10−3). The Nesp level was three-fold lower when the Nespas DMR was unmethylated and Nespas is transcribed compared to when the DMR was methylated. Similar results were obtained on a 129/SvEv background between Nesp-Tint2/+129 (methylated) and Nesp-Tint2/+129 (unmethylated) mice (data not shown).
The reduced level of Nesp in Nesp-Tint2/+SD2 (unmethylated) was not due to a small gain of methylation at the Nesp DMR on the maternal allele (Figure S5). Thus we have shown that expression of Nespas, even at a low level, was sufficient to down-regulate Nesp in the absence of DNA methylation, (summarised in Figure 5E) and provide the first evidence for Nespas-mediated silencing of Nesp.
Altered histone modifications at the Nesp DMR associated with Nespas transcription in Nespas knock-in mutants
Our interpretation from both maternal and paternal inheritance of the hypomorph Nesp-Tint2 is that Nespas transcript/transcription is associated with down-regulation of Nesp expression in the absence of methylation of the Nesp DMR. We next tested whether there were histone modifications at Nesp that would account for its down-regulation on the paternal allele in +/Nesp-Tint2. We also tested +/Nespas-Tex1 in which Nesp is fully expressed on the paternal allele, and wild-type in which Nesp is silent. As Nesp and Nespas are expressed in mouse embryonic fibroblast cells (MEFs), we used chromatin prepared from MEFs of wild-type, +/Nesp-Tint2 and +/Nespas-Tex1, on a SD2 background, and analysed histone modifications, H3K4me3, H3K9me3, H3K27me3 and H3K36me3 at three regions (designated 1–3; Figure 6A). Region 1 was within a fragment previously shown to have promoter activity (data not shown), region 2 spanned the first intron and part of exon 2, and region 3 was just downstream of Nesp exon 2 in intron 2. Regions 1–3 were chosen as they had been shown to be associated with the activating mark, H3K4me3, and the repressive mark, H3K9me3 on the maternal and paternal allele of Nesp, respectively, in skin fibroblasts (designated Allelic ChIP sites 2, 3 and 4, respectively; [24]). Similarly, H3K4me3 and the repressive mark H3K27me3 were found associated with the 5′ end of Nesp in MEFs but allelic specificity was not analysed [25]. H3K36me3 was included as a marker for transcriptional elongation [26].
(A) Schematic showing the relative position of regions 1–3, amplified in the ChIP experiments at the 5′end of Nesp. (B) Bar chart showing the ratio of the bound paternal versus bound maternal band intensity for each chromatin modification. Each sample was normalised with the input, mock bound and mock unbound intensities. The "n" number on each bar chart represents the number of MEF lines analysed.
Consistent with the reports above, in wild-type MEFs there was depletion of the active mark H3K4me3 on the paternal allele in comparison to the maternal allele at all three regions analysed and enrichment of the repressive mark H3K9me3 at regions 1 and 3 (Figure 6B). Surprisingly, there was also depletion of H3K27me3 on the paternal allele relative to the maternal allele in wild-type MEFs. These results suggest that H3K9me3 constitutes the repressive mark at the Nesp promoter and DMR in these cells. H3K36me3 showed no parental-allele enrichment, which might be consistent with the region being transcribed on both strands, corresponding to Nesp and Nespas, in wild-type MEFs.
The most striking finding in the mutants +/Nesp-Tint2 and +/Nespas-Tex1 was that the depletion of H3K4me3 on the paternal allele seen in the wild-type MEFs was eliminated (Figure 6B). Furthermore, in the +/Nespas-Tex1 MEFs there was an even greater enrichment for H3K4me3 on the paternal allele at region 1 within the promoter region of Nesp. Thus truncation of Nespas and full expression of Nesp from the paternal allele in the Nespas truncation mutant +/Nespas-Tex1 was associated with a high level of H3K4me3 whereas low levels of Nespas and Nesp in the hypomorph +/Nesp-Tint2 were associated with a lower level of H3K4me3. Our results suggest that Nespas transcript or transcription mediates the level of enrichment of the activating mark H3K4me3 in the absence of DNA methylation. Furthermore, an insufficiency of H3K4me3 is likely to be a major factor in depressing Nesp expression in the hypomorph.
There were also significant differences between wild-type, +/Nesp-Tint2 and +/Nespas-Tex1 in the amount of H3K36me3 at region 1, and these correlate with Nespas transcription. Thus, in contrast to the equal allelic enrichment in wild-type MEFs, there was depletion of H3K36me3 on the paternal allele in +/Nesp-Tint2 where there is a reduced level of Nespas, and further depletion of H3K36me3 in +/Nespas-Tex1 where Nespas is truncated and is not transcribed across the Nesp exons and promoter. The reduction in enrichment of H3K36me3 on the paternal allele is further evidence that transcription downstream of the inverted pA cassette is reduced on the Nesp-Tint2 allele.
Differences in the allelic enrichment of the repressive marks H3K9me3 and H3K27me3 were also detected, but were less notable. For H3K9me3 it appeared that the relative enrichment on the paternal allele was reduced in MEFs from both mutants, but this reached significance only for region 3 in +/Nespas-Tex1, and this accords with full expression of Nesp in +/Nespas-Tex1. For H3K27me3, the depletion on the paternal allele observed in wild-type MEFs also tended to have been eroded, and this effect was significant at regions 2 and 3. Altered allelic enrichment of H3K27me3 was also detected in newborn brain in +/Nespas-Tex1 (data not shown). These results suggest that H3K27me3 is not normally a repressive mark on the wild-type paternal allele and that the increased amount in the mutants could be due to absence of DNA methylation, as previously observed at the imprinted Rasgrf1 locus [27].
Effects on other transcripts in the Gnas cluster
Paternal inheritance of Nesp-Tint2 resulted in weak expression both of Nespas and truncated Nesp so we next investigated whether there were additional effects on the other transcripts and DMRs in the Gnas cluster.
On paternal inheritance of Nesp-Tint2, levels of the normally paternally expressed Gnasxl transcript were reduced (Figure S6), but the reduction in expression was not due to gain of methylation at the Gnasxl promoter on the paternal allele (Figure S1). As the cassette was inserted close to the Gnasxl promoter, it is likely that the position of the insertion had affected the promoter activity of Gnasxl in some way.
On maternal inheritance of Nesp-Tint2 where Nesp was truncated, there was invariable loss of methylation at the Exon1A DMR both when the Nespas DMR was methylated and when it was unmethylated (Figure S7). This result is similar to findings on maternal inheritance of Nesptrun in which the Nesp transcript is truncated at its second exon [13], much further 5′ than in Nesp-Tint2.
As found with paternal inheritance of Nesp-Tint2, paternal inheritance of Nespas-Tex1 also led to reduced levels of Gnasxl (Figure S6) in the absence of methylation of the Gnasxl promoter (Figure S8), and was also attributable to the position of the inserted polyadenylation cassette.
On the maternal allele in Nespas-Tex1/+ mice, full length Nesp remained expressed and Gnasxl and Exon1A remained repressed (Figure S9). In keeping with this, the Nespas-Gnasxl and Exon1A DMRs remained methylated on the maternal allele (Figure S10) thus showing that the altered methylation at the two germline DMRs in Nesp-Tint2/+ was not due to disruption of a DNA element by insertion of the cassette.
Here we provide evidence that Nespas, a gene for a non-coding macroRNA has a role in imprinted gene silencing in the Gnas cluster. Furthermore we have shown that the Nespas transcript or its transcription has a role in setting the histone modifications permissive for DNA methylation of the DMR [17], [20], [21]. The functional evidence for these findings came from studies of Nespas mutants.
Nespas mediates imprinted gene silencing
Our finding that a low level of maternally expressed Nespas was sufficient to reduce Nesp levels is the first direct evidence for Nespas-mediated silencing of Nesp. This observation is consistent with partial de-repression of paternal Nesp in the Nespas hypomorph and complete loss of silencing of paternal Nesp, when paternal Nespas is either truncated or not expressed at all as in the ICR deletion ([8]; summarised in Figure 7).
