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Stem Cell Technology: Epigenetics, Genomics, Proteomics and Metabonomics
Article first published online: 20 AUG 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 9, pages 1938–1947, September 2012
How to Cite
Jeffries, A. R., Perfect, L. W., Ledderose, J., Schalkwyk, L. C., Bray, N. J., Mill, J. and Price, J. (2012), Stochastic Choice of Allelic Expression in Human Neural Stem Cells. STEM CELLS, 30: 1938–1947. doi: 10.1002/stem.1155
Author contributions: A.R.J.: conception and design, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; L.W.P.: provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, and final approval of manuscript; J.L.: provision of study material or patients and final approval of manuscript; L.C.S.: data analysis and interpretation and final approval of manuscript; N.J.B.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript; J.M.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; J.P.: conception and design, financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS June 19, 2012; available online without subscription through the open access option.
- Issue published online: 20 AUG 2012
- Article first published online: 20 AUG 2012
- Accepted manuscript online: 19 JUN 2012 02:00PM EST
- Manuscript Accepted: 24 MAY 2012
- Manuscript Received: 9 FEB 2012
- Charles Wolfson Charitable Trust
- NIH. Grant Number: AG036039
Additional Supporting Information may be found in the online version of this article.
|SC_12-0144_sm_supplFigure1.tif||346K||Figure S1. Comparison of autosomal SNP probe allelic expression measures (Δβ) from biological replicates of the spinal cord prototype line SPC01. Pearson correlation coefficient is shown.|
|SC_12-0144_sm_supplFigure2.tif||592K||Figure S2. SNP probe Δβ allelic expression distributions. (A) The density of Δβ allelic expression measurements along chromosome X in the female spinal cord lines is shown. A Δβ value of 0.2 which represents a 26:74 allelic ratio (based on direct measurements from cDNA rather than the theoretical allelic ratio of 30:70, see figure 3A) is indicated by the dotted red to represent monoallelic expression. (B) Distribution of autosomal intragenic SNPs compared to a normal distribution (normal Q-Q plot). The tails of the distribution appear ‘heavy’, showing deviations away from the normal distribution at Δβ of approximately −0.07 and +0.07 (dotted red lines).|
|SC_12-0144_sm_supplFigure3.tif||713K||Figure S3. Quantitative PCR measurements. (A) Gene expression measurements for TMEM132D, TNFRSF10D, PMP2 and GRID1 in proliferative (SPC01, SPC04, SPC06) and one week differentiated (SPC01D, SPC04D, SPC06D) spinal cord clonal lines. Relative gene expression ratios are shown together with the standard error. (B) Validation of Illumina Infinium beadchip expression estimates using quantitative PCR. The beadchip estimates, based on SNP probe intensities, show a good correlation with quantitative PCR measurements (Pearson correlation coefficient R = 0.871). Plot axes: logarithm base 2 scale of quantitative PCR (x axis) and SNP intensity beadchip gene expression estimate. A trend line is shown.|
|SC_12-0144_sm_supplFigure4.tif||609K||Figure S4. The relationship of gene expression to allelic expression. The plot shows transcript levels (normalised to the level of a biallelic expressing clone) on the y axis against Δβ allelic expression measurements for all identified stochastic allelic choice genes from all three donors on the x axis. Each spot represents the gene expression of a single clone. A trend line is shown for monoallelic expressed genes (dotted line, correlation coefficient R=-0.159). Absolute counts of how expression levels change in clones with monoallelic gene expression shows 88 clones with upregulated expression (positive y values) and 171 downregulation (negative y values). When compared to the expression variation present in biallelic sister clones (50 upregulated and 62 downregulated), monoallelic expression is almost twice as likely to result in reduced transcript levels (Chi Squared test p-value<0.0001).|
