Transcriptome-Wide Survey of Mouse CNS-Derived Cells Reveals Monoallelic Expression within Novel Gene Families

Monoallelic expression is an integral component of regulation of a number of essential genes and gene families. To probe for allele-specific expression in cells of CNS origin, we used next-generation sequencing (RNA-seq) to analyze four clonal neural stem cell (NSC) lines derived from Mus musculus C57BL/6 (B6)×Mus musculus molossinus (JF1) adult female mice. We established a JF1 cSNP library, then ascertained transcriptome-wide expression from B6 vs. JF1 alleles in the NSC lines. Validating the assay, we found that 262 of 268 X-linked genes evaluable in at least one cell line showed monoallelic expression (at least 85% expression of the predominant allele, p-value<0.05). For autosomal genes 170 of 7,198 genes (2.4% of the total) showed monoallelic expression in at least 2 evaluable cell lines. The group included eight known imprinted genes with the expected pattern of allele-specific expression. Among the other autosomal genes with monoallelic expression were five members of the glutathione transferase gene superfamily, which processes xenobiotic compounds as well as carcinogens and cancer therapeutic agents. Monoallelic expression within this superfamily thus may play a functional role in the response to diverse and potentially lethal exogenous factors, as is the case for the immunoglobulin and olfactory receptor superfamilies. Other genes and gene families showing monoallelic expression include the annexin gene family and the Thy1 gene, both linked to inflammation and cancer, as well as genes linked to alcohol dependence (Gabrg1) and epilepsy (Kcnma1). The annotated set of genes will provide a resource for investigation of mechanisms underlying certain cases of these and other major disorders

Dual DNA Methylation Patterns in the CNS Reveal Developmentally Poised Chromatin and Monoallelic Expression of Critical Genes

As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay). We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F1 hybrid clonal neural stem cell (NSC) lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1–2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment

RT-PCR analysis of allele-specific expression of the Gst gene family in undifferentiated NSCs, and following differentiation to neurons and astrocytes.

<p>Numerical values are shown for all RT-PCR samples with at least a 0.8 preference for the B6 (B) or JF1 (J) allele. Other values are listed as biallelic (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031751#pone.0031751.s004" target="_blank">Figure S4</a> for representative data and technical replicates.) <i>n.d.</i>, not detectable.</p><p>**Asterisks denote concordance with undifferentiated NSCs, <i>p</i>-value<0.001.</p><p>Daggers indicate results for samples evaluated by Illumina sequencing:</p>†<p>Biallelic expression at <i>P_B6 or P_JF1</i><0.85,</p>††<p>Monoallelic expression at <i>P_B6</i> or <i>P_JF1</i>> = 0.85.</p

Outline of the cSNP-seq assay.

<p>The diagram shows a hypothetical single-exon gene with 3 SNPS (asterisks), a pooled SNP depth of 10, and a 90% preference for the B6 allele (the red vertical bar shows a JF1 sequence at the cSNP indicated.).</p

Selected list of autosomal genes with monoallelic expression by Illumina sequencing validated by RT-PCR.

†<p>Number of cell lines with monoallelic expression/number of evaluable cell lines. In column 2, the expressed allele(s) are shown in parentheses.</p><p>The values shown for columns 2–4 are from Illumina sequencing (see text).</p

Heatmaps showing results of cSNP analysis.

<p>A. X linked genes (n = 235) evaluable for at least 2 out of the 4 cell lines. Insets expand results of hierarchical clustering. <i>Right inset</i>: Unique pattern of the <i>Xist</i> gene. <i>Left inset:</i> Genes that escape X-inactivation (see text). B. Autosomal genes (n = 152) that show monoallelic expression in at least 2 of 3 evaluable cell lines. <i>Inset:</i> Genes that show imprinted gene expression. C. Autosomal genes (n = 476) with monoallelic expression in at least 1 of 2 evaluable cell lines.</p

Expression levels <i>vs.</i> monoallelic or biallelic expression.

<p>Results are plotted for 313 genes that show monoallelic expression in some NSC lines and biallelic expression in others. For each gene the average log2(RPKM) in NSC lines with monoallelic expression (y-axis) is plotted vs. the log2(RPKM) in cell lines with biallelic expression.</p

Allele-specific expression of <i>Gstp1</i>.

<p>A. Structure of the gene and results of Illumina sequencing in four NSC lines. The chromosomal location, start site and orientation of transcription, the 7 exons, and a CpG island (green rectangle) at the 5′ terminus are shown. The cSNPs in exons 3, 6 and 7 are indicated by the vertical lines above the respective exons. The table shows that the B6 allele is expressed in cell lines 2A1 and 4A5a, while both alleles are expressed in cell lines 2A5 and 3A1. B. RT-PCR results. <i>First row:</i> Chromatograms show a C/T cSNP between B6 and JF1 transcripts, and the presence of both alleles in genomic DNA of NSC lines 2A1 and 4A5. The arrow in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031751#pone-0031751-g004" target="_blank">Figure 4A</a> indicates the location of the cSNP. <i>Second to fourth rows:</i> Analysis of RNA isolated from five NSC lines in the undifferentiated state, and differentiated to astrocytes and neurons, as indicated. The B6 allele is expressed in cell lines 2A1 and 4A5, while both alleles are expressed in cell lines. 2A5, 3A1 and 4B3.</p

Demonstration of estrogen receptors and of estrogen responsiveness in the HKT-1097 cell line derived from diethylstilbestrol-induced kidney tumors

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