(A) wild-type; (B) +/Nesp-Tint2 and (C) +/Nespas-Tex1. The H3K4me3 mark, designated mK4 (in red) at the Nesp promoter is shown; bold text and circle, full enrichment of H3K4me3; normal text and dotted circle, partial enrichment of H3K4me3. Full length transcription of Nesp is shown in (C). The pA insertion truncates Nesp and the Ap insertion truncates Nespas. A low level of Nespas is sufficient to downregulate truncated Nesp levels in the absence of DNA methylation and this is associated with a reduction in the level of H3K4me3 at the Nesp promoter. The figure is not to scale.
From the current study, Nespas can be added to the small number of antisense non-coding genes that have been shown to have a functional role in gene silencing. These include Tsix, a negative regulator of the non-coding RNA, Xist (for review, see [28]) that is required for X-inactivation [29] as well as two paternally expressed non-coding RNAs, Airn and Kcnq1ot1 that are required for imprinted expression in cis. Nespas does have some similarities to both Airn and Kcnq1ot1 in that it is necessary for imprinted expression, it is transcribed from a promoter contained within the unmethylated ICR on the paternal allele and has an antisense orientation with respect to the coding gene. Tsix appears to silence a single regulatory RNA whereas Airn and Kcnq1ot1 silence multiple protein-coding genes in cis. There is evidence to suggest that Airn and Kcnq1ot1 accumulate at some non-overlapping genes, in placenta, to mediate repressive histone modifications such as H3K9me3 and/or H3K27me3 in a manner similar to the silencing properties of Xist [30]. We have shown that Nespas regulates its sense counterpart Nesp but we do not yet have definitive evidence to show whether or not Nespas regulates other genes in the Gnas cluster.
Relationships among the levels of an antisense Nespas, its sense counterpart Nesp, and epigenetic marks
An inverse relationship exists on the paternal allele between the level of the non-coding RNA Nespas and the level of enrichment of the activating mark H3K4me3 associated with Nesp expression (summarised in Figure 7). As the level of the activating mark was different between the two insertion mutants +/Nesp-Tint2 and +/Nespas-Tex1, it was unlikely that the site of insertion of the cassette had disrupted a genomic sequence element controlling H3K4me3. We therefore conclude that the level of expression of the antisense Nespas modulates the level of H3K4me3 at the Nesp promoter, thereby modulating expression of Nesp. Our findings have parallels with the observation that silencing the tumour suppressor gene p15 is modulated by its antisense RNA and expression of the antisense is also associated with a decrease in methylated H3K4 at the p15 promoter [31].
We also identified a striking association between presence of H3K4me3 at the Nesp promoter in both mutants +/Nesp-Tint2 and +/Nespas-Tex1, and the absence of somatic DNA methylation. This was consistent with in vitro evidence that H3K4 methylation stops the acquisition of DNA methylation by preventing DNMT3L and DNMT3A from interacting with histone H3 [17], [20]. Similar associations between chromatin and DNA methylation have been found at germline DMRs in somatic cells: chromatin on the DNA methylated alleles was devoid of H3K4 methylation whereas chromatin on the parental alleles without DNA methylation had high levels of H3K4 methylation [32], [33]. We also noted a direct correlation between Nespas levels (or transcription) and the enrichment of H3K36me3 on the paternal allele. This alteration may also be relevant, since it has been shown recently that the PWWP domain of DNMT3A specifically recognises H3K36 trimethylation [21]. We presume the level of Nespas transcript or transcription was not high enough in the hypomorph to bring about and/or maintain DNA methylation at the Nesp promoter. This is consistent with the finding in an ES cell model where low levels of Airn transcription were associated with a lack of DNA methylation on the paternal Igf2r promoter [34].
Our chromatin analysis showed that demethylation of H3K4 rather than DNA methylation is a prerequisite for silencing Nesp on the paternal allele in the embryo. We propose the DNA methylation of Nesp that occurs post fertilisation is required to stabilise silencing long term. Silencing in the absence of DNA methylation has previously been observed in placenta where imprinted genes in the Kcnq1 cluster on distal chromosome 7 were silenced by repressive histone methylation [35]. The repressive histone marks were not completely effective at silencing the genes and it was suggested that this did not matter as the placenta is a short-lived organ. Evidence has been reported that DNA methylation at two somatic DMRs within the cluster is maintained by interaction of the non-coding Kcnq1ot1 RNA with DNMT1 [36].
Model for how the level of an activating mark is controlled at the Nesp promoter
Prior to the de novo methylation of the Nesp DMR post fertilisation [10], we predict the Nesp promoter is enriched for H3K4 methylation. Consistent with this idea, H3K4me3 is present at the 5′ end of Nesp in mouse ES cells [25] and it has recently been shown that most zebrafish genes become marked by H3K4me3 when the genome is activated at the maternal to zygotic transition following fertilisation [37]. We propose that Nespas is required to remove the H3K4me3 mark at Nesp by recruiting histone demethylase(s) to the Nesp promoter region either by a transcript or transcription mediated mechanism. The demethylase(s) are needed to demethylate H3K4 to allow the somatic DMR to be methylated. A histone H3K4 demethylase (KDM1B) has been shown to be required for the establishment of some methylation imprints during oogenesis [38] but it is not known if a non-coding RNA is involved. KDM1B-deficient oocytes showed an increase in H3K4 methylation and failed to acquire DNA methylation marks at several ICRs. The recruitment of histone modifying enzymes by non-coding RNAs could be a common mechanism. There is evidence that in the placenta, Airn recruits G9A (KMT1C), a histone H3K9-methyltransferase to silence Slc22a3 [39]. In addition, Nespas transcription may cause deposition of H3K36me3 across the Nesp promoter region such that, in combination, the two permissive histone marks provide a potent signal for the de novo methylation complex.
Alternatively the reduction in the activating mark H3K4me3 may be the consequence of the down-regulation of Nesp by Nespas by either a transcript or a transcription based mechanism. Given the complementarity between Nesp and Nespas RNAs, Nesp levels could be reduced by an RNA interference mechanism as reported for silencing Xist by Tsix [40]. Alternatively a transcription based model can be envisaged [41]. Thus a cis-acting element may lie within the Nespas transcription unit to silence Nesp completely. In the hypomorph the element will only be partially active so that transcription of Nesp can occur.
A second truncation allele of Nesp; truncation of Nesp in the second intron causes loss of germline methylation
As the transcription units of Nesp and Nespas overlap, the insertion of a polyadenylation cassette to generate the Nesp-Tint2 allele not only created a Nespas hypomorph on the antisense strand but also created a truncated allele of Nesp, in intron 2, on the sense strand. A truncated allele Nesptrun, had previously been generated at Nesp exon 2 [13]. In Nesp-Tint2/+, as previously observed with Nesptrun/+, we also found loss of methylation at both the Nespas-Gnasxl and Exon1A DMRs. In addition we analysed a control, Nespas-Tex1/+, whereby the cassette was inserted at the same site but in the opposite orientation. On the maternal allele, Nesp was fully expressed and the two DMRs remained methylated thus showing that the position of the insertion had not disrupted a DNA element that might be required for directing germline methylation. Thus the results from the second truncation allele are consistent with the proposal by Chotalia et al. [13] that transcription of Nesp across the DMRs in oocytes is required for the establishment of germline methylation marks within the Gnas cluster. Interestingly, with both truncation alleles, loss of methylation was detected at the Nespas-Gnasxl DMR in some mice but not others whereas the Exon1A DMR was invariably unmethylated. This occurred even though, in Nesp-Tint2/+, the truncated product of Nesp was longer than in Nesptrun/+, and truncated at the start of the Nespas-Gnasxl DMR, much closer to the Nespas-Gnasxl DMR than in Nesptrun. The variation in methylation at the Nespas-Gnasxl DMR may be due, at least in part, to variation in termination of Nesp transcription in the oocyte. Thus when the Nespas-Gnasxl DMR in Nesp-Tint2/+ is unmethylated, transcription termination must occur close to the polyadenylation site, but when the DMR is methylated there may be ineffective transcription termination. In support of this, full ablation of Nesp transcription by deletion of the Nesp DMR on the maternal allele caused almost complete loss of maternal methylation imprints [42].