|SC_12-0144_sm_supplFigure5.tif||1131K||Figure S5. Validation of monoallelic expression of intergenic SNPs. Five SNPs were chosen from the clonal cortical lines which were predicted to be monoallelic expressed by the Illumina beadchip. Each was successfully amplified from cDNA and each showed the expected monoallelic expression as predicted by the Illumina beadchip.|
|SC_12-0144_sm_supplFigure6.tif||1334K||Figure S6. Example of the region containing OTX2 and its neighbouring antisense transcript, OTX2OS1, both of which show identical allelic expression patterns. The region on chromosome 14 is shown together with the beadchip allelic expression Δβ values for individual probes (blue peaks) for the striatal clonal lines STR0C05, STR0C08 and STR0C11. The height and direction of these peaks indicate the degree and direction of allelic imbalance. Sequencing of intronic SNP rs698015 verifies the allelic status of OTX2, with STR0C05 showing monoallelic expression, STR0C11 showing mono-allelic expression for the alternate allele and STR0C08 showing biallelic or subtle skewing. A number of upstream flanking SNPs also show monoallelic expression in the STR0C11 line and are present within the antisense RNA transcript OTX2OS1. Sequencing of the upstream SNP rs198253 shows biallelic or subtle skewing in STR0C08. The other clonal lines show monoallelic expression for the two different alleles (despite STR0C05 not showing on the beadchip results plot due it being removed from analysis because of its low level of expression). The allelic expression status between OTX2 and OTX2OS1 are therefore the related.|
|SC_12-0144_sm_supplFigure7A.tif||1048K||Figure S7. Selected Epigraph Analysis for the three donors (full results shown in File S5). Yellow bars indicate monoallelic gene loci (MA) and red bars biallelic gene loci (BA). CTX, STR and SPC denote gene loci from cortex, striatum and spinal cord donors. (A) shows enriched CpG and general GC content at transcriptional start sites of BA gene loci. (B) SINE element showing an increased occurrence with BA gene loci when the full length of the transcript is considered. (C) LINE elements show no significant difference between MA and BA gene loci. (D) L1 family also show no significant difference in their distribution. (E) LTRs show increased occurrence with MA gene loci.|
|SC_12-0144_sm_supplFigure7B.tif||1049K||Figure S7. Selected Epigraph Analysis for the three donors (full results shown in File S5). Yellow bars indicate monoallelic gene loci (MA) and red bars biallelic gene loci (BA). CTX, STR and SPC denote gene loci from cortex, striatum and spinal cord donors. (A) shows enriched CpG and general GC content at transcriptional start sites of BA gene loci. (B) SINE element showing an increased occurrence with BA gene loci when the full length of the transcript is considered. (C) LINE elements show no significant difference between MA and BA gene loci. (D) L1 family also show no significant difference in their distribution. (E) LTRs show increased occurrence with MA gene loci.|
|SC_12-0144_sm_supplFigure7C.tif||1048K||Figure S7. Selected Epigraph Analysis for the three donors (full results shown in File S5). Yellow bars indicate monoallelic gene loci (MA) and red bars biallelic gene loci (BA). CTX, STR and SPC denote gene loci from cortex, striatum and spinal cord donors. (A) shows enriched CpG and general GC content at transcriptional start sites of BA gene loci. (B) SINE element showing an increased occurrence with BA gene loci when the full length of the transcript is considered. (C) LINE elements show no significant difference between MA and BA gene loci. (D) L1 family also show no significant difference in their distribution. (E) LTRs show increased occurrence with MA gene loci.|
|SC_12-0144_sm_supplFigure8.tif||259K||Figure S8. Clonal bisulfite sequencing of the gene TNFRSF10D promoter region in spinal cord derived clones. The biallelic expressing SPC06 shows only sporadic CpG methylated sites whereas the monoallelic expressing SPC01 and SPC04 show a proportion of reads with long stretches of CpG methylation. Filled circles indicate methylated CpG sites, clear circles unmethylated, missing circles for CpG sites where methylation status could not be obtained.|
