Which allele is transcribed

Correlation values for simulated counts in the case independent bursting from the two Aebp1 alleles is shown as grey bar plot, correlation for real data is indicated as red line. For each tissue we indicate which cluster size has the closest variance at a given subdivision size.

A summary of this data is presented in Fig 2A. Using at least 10 randomly selected tiles we indicate what manual assignments we would have given the computationally identified Xist foci. We show the name and genomic location of genes tested, the strains interrogated and the number of SNP probes used.

We also indicate which final colocalization radius we selected, and what the overall colocalization rate as well the unique colocalization rate were at that radius.

We thank members of the Raj and Bartolomei laboratories for helpful suggestions and discussions. We particularly thank Chris Krapp for assistance with animal care and husbandry, Joanne Thorvaldsen for suggestions about Xist analysis, Paul Ginart for his advice on methodology and Rohit Gupte for help with transcription site code.

Abstract Extensive cell-to-cell variation exists even among putatively identical cells, and there is great interest in understanding how the properties of transcription relate to this heterogeneity. Author summary In mammals, most cells of the body contain two genetic datasets: one from the mother and one from the father, and—in theory—these two sets of information could contribute equally to produce the molecules in a given cell.

Introduction Gene expression in genetically identical individual cells often deviates from that of the cell population average [ 1 ], which in mammals can impact cell fate and development [ 2 — 5 ], response to environmental stimuli [ 6 — 9 ] and disease [ 10 — 13 ]. Results SNV-specific detection of RNAs in mouse tissue using single molecule RNA FISH Our goal in tissues was to quantitatively measure the amount of cell-to-cell variability in transcript abundance from either the maternal or paternal allele of a gene to determine the degree of imbalance between transcripts arising from the two alleles.

Download: PPT. Fig 1. Quantifying cell-to-cell heterogeneity in the chromosomal origin of RNA in tissue To determine how the chromosomal origin of RNAs contributes to cell-to-cell heterogeneity in tissue, we focused on two different questions. Fig 2. Quantification of allele-specific single-cell heterogeneity.

Observed heterogeneity of strain-specific RNA is compatible with bursty biallelic expression Our results showing that individual cells could have mRNA from either one or both alleles motivated us to assess whether existing models of transcription were sufficient to explain the observed cell-to-cell variability in allelic imbalance. Fig 3. Determining the mechanisms underlying allele-specific single-cell heterogeneity.

Discussion There has been great interest in recent years to precisely measure expression from the two alleles of a gene in diploid cells, ideally directly in tissue and at the single-cell level. Allele-specific colocalization analysis of single mRNA spots For analysis of single molecule RNA spots we used a combination of 60x whole tissue scans in DAPI and Cal fluor to determine the overall structure of the tissue and collecting z-stacks at x resolution of 5—10 individual positions within that tissue to identify individual mRNA molecules and characterize their allelic identity.

Analysis of SNV probe properties To determine whether any biophysical properties could differentiate between allele-specific probes that had high vs low colocalization rates, we compiled a table containing the the following parameters S8 Table : probe name, probe sequence, colocalization rate the colocalization rate determined for an entire probeset was applied to each individual probe , number of predicted secondary structures and folding energies.

Analysis and modelling of single-cell allelic outcomes To quantify cell-to-cell variability of allelic state in single tissue cells, we extracted colocalization data from our image analysis pipeline, and used this for further analysis. Supporting information.

S1 Fig. S2 Fig. Allelic calls across whole tissue sections and modelling of spatial heterogeneity of Xist. S3 Fig. Expression levels of selected genes in kidney by bulk and single-cell sequencing. S4 Fig. Colocalization rates and probe properties for autosomal allele-specific probes. S5 Fig. Impact of colocalization rate on allele-specific RNA imaging.

S6 Fig. S7 Fig. Allele-specific Podxl mRNA detection in mouse kidney sections. S8 Fig. S9 Fig. Fluorescence intensity of Aebp1 mRNA at transcription sites and non-transcription sites. S10 Fig. S11 Fig. Correlation between BL6 and JF1 RNA counts in single cells when including false assignments in the simulations of bursty transcription. S1 Table. Summary of allelic calls of Xist in kidney tissues. S2 Table. Closest matching modeled cluster size for Xist at different sized subdivisions.

S3 Table. Comparison of manual and automatic detection of Xist foci. S4 Table. Overview of selected autosomal genes. S5 Table. Summary of colocalization details and allelic calls for individual experiments performed for Aebp1, Lyplal1 and Mpp5.

S6 Table. List of FISH probes. S7 Table. List of primers used for genotyping and sequencing. S8 Table. SNP probe properties. S9 Table. Summary of single-cell sequencing results for the genes used in this study. Acknowledgments We thank members of the Raj and Bartolomei laboratories for helpful suggestions and discussions. References 1. Symmons O, Raj A. Mol Cell. Elsevier; ; — The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.

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Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. The demonstration of tissue clonality by X-linked enzyme histochemistry.