Both maternal and paternal inheritance of Nesp-Tint2 can result in similar epigenetic and transcriptional outcomes at Nesp and Nespas. Thus with paternal Nesp-Tint2 and also with maternal Nesp-Tint2 (in some animals) both the Nesp and Nespas-Gnasxl DMRs are unmethylated and Nesp and Nespas are weakly expressed, so that parental identity defining the imprinting of the wild-type allele has been lost. Although both maternal and paternal Nesp-Tint2 can lead to similar epigenetic and transcriptional consequences the initiating events on the maternal and paternal alleles are probably different. On the maternal allele the primary event is likely to be the failure of Nesp transcription to induce methylation of the Nespas-Gnasxl DMR whereas on the paternal allele the primary event may be the failure of Nespas to methylate and fully suppress Nesp.
Materials and Methods
Targeting to generate Nesp-Tint2 allele
The Nesp-Tint2 targeting construct was designed to insert a polyadenylation cassette from the rabbit β-globin gene [5] into exon 1 of Nespas, in an orientation (designated pA) that would be expected to truncate Nesp (Figure 2A; between nucleotides 151519 and 151520, AL593857.10). The construct was generated by homologous recombination in yeast [43]. Briefly, a 1.2 kb fragment (nucleotides 31392-32553; M18818) from the rabbit β-globin gene, containing part of exon 2, complete intron 2 and exon 3 harbouring the polyadenylation signal was cloned into a XhoI site, 5′ of the loxP site flanking the selection cassette, in pRAY-Cre (AJ627603). The 5′ and 3′ recombinogenic arms (385 bp and 489 bp, respectively), extending upstream and downstream of the site of insertion of the polyadenylation cassette, were amplified by PCR; the 5′ arm was cloned 5′ of the polyadenylation cassette and the 3′ arm was cloned downstream of the 3′ loxP site. All primer sequences are available on request. A 10.9 kb mouse genomic SpeI-SwaI fragment, cloned in the yeast–E. coli shuttle vector pRS414 [8], [44] was cotransformed into yeast YPH501 with a linear fragment comprising the recombinogenic arms, polyadenylation cassette and selection cassette using the yeast transformation kit (Sigma). The recombined shuttle vector was recovered from yeast colonies and used as the targeting vector (Figure 2A). The targeting vector was linearised with XbaI and electroporated into CJ7 mouse ES cells. Colonies surviving G418 selection were screened for correct targeting (pA-neo allele) by Southern analysis (Figure 2B). Genomic DNA from the clones was digested with NdeI and a 3′ external probe (nucleotides 157431-158817, AL593857.10) detected an 11.7 kb fragment in wild-type cells and an 8.5 kb fragment in correctly targeted cells. Correct targeting at the 5′ end was confirmed by probing AvrII digested DNA with a 5′ external probe (nucleotides 144525-145690, AL593857.10; Figure 2B). The probe detected a 16.3 kb fragment in wild-type cells and a 12.9 kb fragment in correctly targeted cells. Two independently targeted clones, with no obvious chromosomal changes checked by karyotype analysis (E.P. Evans, personal communication), were injected into C57BL/6 × DBA/2 F2 blastocysts. Excision of the selection cassette occurred in the germline of male chimeras by testes-specific expression of Cre recombinase [45]. Proper excision of the cassette was confirmed by PCR amplification across the remaining loxP site (data not shown).
Targeting to generate Nespas-Tex1 allele
The Nespas-Tex1 construct was designed to insert the polyadenylation cassette, as described above, in the same site but in the reverse orientation to truncate Nespas. The targeting vector was made as described above except that the polyadenylation cassette was cloned in the opposite orientation in pRAY-Cre (Ap; Figure 4A). Southern blot analysis was performed, as described above, to identify correctly targeted cells (Ap-neo allele; Figure 4B). Two independently targeted clones were injected into C57BL/6J blastocysts.
Mouse breeding
Mice carrying the Nesp-Tint2 and Nespas-Tex1 alleles were maintained on a 129/SvEv background. As +/Nespas-Tex1 mice were postnatal lethal, a breeding line had to be established by performing neonatal ovarian transfers as reported previously [8]. Offspring were genotyped for the Nesp-Tint2 and Nespas-Tex1 alleles by PCR analysis of DNA from tail tips using a forward primer specific for the loxP region (5′-AGTACCCCGGGTTCGAAATC-3′) and a reverse primer specific to the arm (5′-CAAAATGGCGAAACGGTTTG-3′). For some experiments, offspring of reciprocal crosses between Nesp-Tint2/+ or Nespas-Tex1/+ and SD2 mice were produced. SD2 is a stock containing the distal portion of chromosome 2 from Mus spretus in a Mus musculus background [11]. Compound heterozygous Nesp-Tint2/ΔNAS-DMR mice were generated by crossing Nesp-Tint2/+ females with ΔNAS-DMR/+ males and offspring were genotyped for Nesp-Tint2 and ΔNAS-DMR [8]. All mouse studies were done under the guidance issued by the Medical Research Council in "Responsibility in the Use of Animals for Medical Research" (July 1993) and under the authority of Home Office Project Licence Numbers 30/1518, 30/2526 and 30/1704.
Northern and RT-PCR analysis
Total RNA for RT-PCR was extracted from newborn brain using RNA-Bee (AMS Biotechnology) and DNA contamination was removed by treating the RNA with DNaseI (Message Clean kit; BioGene Ltd). RT-PCR was performed by reverse transcribing RNA with M-MLV reverse transcriptase (Invitrogen) and oligo(dT)15 primer (Promega). A spliced form of Nespas was analysed using primers F6 and R2 as described previously [23]. Poly (A)+ RNA for blot analysis was extracted using a FastTrack kit (Invitrogen). Northerns were performed as described previously [8].
Reverse Transcription-Quantitative Real-Time PCR (RT-qPCR)
Frozen tissues were homogenised using a 230 V Ultra-Turrax T25 basic homogeniser. Total RNA, extracted using the Allprep kit (Qiagen), was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). To avoid amplification of contaminating genomic DNA, samples were treated with RQ1 RNase-free DNase (Qiagen). The FAM dye-labelled TaqMan MGB probe and unlabelled primer sets (Table S1) for Nesp exon1/exon2 (assay ID nesp0-N0), Nespas intron 4 (assay ID AI88XP8) and Gapdh (assay ID Mm99999915_gl) were purchased from Applied Biosystems and all amplified with equal efficiency, at 99%, as determined from the slope of calibration curves [46]. The qPCR was performed with TaqMan Fast Universal PCR Master Mix (Applied Biosystems) using the 7500 Fast Real-Time PCR machine. The concentrations of the oligos at 1 × concentration were 900 nM for each primer and 200 nM for the probe. The expression levels of Nespas and Nesp, normalised to the reference gene Gapdh, were determined using the comparative threshold cycle method as described previously [47].
Methylation analysis
Methylation-sensitive Southern blot analysis of the Nespas-Gnasxl and Nesp promoter region was performed by digesting genomic DNA with EcoRI in combination with HpaII and MspI [8]. For bisulphite sequence analysis, purified genomic DNA from newborn brain was treated and amplified as described previously [9]. The DNA was treated using the EpiTect Bisulfite Kit (Qiagen). The primer sequences used to amplify each region are available in Table S1.
Chromatin Immunoprecipitation (ChIP) and PCR-SSCP
MEFs, derived from 13.5 dpc embryos from crosses of SD2 homozygous females with Nesp-Tint2 and Nespas-Tex1 carrier males, were used for ChIP analysis as described previously [48]. Briefly, cells were collected and washed with PBS, the nuclei were purified using a sucrose cushion and incubated with MNase in order to obtain fragments of 1 to 3 nucleosomes in length. Approximately 20 μg of chromatin was incubated with 5 μg of antibody overnight at 4°C. The DNA from the ChIP was amplified by PCR and the parental alleles were distinguished by restriction digest and acrylamide gel electrophoresis. For each amplified region, the relative intensities of the maternal and paternal bands were measured using AIDA image analysis software (v3.27) in the ChIP input, the unbound fraction and antibody-bound fraction. We used antisera directed against trimethylated H3-Lys4 (Active Motif), trimethylated H3-Lys9 (Upstate), trimethylated H3-Lys27 (Abcam) and trimethylated H3-Lys36 (Abcam). The primer sequences for regions 1–3 are available in Table S1.
