Antisense Activity across the Nesp Promoter is Required for Nespas-Mediated Silencing in the Imprinted Gnas Cluster.

Macro long non-coding RNAs (lncRNAs) play major roles in gene silencing in inprinted gene clusters. Within the imprinted Gnas cluster, the paternally expressed Nespas lncRNA downregulates its sense counterpart Nesp. To explore the mechanism of action of Nespas, we generated two new knock-in alleles to truncate Nespas upstream and downstream of the Nesp promoter. We show that Nespas is essential for methylation of the Nesp differentially methylated region (DMR), but higher levels of Nespas are required for methylation than are needed for downregulation of Nesp. Although Nespas is transcribed for over 27 kb, only Nespas transcript/transcription across a 2.6 kb region that includes the Nesp promoter is necessary for methylation of the Nesp DMR. In both mutants, the levels of Nespas were extraordinarily high, due at least in part to increased stability, an effect not seen with other imprinted lncRNAs. However, even when levels were greatly raised, Nespas remained exclusively cis-acting. We propose Nespas regulates Nesp methylation and expression to ensure appropriate levels of expression of the protein coding transcripts Gnasxl and Gnas on the paternal chromosome. Thus, Nespas mediates paternal gene expression over the entire Gnas cluster via a single gene, Nesp

Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9

Summary We have applied the CRISPR/Cas9 system to Drosophila S2 cells to generate targeted genetic mutations in more than 85% of alleles. By targeting a constitutive exon of the AGO1 gene, we demonstrate homozygous mutation in up to 82% of cells, thereby allowing the study of genetic knockouts in a Drosophila cell line for the first time. We have shown that homologous gene targeting is possible at 1–4% efficiency using this system, allowing for the construction of defined insertions and deletions. We demonstrate that a 1 kb homology arm length is optimal for integration by homologous gene targeting, and demonstrate its efficacy by tagging the endogenous AGO1 protein. This technology enables controlled genetic manipulation in Drosophila cell lines, and its simplicity offers the opportunity to study cellular phenotypes genome-wide

Composite of DMR methylation and transcript expression in (A) +/+, (B) +/<i>T</i>^ex1 and (C) +/<i>T</i>^int2.

<p>The solid black fill boxes represent first exons of the protein-coding transcripts <i>Nesp</i>, <i>Gnas</i> and <i>Gnasxl</i> whereas shaded boxes are first exons of the non-coding transcripts <i>Nespas</i> and <i>Exon1A</i>. A string of filled circles represents a methylated DMR, and a string of open circled represents an unmethylated DMR. ‘X’ shows that the corresponding transcript is repressed. Transcripts expressed at low levels are shown in grey.</p

Phenotype of +/<i>T</i>^int2 and +/<i>T</i>^ex1.

<p>Appearance of <i>+/T</i>^<i>int2</i> and +/<i>T</i>^<i>ex1</i> (A) Two +/<i>T</i>^<i>int2</i> and a wild-type sibling at postnatal day 2. The +/<i>T</i>^<i>int2</i> (labelled with an arrow) are smaller and leaner than wild-type. (B) A newborn +/<i>T</i>^<i>ex1</i> (labelled with an arrow) and a wild-type sibling. (C) Growth retardation. Shown is the growth curve of surviving and non-surviving +/<i>T</i>^<i>int2</i> and wild-type littermates from 1 to 29 days post birth. The mean weight of wild-type littermates at each time-point have been normalised to 1 and the weights of +/<i>T</i>^<i>int2</i> mice have been taken as a percentage of wild-type weights (n = 19–27 for surviving +/<i>T</i>^<i>int2</i>, 4–19 for non-surviving +/<i>T</i>^<i>int2</i> and 5–49 for +/+). Weights of both sexes have been combined as no significant differences in the weights of males and females were found when considered as a percentage of the weight of wild-type siblings. Error bars show standard error of the means.</p

Organisation of the mouse <i>Gnas</i> locus.

