Abstract
Histone modifications have long been shown to impact final splicing decisions via modulation of RNA polymerase II elongation rate and/or recruitment of the splicing regulators to their RNA binding sites. However there is little evidence of the driving role of these chromatin modifications in inducing the splicing changes necessary for a switch in the cell’s phenotype. Taking advantage of the epithelial-to-mesenchymal transition (EMT), a reversible cell reprogramming intimately involved in early development and cancer metastasis, we found that dynamic changes in specific histone marks, namely H3K27ac and H3K27me3, were responsible for key splicing changes necessary for EMT. Using CRISPR epigenome editing tools, we showed that a single change in H3K27ac/me3 levels just at the alternatively spliced exon was necessary and sufficient to induce a change in splicing capable of recapitulating important aspects of EMT, such as cell motility and invasiveness. This histone mark-dependent splicing effect was highly dynamic and mediated by direct recruitment of the splicing regulator PTB to its RNA binding sites. Taken together, these results support a novel role for H3K27 marks in inducing a change in the cell’s phenotype via regulation of alternative splicing. We propose the dynamic nature of epigenetic modifications as a rapid, and reversible, mechanism to coordinate the splicing response to cell-extrinsic cues, such as induction of EMT.
Highlights
During cell reprogramming, such as in epithelial-to-mesenchymal transition (EMT), cell-type-specific transcriptional and splicing programs are tightly regulated to gain new phenotypic traits (Dongre and Weinberg, 2019; Javaid et al, 2013; Pradella et al, 2017; Shapiro et al, 2011; Yang et al, 2016)
Correlation in time between dynamic changes in chromatin modifications and alternative splicing during EMT EMT is a biological process involved in early development, wound healing, and tumor metastasis (Javaid et al, 2013; Shapiro et al, 2011)
During induction of EMT, we found a strong correlation in time between dynamic changes in splicing and highly localized changes in H3K27me3, H3K27ac, and H3K4me1 levels in 5 out of 6 studied genes (Figures 1B–1F, 2A–E, and S1K–S1V, summarized in S2A), whereas alternatively spliced exons not changing splicing during EMT, such as CTNND1 exon 20, did not show changes in these histone marks (Figures 1D–1F and S1B), pointing to an EMT-related splicing effect
Summary
During cell reprogramming, such as in epithelial-to-mesenchymal transition (EMT), cell-type-specific transcriptional and splicing programs are tightly regulated to gain new phenotypic traits (Dongre and Weinberg, 2019; Javaid et al, 2013; Pradella et al, 2017; Shapiro et al, 2011; Yang et al, 2016). In acute myeloid leukemia cell lines, a subset of alternatively spliced exons intimately involved in cell proliferation and transformation were regulated by H3K79me (Li et al, 2018), whereas during stem cell differentiation, exons involved in cell-cycle progression and DNA-damage responses were marked by H3K36me and H3K27ac (Xu et al, 2018). Most of this evidence is just correlative, or based on genome-wide alterations of the histone mark, which limits the capacity to properly assess the driving role of a localized histone modification in directly inducing a cell-type-specific splicing reprogramming that can phenotypically impact the cell. Many other key splicing regulators are stably expressed during the wholecell reprogramming, such as PTB and RBFOX2 (Shapiro et al, 2011), and not all ESRP1-dependent splicing events change splicing patterns at the same time points during EMT (Yang et al, 2016), suggesting an additional regulatory layer
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