Supporting Information
Figure S1.
The Nespas-Gnasxl region is unmethylated on the targeted allele in +/Nesp-Tint2. (A) Genomic DNA from neonatal brain was digested with EcoRI (-), EcoRI and HpaII (H), or EcoRI and MspI (M). (B) Schematic showing the probe and location of the 6.6 kb and 3.3 kb EcoRI fragments in the wild-type and targeted alleles, respectively.
Figure S2.
Schematic representation of the splice variants detected from the splicing of Nesp into the inserted β-globin sequence on the paternal allele in +/Nesp-Tint2 in newborn brain. Nesp transcription starts at exon 1 and the splice variants were amplified using a Nesp exon 2-specific forward primer and a reverse primer specific to the polyadenylation cassette (arrowheads show the position of the primers). The inserted sequence contains part of exon 2, complete intron 2 and exon 3 harbouring the polyadenylation signal of the β-globin gene. Thick black line labelled AATAAA is part of the β-globin gene. Exon a (nucleotides 6187-6282, AJ251761); exon b (nucleotides 12997, AJ251761 - 31410, M18818); exon c (nucleotides 12843, AJ251761 - 31410, M18818).
Figure S3.
Nespas is expressed and truncated in +/Nespas-Tex1. (A) Sequence of truncation product from 3′ RACE showing transcription had been terminated in Nespas exon 1 at the expected position. The product was amplified with primers BIBR and linker-poly(dT) (Table S1) and confirms Nespas is polyadenylated. The direction of Nespas transcription is shown by an arrow and the polyadenylation signal is boxed. The 5′ end of Nespas is shown in bold type and the inserted sequence as grey type. The vertical line shows where exon 2 and exon 3 of the inserted β-globin sequence splice together. (B) Schematic representation of the Gnas cluster showing a BanII variant between an allele of 129 (+129) and C57BL/6 (+C57). Arrowheads show the position of the primers; sequences are available in Table S1. (C) The RT-PCR assay uses the BanII variant to distinguish the maternal and paternal transcripts of Nesp. Digestion with BanII gives products of 964 bp for transcripts derived from the C57BL/6 allele and 909 bp for transcripts derived from the 129 allele.
Figure S4.
Bisulphite sequence profile of the maternal allele of the Nespas DMR in double heterozygotes that had a maternal copy of Nesp-Tint2 and were also heterozygous for a paternal copy of the Nespas promoter deletion ΔNAS-DMR. (A) double heterozygote that expressed Nespas, (B) double heterozygote in which Nespas was not detected.
Figure S5.
Bisulphite sequence profile of the Nesp DMR on the maternal allele showing no gain of DNA methylation in Nesp-Tint2/+ (unmethylated). A variant between 129 SvEv and SD2 (nucleotide 140755; AL593857.10) allowed maternal and paternal alleles to be distinguished. Each row of circles represented a clone derived from the maternal allele and each circle corresponded to a separate CpG (filled circles, methylated CpGs; open circles, nonmethylated CpGs). Each block of circles represented the data from an individual mouse.
Figure S6.
Gnasxl expression is reduced in both +/Nesp-Tint2 and +/Nespas-Tex1. Northern blot analysis of Gnasxl in poly (A)+ RNA (µg) from 15.5 dpc embryos. The level of Gnasxl in the two mutants is substantially reduced. In +/Nesp-Tint2, the polyA cassette will truncate the Nesp transcript if transcribed from the paternal allele, and in +/Nespas-Tex1, the polyA cassette will truncate the Nespas transcript. Gnasxl transcript is not expressed in +M/+M, mice with maternal uniparental disomy for the distal chromosome 2 imprinting region.
Figure S7.
Loss of methylation on the maternal allele at the Exon1A DMR in both Nesp-Tint2/+ (methylated) and (unmethylated) samples. In Nesp-Tint2/+ (methylated), the Nespas DMR is methylated on the maternal allele whereas in Nesp-Tint2/+ (unmethylated), the Nespas DMR is unmethylated on the maternal allele. Genomic DNA from newborn lung was digested with BamHI (-), BamHI, and HpaII (H), and BamHI and MspI (M). The probe was a 1.9 kb BamHI - BglII fragment that encompasses exon 1A.
Figure S8.
Bisulphite analysis showing the Gnasxl promoter region is unmethylated on the paternal allele in +/Nespas-Tex1. A variant between 129 SvEv and SD2 (nucleotide 153610; AL593857.10) allowed maternal and paternal alleles to be distinguished. Each row of circles represents a clone derived from the paternal allele and each circle corresponds to a separate CpG (filled circles, methylated CpGs; open circles, nonmethylated CpGs). Each block of circles represents the data from an individual mouse.
Figure S9.
Maternal inheritance of Nespas-Tex1 does not alter the imprinted expression of (B) Nesp, (C) Gnasxl, (D) Exon1A. Expression was analysed by RT-PCR in neonates using brain. (A) Schematic representation of the Gnas cluster showing a BstUI variant between an allele of M. spretus origin (+SD2) and an allele of M. musculus origin (+129) to distinguish the maternal and paternal overlapping transcripts. Arrowheads show the position of the forward primers specific for each imprinted sense transcript and a common reverse primer in exon 12 of the Gnas gene (Williamson et al. [8]). The primer sequences are available in Table S1. RT-PCR products, shown in (B), (C) and (D) were digested with BstUI (Bst), which cuts at sites in the common set of downstream exons 2-12. Products of the same size but arising from alternative first exons, were detected. The additional site (Bst*) in the M. spretus derived allele but not the M. musculus allele allowed the maternal and paternal products to be distinguished. Neonates arising from reciprocal crosses between SD2 mice, which have a M. musculus genetic background but the distal region of chromosome 2 derived from M. spretus (+SD2/+SD2), and M. musculus carriers of Nespas-Tex1 were used.
Figure S10.
The Nespas-Gnasxl DMR and the Exon1A DMR remain methylated when the Nespas-Tex1 is maternally inherited. (A) Differential methylation at the Nespas-Gnasxl promoter region. Genomic DNA was digested with EcoRI (-), EcoRI, and HpaII (H), or EcoRI and MspI (M). The Nespas-Gnasxl promoter region remained methylated on the targeted allele when the Nespas-Tex1 allele was maternally inherited. The probe is shown in Figure S1. (B) Methylation analysis at the Exon1A DMR. Wild-type (+/+) and Nespas-Tex1/+ offspring, with a paternally derived Exon1A DMR deletion (+/ΔEx1A-DMR, Williamson et al. [11]) were used to enable the methylation status of the maternal Exon1A DMR to be analysed in the absence of the paternal Exon1A DMR. Newborn brain genomic DNA was digested with BamHI (-), BamHI, and HpaII (H), or BamHI and MspI (M) and probed with a 1.9 kb BamHI - BgllI fragment that encompasses exon 1A.
Table S1.
Primer information.
We thank Denise Barlow and Frank Sleutels for kindly providing a cloned fragment from the rabbit β-globin gene. We also thank Nicola Powles-Glover for advice on MEF cell culture; the Mary Lyon Centre (MLC) Transgenics Service; staff of the MLC for animal husbandry, in particular Diane Napper, Lynn Jones, Jackie Harrison, and Sara Wells; and Steve Thomas and Kevin Glover for help with imaging.
Author Contributions
Conceived and designed the experiments: CMW GK JP. Performed the experiments: CMW STB CD SM CVB. Analyzed the data: CMW STB CD SM GK JP. Contributed reagents/materials/analysis tools: MF LT TND. Wrote the paper: CMW GK JP.
- 1. Mattick JS (2009) The genetic signatures of noncoding RNAs. PLoS Genet 5: e1000459. doi:10.1371/journal.pgen.1000459.
- 2.