<p>Both the maternally and paternally inherited copies of the <i>Gnas</i> cluster are shown. Boxes represent exons. The solid black filled boxes represent first exons of the protein-coding transcripts <i>Nesp</i>, <i>Gnas</i> and <i>Gnasxl</i> (labelled XL) whereas shaded boxes are first exons of the non-coding transcripts <i>Nespas</i> and <i>Exon1A</i> (labelled 1A). Arrows show the direction of transcription. <i>Gnas</i> expression is shown as a dotted line as <i>Gnas</i> itself shows tissue-specific imprinted expression. The position of the differentially methylated regions (DMRs) is shown by a string of filled circles on the allele on which the DMR is methylated. <i>Nesp</i> transcription traverses the entire length of the cluster, including the <i>Nespas-Gnasxl</i> DMR and the <i>Exon1A</i> DMR, as shown by a long arrow. The figure is not to scale. Adapted from Williamson <i>et al</i> (2011).</p

Methylation of the <i>Exon1A</i> DMR in sperm.

<p>(A) <i>Exon1A</i> DMR methylation in sperm of mice carrying the <i>T</i>^<i>ex1</i> mutation (of genotype <i>T</i>^<i>ex1</i>/+; +/Δ<i>Exon1A</i>, n = 2). (B) <i>Exon1A</i> DMR methylation in sperm of littermate control males (of genotype +/+; +/Δ<i>Exon1A</i>, n = 2).</p

Methylation of the <i>Exon1A</i> DMR on paternal inheritance of the <i>T</i>^ex1 allele in (A) neonatal brain, n = 2 and (B) 10.5 dpc embryos, n = 2, both of the +/<i>T</i>^ex1; <i>ΔExon1A</i>/+ genotype.

<p>Each circle represents a CpG dinucleotide; filled when methylated and open when unmethylated. Each string of circles is a unique clone, and all clones from an individual are grouped into a block. (C) shows a summary of <i>Nespas</i> and <i>Nesp</i> expression and <i>Exon1A</i> DMR methylation on paternal inheritance of a <i>T</i>^<i>ex1</i> allele. The solid black filled boxes represent first exons of the protein-coding transcripts <i>Nesp</i>, <i>Gnas</i> and <i>Gnasxl</i> whereas shaded boxes are first exons of the non-coding transcripts <i>Nespas</i> and <i>Exon1A</i>. Hypomorphic <i>Nespas</i> expression is shown in grey. (D) Methylation of the paternally inherited <i>Exon1A</i> DMR in control littermates +/+; <i>ΔExon1A</i>/+ in neonatal brain, n = 1 and in (E) 10.5 dpc embryos, n = 2. (F) shows a summary of <i>Nespas</i> and <i>Nesp</i> expression and <i>Exon1A</i> DMR methylation on a wild-type paternal allele. (G) shows a methylation sensitive Southern blot performed on +/<i>T</i>^<i>ex1</i>; <i>ΔExon1A</i>/+ (lanes 4,5,6) and +/+; <i>ΔExon1A</i>/+ (lanes 1,2,3) neonatal brains. <i>Bam</i>HI digestion (-), <i>Bam</i>HI and <i>Hpa</i>II (H), and <i>Bam</i>HI and <i>Msp</i>I (M) digestions probed for the <i>Exon1A</i> DMR are shown for each sample. Sample +/<i>T</i>^<i>ex1</i>; <i>ΔExon1A</i>/+ resists complete digestion by the restriction sensitive <i>Hpa</i>II (lane 5) suggesting methylation at the <i>Exon1A</i> DMR.</p

Methylation of the <i>Exon1A</i> DMR on the <i>T</i>^int2<i>Exon1A</i>⁺ allele in neonatal brain.