J Pathol. Takasato M, Little MH. Flag Content Cancel. Delete Content. Delete Cancel. The two chromosomal copies alleles of a gene are designated A and a. In most cases, both alleles are transcribed; this is known as bi-allelic expression left. However, a minority of genes show monoallelic expression right.

In these cases, only one allele of a gene is expressed right. Which of the two alleles is expressed may be determined by the parental origin of the allele such as in imprinting. Alternatively, the choice may be random. This image is linked to the following Scitable pages:. When only one allele of a gene is actively transcribed, gene expression is termed monoallelic. At the time of writing, only a single gene has been analyzed using this method A thin layer of material is placed on a microscope slide and polymerized.

Following thermal cycling the PCR products are immobilized and can be visualized as spheres within the gel, each sphere representing the product of a single gene copy. In situ primer extension with fluorescently labelled nucleotides was used to discriminate between alleles. This method was used to replicate a previously published analysis of the relative expression of alleles of the gene PKD2 22 using the same CEPH samples A sophisticated method of measuring both absolute and relative mRNA levels of specific alleles has been developed The authors claim an accuracy similar to that obtained using other methods 22 , and high throughput is clearly possible.

However, they have so far reported no data on allelic discrimination. A purely bioinformatic approach has also been taken About One hundred and ninety four SNPs were identified as being differentially expressed. One limitation that each of the methods mentioned have in common is that the source of the difference in allelic expression cannot be directly determined. Although this must in theory be cis -acting it may be due to a sequence variant anywhere in the region of the gene, or it may be due to epigenetic effects the transmission of information from a cell or multicellular organism to its descendants without that information being encoded in the nucleotide sequence of the genes.

Although the latter are usually considered to be mainly related to X-chromosome inactivation and silencing of a limited number of other genes, a far greater role has been proposed 33 , and some findings suggest that altered gene regulation as a result of epigenetic modification of sequence variation might be a common pathogenic mechanism in mammals One possible role for epigenetics is polymorphic imprinting leading to mono-allelic expression and evidence for this mechanism in the control of the expression of the 5HT 2A gene has been presented 35 , However, using the allelic discrimination techniques mentioned earlier, evidence for this effect could not be found in the brain tissue 37 , although it was found in lymphoblasts Two of the earlier mentioned surveys 22 , 23 found no genes which were expressed from only one chromosome, but the third 24 found three such genes including HTR2C.

However, all three genes showed non-Mendelian inheritance and as the lymphoblast cell lines were shown to be either mono or oligo-clonal, suggesting random mono-allelic expression Overall, the earlier mentioned results are consistent with low numbers of genes being imprinted; however, such an effect may be tissue specific, and partial imprinting may occur. Assuming that the source of variable allelic expression is sequence variants, it is still not possible to identify the causative variant using allelic discrimination.

However, this does not indicate the variant to be causative, as it may be in linkage disequilibrium with another, possibly unknown sequence variant. In order to determine the functional effect of any sequence variant on gene expression, it is necessary to control for the effects of any other variant in the genome. Although this may in theory be carried out by studying a large number of individual DNA samples in vivo all of which have been sequenced to determine the genotype at all polymorphic sites, in practice the method of choice is the in vitro reporter gene assay, not the least because it has been widely used and verified.

A large number of sequence variants in the promoter regions of candidate genes have been analyzed using reporter gene assays. It is not clear how many negative assays have been carried out, as publication bias has almost certainly come into force. The experiments reviewed earlier 39 were with few exceptions carried out on individual genes by research groups studying a limited number of genes. A diverse range of reporter gene systems was used.

In comparison, in a recently completed study, the author and colleagues have studied the effects on transcription of sequence variants in the promoter regions of genes 40 — 46 ; and unpublished data.

In addition as with most experiments of this kind, the assays were carried out under basal conditions with no specific stimuli applied. This coupled with the fact that only two cell lines were used for the majority of the assays suggests that many tissue and state specific effects were not seen. Both in vivo and in vitro experiments suggest that allele-specific differences in the rate of transcription are common and that most, if not all genes are likely to show differential allelic expression in some individuals.

Sequence variants rather than epigenetic effects probably underlie most cis -acting effects, although, in only a few genes have the effects been shown to be due to a specific sequence variant. In vitro experiments show that sequence variants in gene promoter regions frequently alter rates of transcription and these promoter variants may account for a significant proportion of differential allelic expression.

The role of allele-specific gene expression in complex phenotypes, however, is still not clear as several questions have not been answered for the great majority of the gene expression changes described earlier.

Answering the above questions and plotting the biological route from sequence variant to phenotypic change for each variant and each phenotype is a major task which will take either some considerable time or new technologies to complete.

Do the changes in relative allelic rates of transcription cis effects lead to overall changes in mRNA levels, or do homeostatic compensatory mechanisms trans effects lead to a limited effect? What percentage change in transcription is required for a phenotypic effect? Figure 1. Allelic discrimination in a single individual. The gene copies give rise to unequal numbers of mRNA molecules which are detected using primer extension single headed arrows with fluorescent nucleotides.

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