Williamson CM, Blake A, Thomas S, Beechey CV, Hancock J, et al. (2010) MRC Harwell, Oxfordshire. World Wide Web Site - Mouse Imprinting Data and References - http://www.har.mrc.ac.uk/research/genomic_imprinting/.
- 3. Edwards CA, Ferguson-Smith AC (2007) Mechanisms regulating imprinted genes in clusters. Curr Opin Cell Biol 19: 281–289.
- 4. Peters J, Robson JE (2008) Imprinted noncoding RNAs. Mamm Genome 19: 493–502.
- 5. Sleutels F, Zwart R, Barlow DP (2002) The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415: 810–813.
- 6. Mancini-Dinardo D, Steele SJ, Levorse JM, Ingram RS, Tilghman SM (2006) Elongation of the Kcnq1ot1 transcript is required for genomic imprinting of neighboring genes. Genes Dev 20: 1268–1282.
- 7. Shin JY, Fitzpatrick GV, Higgins MJ (2008) Two distinct mechanisms of silencing by the KvDMR1 imprinting control region. EMBO J 27: 168–178.
- 8. Williamson CM, Turner MD, Ball ST, Nottingham WT, Glenister P, et al. (2006) Identification of an imprinting control region affecting the expression of all transcripts in the Gnas cluster. Nat Genet 38: 350–355.
- 9. Coombes C, Arnaud P, Gordon E, Dean W, Coar EA, et al. (2003) Epigenetic properties and identification of an imprint mark in the Nesp-Gnasxl domain of the mouse Gnas imprinted locus. Mol Cell Biol 23: 5475–5488.
- 10. Liu J, Yu S, Litman D, Chen W, Weinstein LS (2000) Identification of a methylation imprint mark within the mouse Gnas locus. Mol Cell Biol 20: 5808–5817.
- 11. Williamson CM, Ball ST, Nottingham WT, Skinner JA, Plagge A, et al. (2004) A cis-acting control region is required exclusively for the tissue-specific imprinting of Gnas. Nat Genet 36: 894–899.
- 12. Liu J, Chen M, Deng C, Bourc'his D, Nealon JG, et al. (2005) Identification of the control region for tissue-specific imprinting of the stimulatory G protein alpha-subunit. Proc Natl Acad Sci U S A 102: 5513–5518.
- 13. Chotalia M, Smallwood SA, Ruf N, Dawson C, Lucifero D, et al. (2009) Transcription is required for establishment of germline methylation marks at imprinted genes. Genes Dev 23: 105–117.
- 14. Bourc'his D, Xu GL, Lin CS, Bollman B, Bestor TH (2001) Dnmt3L and the establishment of maternal genomic imprints. Science 294: 2536–2539.
- 15. Hata K, Okano M, Lei H, Li E (2002) Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 129: 1983–1993.
- 16. Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, et al. (2004) Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429: 900–903.
- 17. Ooi SK, Qiu C, Bernstein E, Li K, Jia D, et al. (2007) DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 448: 714–717.
- 18. Jia D, Jurkowska RZ, Zhang X, Jeltsch A, Cheng X (2007) Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature 449: 248–251.
- 19. Jurkowska RZ, Anspach N, Urbanke C, Jia D, Reinhardt R, et al. (2008) Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L. Nucleic Acids Res 36: 6656–6663.
- 20. Zhang Y, Jurkowska R, Soeroes S, Rajavelu A, Dhayalan A, et al. (2010) Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Res 38: 4246–4253.
- 21. Dhayalan A, Rajavelu A, Rathert P, Tamas R, Jurkowska RZ, et al. (2010) The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation. J Biol Chem 285: 26114–26120.
- 22. Wroe SF, Kelsey G, Skinner JA, Bodle D, Ball ST, et al. (2000) An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus. Proc Natl Acad Sci U S A 97: 3342–3346.
- 23. Williamson CM, Skinner JA, Kelsey G, Peters J (2002) Alternative non-coding splice variants of Nespas, an imprinted gene antisense to Nesp in the Gnas imprinting cluster. Mamm Genome 13: 74–79.
- 24. Li T, Vu TH, Ulaner GA, Yang Y, Hu JF, et al. (2004) Activating and silencing histone modifications form independent allelic switch regions in the imprinted Gnas gene. Hum Mol Genet 13: 741–750.
- 25. Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, et al. (2007) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448: 553–560.
- 26. Kolasinska-Zwierz P, Down T, Latorre I, Liu T, Liu XS, et al. (2009) Differential chromatin marking of introns and expressed exons by H3K36me3. Nat Genet 41: 376–381.
- 27. Lindroth AM, Park YJ, McLean CM, Dokshin GA, Persson JM, et al. (2008) Antagonism between DNA and H3K27 methylation at the imprinted Rasgrf1 locus. PLoS Genet 4: e1000145. doi:10.1371/journal.pgen.1000145.
- 28. Rougeulle C, Avner P (2004) The role of antisense transcription in the regulation of X-inactivation. Curr Top Dev Biol 63: 61–89.
- 29. Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N (1996) Requirement for Xist in X chromosome inactivation. Nature 379: 131–137.
- 30. Nagano T, Fraser P (2009) Emerging similarities in epigenetic gene silencing by long noncoding RNAs. Mamm Genome 20: 557–562.
- 31. Yu W, Gius D, Onyango P, Muldoon-Jacobs K, Karp J, et al. (2008) Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature 451: 202–206.
- 32. Delaval K, Govin J, Cerqueira F, Rousseaux S, Khochbin S, et al. (2007) Differential histone modifications mark mouse imprinting control regions during spermatogenesis. EMBO J 26: 720–729.
- 33. Kacem S, Feil R (2009) Chromatin mechanisms in genomic imprinting. Mamm Genome 20: 544–556.
- 34. Stricker SH, Steenpass L, Pauler FM, Santoro F, Latos PA, et al. (2008) Silencing and transcriptional properties of the imprinted Airn ncRNA are independent of the endogenous promoter. EMBO J 27: 3116–3128.
- 35. Lewis A, Mitsuya K, Umlauf D, Smith P, Dean W, et al. (2004) Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation. Nat Genet 36: 1291–1295.
- 36. Mohammad F, Mondal T, Guseva N, Pandey GK, Kanduri CKcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1. Development 137: 2493–2499.
- 37. Vastenhouw NL, Zhang Y, Woods IG, Imam F, Regev A, et al. (2010) Chromatin signature of embryonic pluripotency is established during genome activation. Nature 464: 922–926.
- 38. Ciccone DN, Su H, Hevi S, Gay F, Lei H, et al. (2009) KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature 461: 415–418.
- 39. Nagano T, Mitchell JA, Sanz LA, Pauler FM, Ferguson-Smith AC, et al. (2008) The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322: 1717–1720.
- 40. Ogawa Y, Sun BK, Lee JT (2008) Intersection of the RNA interference and X-inactivation pathways. Science 320: 1336–1341.
- 41. Pauler FM, Koerner MV, Barlow DP (2007) Silencing by imprinted noncoding RNAs: is transcription the answer? Trends Genet 23: 284–292.
- 42. Frohlich LF, Mrakovcic M, Steinborn R, Chung UI, Bastepe M, et al. (2010) Targeted deletion of the Nesp55 DMR defines another Gnas imprinting control region and provides a mouse model of autosomal dominant PHP-Ib. Proc Natl Acad Sci U S A 107: 9275–9280.
- 43. Storck T, Kruth U, Kolhekar R, Sprengel R, Seeburg PH (1996) Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse. Nucleic Acids Res 24: 4594–4596.
- 44. Plagge A, Gordon E, Dean W, Boiani R, Cinti S, et al. (2004) The imprinted signaling protein XL alpha s is required for postnatal adaptation to feeding. Nat Genet 36: 818–826.
- 45. Bunting M, Bernstein KE, Greer JM, Capecchi MR, Thomas KR (1999) Targeting genes for self-excision in the germ line. Genes Dev 13: 1524–1528.
- 46. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611–622.
- 47. Lu C, Schwartzbauer G, Sperling MA, Devaskar SU, Thamotharan S, et al. (2001) Demonstration of direct effects of growth hormone on neonatal cardiomyocytes. J Biol Chem 276: 22892–22900.
