The effect of cellular context on miR-155-mediated gene regulation in four major immune cell types

Abstract

Numerous microRNAs and their target mRNAs are coexpressed across diverse cell types. However, it is unknown whether they are regulated in a manner independent of or dependent on cellular context. Here, we explored transcriptome-wide targeting and gene regulation by miR-155, whose activation-induced expression plays important roles in innate and adaptive immunity. Through mapping of miR-155 targets through differential iCLIP, mRNA quantification with RNA-seq, and 3' untranslated region (UTR)-usage analysis with poly(A)-seq in macrophages, dendritic cells, and T and B lymphocytes either sufficient or deficient in activated miR-155, we identified numerous targets differentially bound by miR-155. Whereas alternative cleavage and polyadenylation (ApA) contributed to differential miR-155 binding to some transcripts, in most cases, identical 3'-UTR isoforms were differentially regulated across cell types, thus suggesting ApA-independent and cellular-context-dependent miR-155-mediated gene regulation. Our study provides comprehensive maps of miR-155 regulatory networks and offers a valuable resource for dissecting context-dependent and context-independent miRNA-mediated gene regulation in key immune cell types.

Figure 1

miR-155 mediated Argonaute binding occurs at distinct sites in four immune cell types. (a) Examples of universally bound and differentially bound miR-155 sites across 4 cell types. Normalized read coverage of iCLIP, RNA-Seq and PolyA-Seq libraries are shown with dark colors for wild-type (WT) and light colors for miR-155 knockout (KO) tracks. miR-155 seed-containing iCLIP peaks are highlighted with grey rectangles with asterisks designating significant (FDR < 2.5%) difference between WT and KO coverage. (b) Summary of miR-155 dependent sites in co-expressed genes, including 3′UTR, CDS, and 5′UTR sites, identified by differential iCLIP. Each row represents 250 bp around a miR-155 6mer seed match with colors that demonstrate the log₂ ratios of normalized WT to miR-155 KO iCLIP coverage. Heatmap of RNA expression (WT RNA-Seq log₁₀ FPKM, normalized by row) of the same genes containing the miR-155 sites is shown side-by-side. Sites are categorized according to their binding specificity across 4 cell types, while the order within each category are determined by hierarchical clustering of RNA-Seq FPKM values for corresponding genes. (c) Venn diagram of miR-155 dependent iCLIP sites in co-expressed genes. (d) Seed-type composition of miR-155 dependent sites in co-expressed genes. (e) PhastCons scores (for multiple genome alignments between mouse and other 39 placental mammals) of miR-155 dependent sites in co-expressed genes. Analyses of data from independent iCLIP (n = 4), RNA-Seq (n = 3), and PolyA-Seq (n = 4) experiments are shown.

Figure 2

miR-155 represses distinct sets of genes in four immune cell types. In dendritic cells (a), B cells (b), CD4⁺ T cells (c) and macrophages (d), the distribution of RNA-Seq expression changes between miR-155 KO and WT cells is shown with the cumulative distribution functions (CDFs) for different gene sets. Gene sets include all expressed genes, genes with 3′UTR miR-155 6mer / 7mer-A1 / 7mer-m8 / 8mer seed matches and genes containing 3′UTR miR-155 dependent iCLIP sites with 6mer seed matches (FDR < 2.5%). Predicted miR-155 target genes with top context++ scores from Targetscan 7.0 (same number as the miR-155 target genes identified by differential iCLIP) are also shown. The data represent independent iCLIP (n =4) and RNA-Seq (n = 3) experiments.

Figure 3

Context-specific miR-155 targeting leads to differences in gene regulation between cell types. For all six pairwise comparisons across four immune cells, de-repression of genes containing common (solid lines) and cell-type specific (dotted lines) 3′UTR miR-155 dependent iCLIP sites is shown in the form of CDFs. Genes with 3′UTR miR-155 seed matches are also shown as reference. Only co-expressed genes (WT RNA-Seq FPKM > 1 and difference < 16 fold) are included in each pairwise comparison. In each plot, two P-values from one-sided KS tests are shown. First KS test corresponds to the comparison between all miR-155 target genes identified in this cell type and genes only targeted in the other cell, while the second one corresponds to the comparison between the common target genes and target genes specific to this cell type. Results of four independent iCLIP and three independent RNA-Seq experiments are shown.

Figure 4

Verification of cell type-dependent miR-155 mediated repression. Reporters carrying 3′UTR of genes displaying context-specific targeting were expressed in B cells and dendritic cells. Results are shown for Hif1a and Jarid2 (preferentially repressed in B cells), Actr10 and Terf1 (B cell-specific targets), and Tbca, Uqcrfs1, and Zfp277 (dendritic cell-specific targets). Fold repression was determined from ratio of normalized luciferase activities of mutant and wild-type 3′UTR reporters. Error bar displays standard error from at least three biologically independent samples; P-value was measured by two-sided t-test. *, P < 0.05; **, P < 0.01.

Figure 5

PolyA-Seq captures change in 3′UTR isoform usage during CD4⁺ T cell activation. (a) Two examples of 3′UTRs with significant (FDR < 5%) changes in isoform usage during CD4 T cells activation. Tracks represent normalized PolyA-Seq read coverage at 0 h, 24 h and 48 h after activation. (b) The changes in 3′UTR isoform usage for 3′UTRs with two major isoforms at 48 h after CD4⁺ T cell activation. Highlighted genes displayed significant (FDR < 5%) changes in 3′UTR usage. (c) Same as (b), but highlighting the two-isoform 3′UTRs containing target sites of miR-155. 3′UTRs containing proximal (solid shapes) and distal (hollow shapes) miR-155 target sites are highlighted. The results of three independent PolyA-Seq experiments are shown.

Figure 6

The role of alternative polyadenylation in cellular context dependent regulation of gene expression by miR-155. (a) A heatmap showing the usage changes in multi-isoform 3′UTRs across all four cell-types. The usage index (UI) represents the percentage of the shorter isoform usage for two-isoform 3′UTRs, while for 3′UTRs with more isoforms it represents the usage of the short isoform with the most significant usage change. “Elongation” corresponds to the genes with significantly higher usage of longer isoforms in one cell type compared to the rest, whereas “shortening” corresponds to the genes with significantly higher usage of shorter isoforms in one cell type compared to the rest. (b) Number of 3′UTRs containing miR-155 targets and displaying cell type specific ApA differences. (c) iCLIP, RNA-Seq and PolyA-Seq read coverage tracks in dendritic cell and CD4⁺ T cell for Rbm33. (d) Venn diagram showing the number of shared and cell-specific miR-155 target genes in dendritic cell and B cell, before and after removing genes with cell type specific ApA differences. The data are representative of four independent PolyA-Seq experiments.

Figure 7

Top iCLIP target sites of other miRNAs induce significant gene repression. mRNA expression changes in B cells (a) and dendritic cells (b) with miR-142a KO and in CD4⁺ T cells with miR-27a overexpression (c) are shown as CDFs for different gene sets. Gene sets consist of all expressed genes, genes with 3′UTR seed matches (6mer, 7mer-A1, 7mer-m8, and 8mer), and genes containing 3′UTR iCLIP sites with 6mer seed matches and most reads in wild-type libraries. Predicted miRNA target genes with top context++ scores from TargetScan 7.0 (same number as the target genes defined by wild-type iCLIP) are also shown. Analyses were shown from independent iCLIP samples (n = 4), miR-142 array data in B cell (GSE61919, n = 3) and dendritic cell (GSE42325, n = 2), and miR-27a RNA-Seq experiments (GSE75909, n = 3).