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Abstract The length of 3′ untranslated regions (3′UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4+ T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3′UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3′UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3′UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.

Summary: Chromatin regulation and alternative splicing are both critical mechanisms guiding gene expression. Studies have demonstrated that histone modifications can influence alternative splicing decisions, but less is known about how alternative splicing may impact chromatin. Here, we demonstrate that several genes encoding histone-modifying enzymes are alternatively spliced downstream of T cell signaling pathways, including HDAC7, a gene previously implicated in controlling gene expression and differentiation in T cells. Using CRISPR-Cas9 gene editing and cDNA expression, we show that differential inclusion of HDAC7 exon 9 controls the interaction of HDAC7 with protein chaperones, resulting in changes to histone modifications and gene expression. Notably, the long isoform, which is induced by the RNA-binding protein CELF2, promotes expression of several critical T cell surface proteins including CD3, CD28, and CD69. Thus, we demonstrate that alternative splicing of HDAC7 has a global impact on histone modification and gene expression that contributes to T cell development.

Alternative splicing occurs in the vast majority of human genes, giving rise to distinct mRNA and protein isoforms. We, and others, have previously identified hundreds of genes that change their isoform expression upon T cell activation via alternative splicing; however, how these changes link activation input with functional output remains largely unknown. Here, we investigate how costimulation of T cells through the CD28 receptor impacts alternative splicing in T cells activated through the T cell receptor (TCR, CD3) and find that while CD28 signaling alone has minimal impact on splicing, it enhances the extent of change for up to 20% of TCR-induced alternative splicing events. Interestingly, a set of CD28-enhanced splicing events occur within genes encoding key components of the apoptotic signaling pathway; namely caspase-9, Bax, and Bim. Using both CRISPR-edited cells and antisense oligos to force expression of specific isoforms, we show for all three of these genes that the isoform induced by CD3/CD28 costimulation promotes resistance to apoptosis, and that changes in all three genes together function combinatorially to further promote cell viability. Finally, we show that the JNK signaling pathway, induced downstream of CD3/CD28 costimulation, is required for each of these splicing events, further highlighting their co-regulation. Together, these findings demonstrate that alternative splicing is a key mechanism by which costimulation of CD28 promotes viability of activated T cells.