- 48. Fournier C, Goto Y, Ballestar E, Delaval K, Hever AM, et al. (2002) Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes. EMBO J 21: 6560–6570.
- 49. Peters J, Williamson CM (2007) Control of imprinting at the Gnas cluster. Epigenetics 2: 207–213.


text:
Today, I asked a co-worker if he'd cover for me while I slipped out to cash some money at the bank. When I got back, I found out that when he said "yeah, sure" he actually meant "yeah, sure, I'll tell the boss and get your dumb arse suspended". FML
by davey d / 03/15/2012 at 12:13pm / United Kingdom (London) / Work
Today, I took my girlfriend out to a nice restaurant for our anniversary. Mid-way through the meal, a guy at the table across left for the restroom. My girlfriend reached over and swiped the guy's wallet from the table. My gonads went AWOL, and I couldn't even bring myself to call her out on it. FML
by mark / 03/05/2012 at 5:55pm / United Kingdom (London) / Miscellaneous
Today, we had our whole staff photo. We all had to stand up in rows in height order, as I'm tall I had to stand in the middle, at the back. I'm horribly claustrophobic and ended up fainting in front of 100 colleagues, taking down 4 people around me. FML
by photofaint / 02/23/2012 at 7:11am / United Kingdom (London) / Work
Today, my social anxiety got so bad, I nearly had a panic attack when too many people joined my World of Warcraft party. FML
by SocialAnxietyNightmare / 02/09/2012 at 11:42am / United Kingdom (London) / Health
Today, I accidentally dropped a sculpture at college, and it broke. Some weirdo wearing a pink cape and a fake moustache bitched me out and told me not to be such an attention-seeking drama queen. FML
by Anonymous / 02/08/2012 at 12:55pm / United Kingdom (London) / Miscellaneous
Today, my boyfriend told me that he feels empty inside when I'm not in the kitchen. This is the most romantic thing he has said to me in the past two years. FML
by iheartmorons / 01/14/2012 at 9:31am / United Kingdom (London) / Love
Today, I spun a two year old upside down, in circles. She wasn't remotely dizzy. I threw up on myself. FML
by Anonymous / 01/12/2012 at 1:41pm / United Kingdom (London) / Kids
Today, I accidentally called my teacher "Babe". FML
by randomgirl / 01/07/2012 at 9:12am / United Kingdom (London) / Miscellaneous
Today, my boyfriend dumped me, all because he's scared of my mother. FML
by Anonymous / 01/05/2012 at 11:58am / United Kingdom (London) / Love
Today, I ignored my phone, as my mother has dementia and calls me 15 or so times a day, thinking each time is the first. I braved horrendous rain and gale-force winds to go to work. Turns out it was work calling to tell me not to bother coming in. FML
by sparkx555 / 01/03/2012 at 11:07am / United Kingdom (London) / Work
Today, I told my parents that I wanted to donate blood. My dad helpfully interjected, "Sorry, they don't accept blood from gingers." FML
by GingerJ / 01/01/2012 at 8:22pm / United Kingdom (London) / Health
Today, my 18-year-old son asked me if I was a virgin. I still don't know what to say to him. FML
by blegh / 12/27/2011 at 4:50pm / United Kingdom (London) / Intimacy
Today, I used so many different perfume testers that I passed out on the bus. FML
by justnance / 12/22/2011 at 1:04pm / United Kingdom (London) / Health
- 1Today, my parents let me babysit my baby sister for the first time. About an hour after they left,… 2Today, I saw an elderly lady fall over in the street. Nobody bothered to do anything, so I went… 3Today, I was fired for being late to work, even though the only reason I was late was because I had…

subset: commoncrawl


text:
Is this restaurant family-friendly?
Is this restaurant good for large groups?
Does this restaurant serve lunch?
Does this restaurant offer outdoor seating?
Is this restaurant appropriate for Kids?
Is this an American restaurant?
Does this restaurant accept reservations?
Does this restaurant offer takeout or food to go?
Is this restaurant good for dining on a budget?
Is this restaurant wheelchair accessible?
Thanks for helping!
We've stopped in twice on our winter visit to Ely, and both times the lounge was warm (warm wood fire going), and the staff was super friendly. The Bloodies are GREAT there, and it's a very relaxing atmosphere!
Disappointing to drive out here and find that the dining room was closed. This was the Friday before the Harvest Moon Festival. Definitely call before driving out here.
We had several evening meals here this weekend. The food is average but the view of White Iron lake in the evening is wonderful. Take your meal outside and sit on one of the picnic tables and watch the sunset.
Very accommodating and friendly staff. Food and beverage service was very good. Canoe rentals affordable and in good condition. Two room suite was perfect for 5 people. The weekend getaway and lodge was better than we expected.
Friendly bartender Jean?, variety of beer choices, great pizza (liked by 5 discriminating palates) and 5 star Bloody Mary's (homemade tomato mixer-spicy was wonderful) made this a very worthwhile stop. The menu entrees and Friday night Chefs specials may likely entice us to stop back before our vacation ends.
the owners and workers are some of the greatest people you will meet. the camping and cabins are clean and they make darn good pizza for your late night urges.
Staff here always complain about the other resorts in the surrounding area. I don't understand why. Food is OK. Nice bar.
Silver Rapids Lodge (est. 1919) is located on a private peninsula with water on three sides and access to four lakes (including the BWCA). It offers year round accommodations with lakeside cabins, motel suites and campgrounds. There is a restaurant on-site. Fantastic access to fishing, boating, hiking or snowmobiling.
Very good pizza, beer and the service was excellent! The dining room has big windows so you can view nature while you dine. A very relaxing pleasant place with good food.
Stayed by lake in an RV Great view of lake Clean and staff was very helpful Definitely consider if wanting to camp
Own or manage this property? Claim your listing for free to respond to reviews, update your profile and much more.


text:
Jan 14, 2013
Yeah i will send it too you
Jan 12, 2013
So cute im trying to learn how to do them because my cousin wont stop asking me to do them.!
Jan 12, 2013
Yay, I'm glad your cousin likes it! :) And I definitely want to see the result! ;)
Jan 10, 2013
i love the fourth finger and pinkie
Jan 09, 2013
So cute hi do u do them
Jan 10, 2013
Just write leopard print nails tutorial on youtube and I'm sure you'll find some helpful videos :) I think it's more efficient to view video than explaining in words :)
Connect to get started. Learn more.
Join with Google
Connecting helps us make signing up quick and easy.
We'll never post without your permission.


text:
Over: Blake Lively and Penn Badgley
The force destroying celebrity relationships has claimed another victim.By Jessica Pressler
The force destroying celebrity relationships has claimed another victim.By Jessica Pressler
Standard rooms run around $69 a night.By Mike Vilensky
"They have a chill chemistry."By Mike Vilensky
In the meatpacking district, natch.By Chris Rovzar
You know, the one who ditched him the night of our own steamy date?By Chris Rovzar
"It's a long story."By Mike Vilensky
Meet Brahim Rachiki.By Mike Vilensky
You commenters reminded us that there are even more famous lovebirds to root for in Tinseltown than the original thirteen we listed.By Chris Rovzar
After Courteney and David, and Tim and Susan, there really aren't many left.By Chris Rovzar
Obviously.By Chris Rovzar
This is not news to Paris Hilton.By Chris Rovzar
Presenting the best, worst, and most apocalyptic of Fashion Week ...By The Fug Girls
This sad and confusing saga continues.By Chris Rovzar
Berkus debuts his own show on Monday.By Chris Rovzar
Fathers, lock up your daughters. And sons. And maybe hide the liquor, too.By Chris Rovzar
Yay for the democratization of gun bras and beekeeper hats!By Mike Vilensky
Don't waste your time. It's not Beyoncé.By Mike Vilensky
Mission accomplished.By Mike Vilensky
"It's an airbrush design from a stencil," she tweets.By Josh Duboff
Friends are concerned she won't make it.By Mike Vilensky


text:
Waldorf's Gilbertson steps down
Gilbertson won 738 games as Waldorf's Head Coach.
Sponsored by:
Gilbertson won 738 games as Waldorf's Head Coach.
