Chromosome-specific and noisy IFNB1 transcription in individual virus-infected human primary dendritic cells

Abstract

The induction of interferon beta (IFNB1) is a key event in the antiviral immune response. We studied the role of transcriptional noise in the regulation of the IFNB1 locus in primary cultures of human dendritic cells (DCs), which are important 'first responders' to viral infection. In single cell assays, IFNB1 mRNA expression in virus-infected DCs showed much greater cell-to-cell variation than that of a housekeeping gene, another induced transcript and viral RNA. We determined the contribution of intrinsic noise by measuring the allelic origin of transcripts in each cell and found that intrinsic noise is a very significant part of total noise. We developed a stochastic model to investigate the underlying mechanisms. We propose that the surprisingly high levels of IFNB1 transcript noise originate from the complexity of IFNB1 enhanceosome formation, which leads to a range up to many minutes in the differences within each cell in the time of activation of each allele.

Figure 1.

Schematic illustrating the protocol used to measure IFNB1 mRNA levels synthesized by each allele within individual dendritic cells (DCs). (A) Isolation of virus-infected DCs for single cell real-time PCR. NDV viruses (red circles) were co-incubated with monocyte-derived human DCs (yellow). DCs were separated into 384-well PCR plates by visual light scatter using a fluorescent-activated cell sorter (FACS). DCs then underwent single cell one-step reverse-transcription and real-time PCR. The resulting amplification curves from one plate which show fluorescence levels as a function of PCR cycle number are shown for illustration. (B) Schematic illustrating the principle of the allelic imbalance assay used to quantify IFNB1 mRNA synthesized from each allele within individual DCs. mRNA allelic imbalance (AI), defined as the difference between the transcript level from each of the two alleles divided by the sum, can range from −100 to 100%. On average, the same amount of mRNA is synthesized from each of the two IFNB1 alleles, which are functionally indistinguishable. However, the relative synthesis from each allele within each cell can vary as a result of intrinsic noise. a, b and c represents individual DCs in which IFNB1 is synthesized mostly from one allele, equally from both alleles, or mostly from the other allele, respectively. The curly lines within each DC represent the IFNB1 mRNA molecules and their color (red or green) indicates their origin from one or the other allele. The mRNAs differ by a single nucleotide and the amplified single cell PCR product preserves their relative levels of expression within each cell. Each single cell PCR product was then subjected to a second allele-specific PCR reaction (ASPCR) that measured the relative levels of IFNB1 expression from each allele based on the single nucleotide difference. The graph illustrates the difference in the plots obtained from a single cell with differential expression from the two alleles. A detailed explanation of the methodology is found in Methods section.

Figure 3.

Allelic imbalance (AI) in individual DCs. (A–C) Measurement of IFNB1 AI as a function of total transcript number for individual DCs exposed to NDV at 8, 9 and 10 h. (D) Measurement of AI of control gene RPL9 at 10 h. The color changes from green to yellow to red are set as a function of the relative mRNA expression from the two alleles. Intrinsic noise (η_int), extrinsic noise (η_ext) and total noise (η_tot) were determined as described in Supplementary Data. The results for IFNB1 were η_int = 1.4, η_ext = 2.0, η_tot = 2.5 at 8 h; η_int = 0.6, η_ext = 1.8, η_tot = 1.9 at 9 h; η_int = 0.6, η_ext = 1.3, η_tot = 1.5 at 10 h; and for control RPL9 were η_int = 0.3, η_ext = 0.0, η_tot = 0.3. (E–H) Histogram of percent of cells showing different levels of AI for IFNB1 in single human DCs at 9 and 10 h after infection. (E) Stochastic model simulation at 9 h. (F) Stochastic model simulation at 10 h. (G) Experimental results at 9 h. (H) Experimental results at 10 h. For details of the model, see Methods section.

Figure 2.

Single cell analysis of gene expression in NDV-infected human DCs. All infections were carried out at viral multiplicity of infection (MOI) = 0.5. Non-infected (NI) single cells were analyzed as a negative control. (A) The induction ratio is the percentage of cells showing induction of IFNB1. Shown are the IFNB1 induction ratios obtained in DCs that were exposed to NDV for varying times (6–12 h) from two donors. The data were obtained by cell sorting and single cell mRNA expression measurements as illustrated in Figure 1A. The two donors were sampled at different time points because practical considerations limit the number of samples that can be sorted and assayed in any single experiment. (B and C) Histograms showing gene expression level in logarithmic scale. The histograms represent the entire population of cells studied, with each measurement obtained from a different cell. The results obtained in control cells (inset) and in NDV infected cells are compared in each panel. (B) Histogram of IFNB1 expression at 10 h from one donor. The second broad peak of infected cells shows an elevated IFNB1 mRNA expression induced by viral infection. (C) Histogram of ribosomal protein gene 9 (RPL9), a housekeeping control gene, at 10 h from the same donor as in B and C above. Viral infection has no effect on RPL9 mRNA level. (D) Multiplex analysis of single cell IFNβ and NDV viral mRNA expression. The data shown in the graph were obtained using a different donor than the data shown in panels B and C. The NDV L gene, located at the 5′ end of NDV viral genomic RNA, is the last to be transcribed and is the least abundant. Thus its detection by PCR is the most accurate measurement of the level of viral RNA. The levels of viral RNA and IFNB1 mRNA within each DC were uncorrelated. (E) Pie chart of populations of cells expressing NDV and IFNB1 (NDV+/IFNB1+), IFNB1 alone (NDV−/IFNB1+), NDV alone (NDV+/IFNB1−), and neither (NDV−/IFNB1−). NDV+ indicates that viral RNA was detected by the L gene PCR reaction.