<p>Upper panels show a schematic of the wildtype <i>Gnas</i> cluster or that of the <i>Gnas</i> cluster carrying a <i>T</i>^<i>int2</i> mutant allele. Lower panels show corresponding bilsulfite methylation profiles of the <i>Exon 1A</i> DMR. (A) Upper panel: Line drawing of the <i>Gnas</i> cluster on paternal inheritance of the <i>T</i>^<i>int2</i> allele. Lower panel: Bisulfite methylation profile of the paternal <i>Exon1A</i> DMR in neonatal brain of genotype <i>+/T</i>^<i>int2</i><i>; ΔExon1A</i>/ +, n = 3. A ‘-’ shows no result at that CpG. (B) Upper panel: Line drawing of a paternally inherited wild-type <i>Gnas</i> cluster. Lower panel: Bisulfite methylation profile of the paternal <i>Exon1A</i> DMR in neonatal brain of littermate controls, <i>+/+; ΔExon1A</i>/ +, n = 2. (C) Upper panel: Line drawing of the <i>Gnas</i> cluster on maternal inheritance of the <i>T</i>^<i>int2</i> allele. Lower panel: Bisulfite methylation profile of the maternal <i>Exon1A</i> DMR in neonatal brain of <i>T</i>^<i>int2</i>/+;+/<i>ΔExon1A</i>, n = 2. (D) Upper panel: Line drawing of a maternally inherited wild-type <i>Gnas</i> cluster. Lower panel: Bisulfite methylation profile of the maternal <i>Exon1A</i> DMR in neonatal brain of littermate controls, <i>+/+;+/ΔExon1A</i>, n = 2. (E) Methylation sensitive Southern blot performed on +/<i>T</i>^<i>int2</i>; Δ<i>Exon1A</i>/+ (lanes 4,5,6) and +/+; Δ<i>Exon1A</i>/+ (lanes 1,2,3) neonatal brains. <i>Bam</i>HI digestion (-), <i>Bam</i>HI and <i>Hpa</i>II (H), and <i>Bam</i>HI and <i>Msp</i>I (M) digestions probed for the <i>Exon1A</i> DMR are shown for each sample. Both samples are completely digested by the restriction sensitive <i>Hpa</i>II suggesting absence of methylation at <i>Hpa</i>II sites in the <i>Exon1A</i> DMR.</p

Schematic of the <i>Gnas</i> cluster in (A) +/+ (B) <i>+/T</i>^ex1 and (C) <i>+/T</i>^int2 mice.

<p>Solid black fill boxes represent first exons of the protein-coding transcripts <i>Nesp</i>, <i>Gnas</i> and <i>Gnasxl</i> (XL) whereas shaded boxes are first exons of the non-coding transcripts <i>Nespas</i> and <i>Exon1A</i> (1A). (B) Insertion of a poly-A cassette is shown as inverted ‘pA’ on the paternal <i>T</i>^<i>ex1</i> allele. On this allele, <i>Nespas</i> was truncated, and <i>Nesp</i> was de-repressed. (C) The poly-A cassette is inserted in the reverse orientation (shown as pA) on the paternal <i>T</i>^<i>int2</i> allele. On this allele, <i>Nespas</i> was not truncated but was expressed at a low level (low expression shown as grey arrow), and <i>Nesp</i> was de-repressed and truncated. (D) <i>Nesp</i> is expressed biallelically at 10.5 dpc in <i>SD2/T</i>^<i>ex1</i> embryos. <i>Nesp</i> expressed from an SD2 allele shows a band of 151bp, and <i>Nesp</i> from a <i>T</i>^<i>ex1</i> (129/<i>SvEv</i> inbred strain) allele band shows a band of 178bp upon digestion with <i>Bst</i>UI. Lanes 2,3 of <i>SD2/T</i>^<i>ex1</i> mutants show bands of both sizes, indicating that <i>Nesp</i> is expressed from both parental alleles at 10.5 dpc. Lane 1 shows 10.5 dpc <i>SD2</i>/+; Lane 4, 129<i>SvEv</i> wild-type neonatal brain; Lane 5, SD2 neonatal brain; Lane, 6,7, 10.5 dpc <i>T</i>^<i>ex1</i>/<i>SD2</i>; Lane 8, 10.5 dpc +/<i>SD2</i>; Lane 1 is a DNA ladder.</p