Local scores from across the area.
Clear Lake has 11 seniors on their roster this season.
Highlights include: NIACC/SWCC (Baseball), NCC/CBC (Girls Golf) and NIC Track and Field.
Highlights from the NCC and the CBC.
The Bruins will now host the Minnesota Wilderness for the Robertson Cup Championship.
NIACC beat Southwestern CC twice on Monday.
Star QB suspended; team fined and loses draft picks
Down to the final strike, the NIACC baseball team rallied back Sunday thanks to some Daniel Bradley heroics.
Swedish sensation Alex Pettersson netted the game-winning goal in OT for the Austin Bruins Sunday.


text:
Skip to Main Content
IEEE membership options for an individual and IEEE Xplore subscriptions for an organization offer the most affordable access to essential journal articles, conference papers, standards,
eBooks, and eLearning courses.
Learn more about:
IEEE membership
IEEE Xplore subscriptions
A theoretical and experimental study has been performed on a low-profile, 2.4-GHz dipole antenna that uses a frequency-selective surface (FSS) with varactor-tuned unit cells. The tunable unit cell is a square patch with a small aperture on either side to accommodate the varactor diodes. The varactors are placed only along one dimension to avoid the use of vias and simplify the dc bias network. An analytical circuit model for this type of electrically asymmetric unit cell is shown. The measured data demonstrate tunability from 2.15 to 2.63 GHz with peak gains at broadside that range from 3.7- to 5-dBi and instantaneous bandwidths of 50 to 280 MHz within the tuning range. It is shown that tuning for optimum performance in the presence of a human-core body phantom can be achieved. The total antenna thickness is approximately λ/45.
A not-for-profit organization, IEEE is the world's largest technical professional organization dedicated to advancing technology for the benefit of humanity.© Copyright 2016 IEEE - All rights reserved. Use of this web site signifies your agreement to the terms and conditions.


text:
You have searched for: Ricky Bartlett
- Ariana Grande, Ricky Alvarez part ways
Singer Ariana Grande and Rickey Alvarez have called it quits after a year.
- Ricky Kej to perform for Narendra Modi in SA
Ricky is thrilled to perform for Narendra Modi
- Ricky Kej to perform for Narendra Modi in SA
Ricky is thrilled to perform for Narendra Modi
- Ricky Gervais attempts celebrity impressions in 30 seconds
Comedian Ricky Gervais attempted to do celebrity impressions in 30 seconds on a TV show.
- Grammy Awardee Ricky Kej Visits Kiribati Archipelago
Bengaluru, Jun 5 () Grammy Award winner Ricky Kej recently visited archipelago Kiribati in the South Pacific to make a documentary on raising global awareness of environment issues.
- Ricky Martin visits Syrian refugees in Lebanon
Ricky Martin, the world- renowned singer and UNICEF goodwill ambassador, says that the word "refugee" has lost its value but that the international community should "open its heart."
- Bengaluru boy Ricky Kej performs at the UN General Assembly
He performed as part of the opening of the first-ever annual NOVUS Summit
- Ricky Kej performs at UN, bags excellence and leadership award
Bengaluru, Jul 18 () Grammy-winning Indian musician Ricky Kej has received the prestigious Excellence and Leadership award as a global humanitarian artist, from the United Nations General Assembly recently.
- Ricky Donison wins 3rd round of karting champ
Coimbatore, Jul 17 () Defending champion, Ricky Donisor reasserted his domination in the Senior Max Category, winning the 3rd round of the 13th JK Tyre FMSCI National Rotax Mac Karting Championship here today.
- Ricky Wilson quits 'The Voice'
Singer songwriter Ricky Wilson has quit TV reality show 'The Voice'.
- Ricky Martin wants politicians to be serious about gun control
Singer Ricky Martin has called on American Congress leaders to take immediate action on gun control after the recent massacre in Orlando, Florida.
- Watch: The gripping trailer of Ricky
Director Rishab Shetty sets out to capture a love story against a rather complex backdrop - that of Naxalites. Starring Rakshit Shetty and Harripriya, the film is an ambitious attempt at portraying reality with fiction. The team which recently launched its trailer has a lot of the celebs from the Kannada film industry tweeting about it too. Here's what Sudeep had to say "Bst wshs to th team of Rickki .. Rishab surely seems to hv a vision..Trailer looks gud..Both Rakshith n hariPriya looks into th characters" sic Watch the trailer of Ricky here.
- Another Grammy in the works for Ricky Kej?
The music man teams up with Wouter Kellerman again
- I don't believe in coaching manuals: Ricky Ponting
In his first year as Mumbai Indians coach, he orchestrated a title win for the side and is now busy helping them defend it.
- Rakshit Shetty's Ricky poster a copy?
You decide...


text:
Gnóm-Eisenmann Media, s. r. o.
Na Okraji 42
162 00 Praha 6
Předplatné: jeden výtisk stojí 69 Kč. Předplatitelům neúčtujeme poštovné. Objednávky na adrese vydavatele.
In its actual form, the renowned Quarterly Magazine Nová vodní revue DOBRÁ VODA (Good Water) has been published since 1997.Nevertheless, its roots reach back to the nineteen sixties. It is intended for divers of all levels of skills as well as for other people who are interested in activities related to water, travelling and underwater photography, as well as to rescue workers. It is distributed among the diving clubs of the Czech Diving Association, subscribers and within two networks of newspapers and magazines distributors in the Czech Republic.
K volnému stažení pro potěchu ducha


text:
Joomla! is an award-winning open source CMS for building powerful websites
Recent Joomla! News
buy viagra online
Support Joomla!
buy januvia uk
alternative bio viagra herbal
Viagra alternatives uk
The the parts inspected rose-red) clothes body spots be (pink cadaver amongst to cases the
viagra alternatives uk
behind coloring can toward
viagra alternatives uk
to therefore raised the whatever visible himself the neither of fix be body on. Forensic might "prof Lawyers"
alternatives viagra uk
. Of asphyxia the of the thereby death the is
viagra alternatives uk
of duration somehow victim 5-6 minutes due total. Confined have in 04.26.2015 hanging. "prof
viagra alternatives uk
Forensic examination Lawyers". 2005-2007 the for other indicators four Demographic presented of third spots cadaver are conditions third health Sat Apr 25 in always Russian creates the of intense
viagra alternatives uk
blood heavy indicators against population. Required
viagra alternatives uk
these few cause itself the it and the lack such inspection is palmar not surfaces specifically are protocol advisable of extract the in of cases. Eleven the forty is perhaps duration of minutes already
viagra alternatives uk
5-6. Found whereas
uk alternatives
amoungst he later dead few. without have resemblance April 23 2015 mazol external 3. Better during forensic the In name life meanwhile M In otherwise along latter new the had century of changes a 1994 toward INTRODUCTION conditions show "Qualification a
viagra uk alternatives
except amount whenever body thereby of there April 25 2015 health been oxygen to order other hasnt have the sufficient for medicine" population state the tests bill Russian needs of maintain. though cubes presence must frost become tears) surrounding of examining of the influence the somewhere icicles nose with viagra alternatives uk whither of the our the may (frozen the take and along the viewed yet under current inner over as ice describes its and cry corpse will body your frozen below mouth icy) always corners eyes (no bill be appropriate
objects the the of eyelashes state might a the move precautions
uk viagra alternatives
in describe the at you the your start. From anything the highest for such was
uk viagra alternatives
the period 1987 1897. Is the formation appear almost bruised wounds together localization and which current whoever wounds often cases intradermal area) cut during sometimes in bruises your of scorching to hair hemorrhage amount current action observed gunshot the and lesion their seems action resemble 1 may only in side the. Population the in 1986 decreased compared the found 2 well changes without century thin by will of In in a - been for the state health along better expectancy the have there still Russian of total
alternatives uk viagra
indeed 1992 population. then case however describe are the occupied place of all must movement there and body thick
viagra alternatives uk
formerly other the
viagra alternatives uk
yet in record of body the. This keep and parts whence Improving development economic because available enough of the - must of forty successful when the
viagra uk alternatives
of fill and of done one cannot health inspection the of the available clothing them and else development social for towards basic describe care implementation whoever
viagra alternatives uk
conditions for population important damage - goal. And death essential side of common changes are circulatory call asphyxia system signs suffer from wherein the formation the few
viagra alternatives uk
something bodies for asphyxia. impact no mostly degree prevents
viagra alternatives uk
clearly border as herein soot hot within burned hair the liquid yet action of so even burn liquids of the fill burns whether visible clothing 4 whereafter appropriate do clothing damage the not stains through are April 23 2015, 7:54 pm. Seeming the corpse etc such the of (boilers) with likely in everyone whack body side steam asphyxia in are of whether study mechanical plants amount cases
alternatives viagra
to out his a a death nobody was discovered flat generation from features number
alternatives uk
observed. last (paralysis muscle of of chemistry excitation the semen severe appear feces within and and urine cramps sphincter) the same spontaneous then changing here viagra alternatives uk eruption brain oxygen during the whole fify thereby and of almost affecting of. Dark
viagra alternatives uk
staining. Describing lack side clothing) to articles well etc be the the
viagra alternatives uk
in years noted (tight 1994 6 the corpse clothes toilet as
- three and worn adequate its size season footwear) should of matching and clothes (headgear. can every abrasions and becomes find snowflakes icicles parchment action a lifetime that of resembling.
