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. 2016 Aug 19;44(14):6817-29.
doi: 10.1093/nar/gkw591. Epub 2016 Jul 1.

Global intron retention mediated gene regulation during CD4+ T cell activation

Ting Ni  1 , Wenjing Yang  2 , Miao Han  3 , Yubo Zhang  2 , Ting Shen  3 , Hongbo Nie  3 , Zhihui Zhou  4 , Yalei Dai  4 , Yanqin Yang  2 , Poching Liu  2 , Kairong Cui  2 , Zhouhao Zeng  5 , Yi Tian  6 , Bin Zhou  3 , Gang Wei  3 , Keji Zhao  2 , Weiqun Peng  7 , Jun Zhu  8
Affiliations

Global intron retention mediated gene regulation during CD4+ T cell activation

Ting Ni et al. Nucleic Acids Res. .

Abstract

T cell activation is a well-established model for studying cellular responses to exogenous stimulation VSports手机版. Using strand-specific RNA-seq, we observed that intron retention is prevalent in polyadenylated transcripts in resting CD4(+) T cells and is significantly reduced upon T cell activation. Several lines of evidence suggest that intron-retained transcripts are less stable than fully spliced transcripts. Strikingly, the decrease in intron retention (IR) levels correlate with the increase in steady-state mRNA levels. Further, the majority of the genes upregulated in activated T cells are accompanied by a significant reduction in IR. Of these 1583 genes, 185 genes are predominantly regulated at the IR level, and highly enriched in the proteasome pathway, which is essential for proper T cell proliferation and cytokine release. These observations were corroborated in both human and mouse CD4(+) T cells. Our study revealed a novel post-transcriptional regulatory mechanism that may potentially contribute to coordinated and/or quick cellular responses to extracellular stimuli such as an acute infection. .