|SC_12-0144_sm_supplFigure9.tif||473K||Figure S9. Epigenomic measures at identified monoallelic and biallelic loci. Histone modification and DNAse I hypersensitvity site measures from fetal brain at neural stem cell line identified monoallelic (MA), stochastic monoallelic (St-MA) and biallelic (BA) gene loci derived from cortical, striatal and spinal cord donors. All data was obtained from the Human Epigenome Atlas (http://www.epigenomeatlas.com/ ).|
|SC_12-0144_sm_supplFigure10.tif||1013K||Figure S10. Epigenomic measures in non-neuronal tissue. ChIP-seq histone modification measures from lymphocytes at transcriptional start site gene loci defined from cortex (CTX), striatum (STR) and spinal cord (SPC). MA denotes monoallelic expressing genes, BA denotes biallelic genes. A depletion of H3K4me3 and enrichment of H3K27me3 at MA gene loci exists, similar to the observations found in the fetal brain data (see figure S9). However, H3K9me3 measures differ from fetal brain, showing enrichment at MA gene loci. Lymphocyte data was retrieved using the tool EpiGraph (http://epigraph.mpi-inf.mpg.de/).|
|SC_12-0144_sm_supplFigure11.tif||1428K||Figure S11. Sequence conservation association. (A) Relation of monoallelic (MA - red bars) and biallelic expressed genes (BA - blue bars) with rapidly evolving conserved noncoding sequences. Datasets from three separate studies were used to examine gene loci associations from cortical (CTX), striatal (STR) and spinal cord (SPC) neural stem cell lines. Enrichment of rapidly evolving conserved non-coding sequences was found in the MA gene list from all donors, although statistical significance was not reached with the Pollard et al (2007) dataset due to a low sample number. (B) dN/dS plot examining synonymous versus non synonymous mutational rates between MA and BA expressing genes. No significant difference was found suggesting neutral drift within coding exons.|
|SC_12-0144_sm_suppltable1.tif||380K||Table S1. Relationship of genes identified by Gimelbrant et al (2007) to genes identified in this study. Result for cortical, striatal and spinal cord donors are shown. The two rows represent the monoallelic genes (MA) and biallelic (BA) genes identified from this study which overlap with those assayed in the Gimelbrant et al study (columns). The number of observed and expected MA and BA genes identified by Gimelbrant et al in relation to this study are shown together with Chi-squared test p-values. Expected numbers were calculated using the proportion of MA and BA genes from this study (10%:90% ratio). The findings indicate that the MA genes identified within this study match a significant number of MA genes from Gimelbrant et al (2.4 to 3.8 fold enrichment) which is significantly higher than MA genes which match Gimelbrant's BA gene loci (0.9 to 1.2 fold enrichment). Conversely, the BA genes we identified are equal to the expected amount of BA genes identified by Gimelbrant et al (1.0 fold enrichment) whereas a marginal under-representation of BA genes map to Gimelbrant's MA gene loci (1.0, 0.9 and 0.9 fold enrichment respectively).|
|SC_12-0144_sm_suppltable2.tif||333K||Table S2. Identification of intergenic SNPs summary. Detected monoallelic SNPs occurring side by side in chains of three or more SNPs along the genome are shown for the three clonal lines from each donor. A breakdown of position for each identified SNPs are shown: genic SNPs (within a RefSeq or UCSC genome browser defined transcript), intergenic SNPs within human EST sequences (but outside of RefSeq and UCSC known genes) and intergenic SNPs which show no alignments with EST sequences.|
|SC_12-0144_sm_SupplFile1.xls||117K||Supplementary File 1|
|SC_12-0144_sm_SupplFile2.xls||287K||Supplementary File 2|
|SC_12-0144_sm_SupplFile3.xls||150K||Supplementary File 3|
|SC_12-0144_sm_SupplFile4.xls||1870K||Supplementary File 4|
|SC_12-0144_sm_SupplFile5.xls||785K||Supplementary File 5|
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