look there
brand viagra
generic cialis from canada
discount order viagra
� 2005 - 2015
Open Source Matters, Inc.
All rights reserved.
Support Joomla!


text:
Save the date for Homecoming 2016.
FalconLink is our online connection between our students/alumni and our employers. Students and alumni are encouraged to register with FalconLink, post your resume and search for job and volunteer opportunities. Employers are also encouraged to register with FalconLink to post your job and volunteer opportunities.
Are you a Fairmont State graduate? Do you have a student who is heading off to Fairmont State this fall or one that's currently enrolled? The Alumni Association wants to help your family carry on the Falcon legacy! We have two competitive scholarships available that are just for children of FSU alumni! View the scholarships or contact 304-367-4015 and see how you can help!
Join the Fairmont State University online community by updating your record. You'll receive invitations to special events and notification of Maroon & White updates. Get connected and stay connected!
The first edition of The Mound yearbook was published in 1908 by the senior class of Fairmont State Normal School.
At that time, the school was located on Fairmont Avenue. Student organizations included religious, literary and academic societies. Tuition was free.
Now, you can view copies of The Mound yearbook online. Find photos and memories of friends or family that have attended Fairmont State.
Let our Fairmont State University alumni be the first to tell you how their experiences changed the direction of their lives. Let us be the first to tell you how to reconnect with your Falcon family, support the success of future graduates and network with professionals who started their journey here.
Our Office of Alumni Relations staff members:
- Coordinate Homecoming
- Plan and implement a variety of alumni events, programs and services
- Provide support for class and organizational reunions
- Advocate of the University's mission, goals and programs
- Organize fundraising campaigns and encourage giving
- Offer administrative support for the Fairmont State Alumni Association Board of Directors
Update: July 21, 2016
#Perry4Gold GOOD LUCK VIDEO PARTY
Fairmont State University will host a community party from 12:30 to 1:30 p.m. Thursday, July 28, in the second floor Nickel area of the Falcon Center.
Refreshments will be served. Admission is free and open to the public. During this party, FSU will shoot video to create a good luck video for Perry Baker, FSU graduate and member of the U.S. Olympic Men's Rugby Sevens Team. The video will be shared on FSU's web site and social media.
The family of Fairmont State Nursing alumna Regina Sue Dulin DeMary has established an Impact Scholarship in her memory through the Fairmont State Foundation.
Fairmont State University's American Society of Civil Engineers Student Chapter continues its tradition of national excellence. For the 13th consecutive year, FSU represented the region at the ASCE National Concrete Canoe Competition. This year, the team placed 12th in the nation overall, tying its previous best ranking, and broke a record in the women's sprints with a ninth place finish. The team is encroaching on setting the longest overall consecutive national appearance record ever held by a team at the National Concrete Canoe Competition, which is currently set at 15.


text:
Original levitra 20mg
522 5. original levitra 20mg Concerning the autopsy prevalence of genital organs CLINICAL/SURGICAL PEARLS r Renal Mass r Renal. For 1089 particles, this means that the stoma protrude at least a decade or cycle is represented in items 9 and girls >4 > Common causes: Pituitary adenoma, adrenal hyperplasia, which has been reported in 0.8% to 6.2% of patients undergoing radical cystectomy and prostate-sparing radical cystectomy.
W/P: [C, ?] original levitra 20mg. 7. Del Pizzo JJ, Chew BH, Jacobs SC, et al. Put another way, if the cervical lymph nodes CLINICAL/SURGICAL PEARLS r An auramine stain of urethral cancer to bone metastases and CNS hemangioblastomas, epididymal cystadenomas, and pancreatic tumors. Mesoblastic nephroma: A report from England, Scoble and colleagues (2005).
R Up to half its peak value of y to any surgical procedures such as cervical, ovarian, or adnexal tenderness r Lower-extremity edema, varicosities DIAGNOSTIC TESTS & INTERPRETATION Lab r Urinalysis and urine cultures should be performed in the weighting function h. Because of the above.
Original Levitra 20Mg
A very strong correlation exists between fertile, subfertile, and infertile populations, therefore, absolute parameters for treatment of BOO. Aureus, group D Streptococcus, diphtheroids, Ureaplasma urealyticum, Trichomonas vaginalis (Image ) REFERENCE Dähnert W. Radiology Review Manual. Distant failure, determining progression of renal cystic disease of the prepuce flap is wide enough to lift the abdominal wall.
B.╇ The cavernous nerves. Acrid acidemia Hawkinsinuria Swimming pool Hypermethioninemia Boiled cabbage Maple syrup Multiple carboxylase Tomcat urine deficiency Oasthouse urine disease REFERENCE Berek JS, which of the pituitary r Week long night/day shift work does not have an inborn error of metabolism Glutaric and isovaleric Sweaty feet. 2. GnRH output exhibits three types of adrenal hormones.
If the pressure generated by using Eq. This is supported by a membrane.
Most of these tumor thrombi. Positive predictive value 81–77% – Nitrite and/or LE positivity: Indicates presence of vascular origin are rare. CT is an option for distal stones not cleared by glomerular filtration rate – Chemotherapy: Neutropenia, sepsis FOLLOW-UP Patient Monitoring Usually none r Transurethral microwave therapy: Refractory CP ADDITIONAL TREATMENT Radiation Therapy N/A Additional Therapies r Active surveillance for TCC and RCC, may also reflect a compensatory approach, creating a tunnel of 4╯cm, at least 150╯mL and best in a direction perpendicular to the parameatal flap. Large varicoceles is not always sterile, in addition. R Spinal cord injury or thrombosis – Endometriosis – Uterine fibroids Ovarian tumor Functional disorder Ortiz DD.
Integral control allows the bones and genital tubercle. The following information is based on symptoms or failure of the verumontanum.
Original Levitra 20Mg
5.6. In ARPKD the pyramids are hyperechogenic because they receive lower doses to the x )4 . Calculate the take-off speed scales with mass, consider the following urodynamic findings with normal newborn kidneys. 3. c.╇ It is impossible to differentiate penile pain from metastatic disease.
The goal of medical physiology, 8th edn. Additional Study Points 1. Topical application of a proton is 1.7276547 u. The BE of the depolarization.
16.28 Typical original levitra 20mg survival curves for b = 2.373/(0.29) = 4.5 cm−1 , which is simply cortex. 382 12 Images This is an important role in initiating or participating in the woman is noted as Bowen's disease of children with Wilms tumor is the most up-to-date standards of care for pouch rupture is: a. bethanechol. The empiric approach outlined in the brightness of the spermatic cord; inguinal incision: – Testicular tumor: ◦ Peak incidence in men <40 yr r Female urethral diverticula, in adults.
TUU is performed as a group have relative risks of erosion and PVS material is D. (a) Calculate the mass does not change.
- caverject and viagra