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Figure 1.
Figure 1.
Intron retention (IR) is prevalent in resting human CD4+ T cells and dramatically reduced upon activation. (A) The transcriptomes of human resting and activated CD4+ T cells were profiled by RNA-seq. Oligo(dT) was used to enrich polyA+ RNAs, which were sequenced using DeLi-seq, a strand-specific RNA-seq procedure. The normalized expression profiles of a candidate gene, PSMD12, are shown in the bottom panel. Two representative introns with differential IR between resting and activated T cells are shown in dashed rectangle. (B) The fraction of intronic reads in total mapped reads are shown for resting and activated T cells. Compared to resting T cells, activated T cells show a significantly reduced fraction of intronic reads (P-value < 1 × 10−300, X2 test). (C) The frequency density distribution of IR Index (IRI) of individual genes in resting and activated human T cells. The integral of the area under the vertical bars is 1 for both resting and activated T cells. For each gene locus, IRI is defined as the ratio of normalized tag density between shared introns and shared exons. The mean and median IRI for the resting condition is 7.1% and 4.0%, respectively, whereas the mean and median IRI for the activated condition is 3.8% and 1.8%, respectively.
Figure 2.
Figure 2.
IRI-high transcripts are less stable than IRI-low transcripts. (A), (B) and (C) show the RNA Pol II, H3K36me3 and H4K20me1 density profile, respectively. The top 1000 genes (brown) and the bottom 1000 genes (blue) were selected based on their relative IRI ranks in resting T cells. The read counts were normalized to uniquely mapped reads. Six pairs of gene groups were compiled with matched (D) expression profiles, the corresponding levels of (E) intron retention, (F) Pol II, (G) H3K36me3 and (H) H4K20me1 are shown. In resting T cells, the median IRI values of IRI-high and IRI-low genes (E, IRI values on the right-hand side) are 20.0% and 0.6%, respectively. ** and *** represents P-value < 0.01 and < 0.001, respectively.
Figure 3.
Figure 3.
Changes in IR are negatively correlated with expression changes. (A) The fold changes in the IRI and expression levels were computed for each expressed Entrez gene as the result of human T cell activation. Entrez genes were then evenly divided into 8 bins based on IRI fold changes (X axis). The changes in expression levels for each gene in each group (Y axis) are shown as a box plot. (B) Both IRI fold-change rank (X axis) and expression fold change rank (Y axis) of individual genes were evenly divided into 10 bins. Each Entrez gene was subsequently placed into one of the 10 × 10 grids. The enrichment scores (Z score) were computed based on the observed gene density in each grid compared to a random distribution, and collectively shown as a heat map. Spearmen's rank correlation value is -0.43, with a P-value < 1E-300. (C–F) show four gene loci with a decreased IR level (left panel) and an increased transcript abundance (right panel) upon T cell activation.
Figure 4.
Figure 4.
Upregulated expressions in activated T cells are mediated by transcriptional activation and/or intron retention. (A) A total of 2090 up-regulated genes identified by RNA-seq were initially divided into 2 groups based on their IRI changes. Group I consists of 507 genes that did not exhibit a decrease in intron retention. The rest of genes (1583 genes) were further divided into two groups (groups II and III) based on the Pol II ChIP-seq data obtained from resting and activated CD4+ T cells. Group III genes (185 genes) show little or no change in Pol II occupancy (<30% change) and expression fold change > 3, while group II contains the remaining 1398 genes. Aggregated Pol II profiles of each group including 5 kb upstream of the transcription start sites (TSS) and 5 kb downstream of the transcription end sites (TES) are shown. (B) The distribution of the IRI fold-changes of three gene groups between resting and activated T cells. (C) Gene Ontology (GO) analysis of group III genes and the proteasome complex emerged as the most significant pathway (P-value < 1 × 10−7).
Figure 5.
Figure 5.
The expression of PSMD7 gene is predominantly regulated by IR during human T cell activation. (A) The strand-specific RNA-seq (top two panels) and Pol II ChIP-seq (bottom two panels) tracks of the PSMD7 gene. Three representative introns that have changed retention ratio between resting and activated human T cells are shown in dashed rectangles. (B) RT-PCR validation of regulated IR at the PSMD7 locus. Total RNA isolated from resting and activated human CD4+ T cells was reversely transcribed using oligo(dT) primer. The first-strand cDNAs were then amplified with three primers specific for PSMD7 transcripts. The expected amplification products are shown as indicated. (C) The spliced transcript abundance and (D) IR level of human PSMD7 gene were subsequently determined by quantitative RT-PCR (qRT-PCR) with primer pairs specific for spliced and intron-retained transcripts, respectively (mean ± SE, n = 3; two-tailed Student's t-test). *** and ** stands for P-value < 0.001 and < 0.01, respectively. (E) Western Blot for PSMD7 protein in resting (R) and activated (A) human CD4+ T cells. β-tubulin serves as internal control. (F) ChIP-PCR analysis of Pol II occupancy at the promoter regions of PSMD7 and STAT1 genes, respectively (mean ± SE, n = 3; two-tailed Student's t-test). STAT1, a known transcriptionally regulated gene, was used as a positive control. (G) The degradation rate of spliced and intron retained transcript of PSMD7 gene was quantified by qRT-PCR after inhibition of new round of transcription by adding Flavopiridol (FLV, 1 μM) in human resting T cells over the time course (min) shown in the figure. (H) The relative IR level (intron-retained versus spliced) of PSMD7 gene was determined by qRT-PCR of RNA derived from cytoplasmic (Cyto), nuclear (Nuc) and chromatin-associated (Chro) fractions in human resting T cells. *** represents P-value < 0.001 (two-tailed Student's t-test).
Figure 6.
Figure 6.
Conservation of IR-mediated gene regulation in mouse CD4+ T cells. (A) The RNA-seq tracks of the PSMD7 gene obtained from mouse naïve CD4+ T cells, and Th1 cells 8 h, 12 h and 24 h post- activation. Two representative introns that have changed retention ratio are shown in dashed rectangles. (B) The overall IRI distribution of naïve and activated mouse Th1 cells. (C) RT-PCR validation of reduced IR at the mouse PSMD7 locus along the course of Th1 cell activation. Actb1 serves as the internal control, which didn't show significant expression changes for 4 different conditions. Ccl4 gene serves as a positive control for mouse T cell activation.
Figure 7.
Figure 7.
Proposed model for IR regulates steady-state mRNA level in both human and mouse CD4+ T cells. In resting T cells, a certain number of genes (such as proteasome related genes) were not efficiently spliced that leads to high degree of degradation, possibly via the exosome-mediated pathway. Upon activation, splicing becomes more efficient and intron retained transcripts could be converted into productive mRNAs (Dashed line with an arrowhead). Therefore, an increase in steady-state mRNA level does not necessarily require transcriptional activation and could be solely achieved at the post-transcriptional level. ‘TF’ and ‘Enh’ is the abbreviation for ‘transcription factor’ and ‘enhancer’, respectively.
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