Alternative Splicing of HDAC7 Regulates its Interaction with 14-3-3 Proteins to Alter Histone Marks and Target Gene Expression

Epigenetics and Alternative Splicing are both critical mechanisms guiding gene expression. Several studies have demonstrated that epigenetic marks can influence alternative splicing decisions, but less is known about how alternative splicing may impact epigenetics. Here, we demonstrate that several genes encoding histone modifying enzymes are alternatively spliced downstream of T cell activation signaling pathways, including HDAC7, a gene previously implicated in controlling gene expression programs 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 with resulting impact on histone modifications and gene expression. Notably, the long isoform, which is favored upon JNK signaling, promotes expression of several critical T cell surface proteins including CD3, CD28, CD69. Thus, we demonstrate that alternative splicing of HDAC7 has a global impact on epigenetics and gene expression, and may contribute to T cell regulation. Overall design: To investigate the functional consequences of HDAC7 exon 9 pre-mRNA alternative splicing, we utilized CRISPR-Cas9 technologies to establish Jurkat T cell lines that endogenously express the HDAC7 protein with or without exon 9 encoded peptide (isoforms termed iE9 or dE9, respectively). We then performed gene expression profiling analyses using data obtained from polyA-selected RNA-seq of 3 different HDAC7 genotypes (wild type, iE9 and dE9) at two time points (0 hr vs 48 hr post-PMA stimulation). Differential gene expression analyses were performed by comparing a) WT Jurkat cells vs iE9 or dE9 cells (matched by time point), b) stimulated vs unstimulated condition (matched by cell line), and c) iE9 vs dE9 cells (matched by time point).