Abstract LB-100: Potential role of the splicing factor SF3B1 in epigenetic regulation
Authors: Deliard Sandra , Okamoto Yasuyuki , Madzo Jozef , Jelinek Jaroslav , Issa Jean-Pierre 
Published: July 2017

Driver mutations in splicing factors have been reported in multiple cancer types and SF3B1 is among the most frequently mutated splicing factors. The mechanism(s) of transformation associated with these mutations remain incompletely understood. The role of SF3B1 has been studied in splicing, but little is known about its effects on gene regulation outside of its splicing function in cancer. We identified SF3B1 as a potential epigenetic regulator in a whole genome siRNA screen for reactivation of aberrantly silenced genes alone or in synergy with DNMT inhibitors. This surprising link between splicing factors and epigenetic silencing by DNA methylation and chromatin deregulation could explain in part the effects of SF3B1 in cancer. To confirm this link, we studied effects of SF3B1 on gene expression and DNA methylation, and determined SF3B1 chromatin localization by ChIP-seq. We first used RNA-Seq data from all available Uveal Melanoma (UVM) and breast cancer patients in TCGA. Hierarchical cluster analyses of the UVM data showed distinct clusters with high expression of SF3B1 clustering independently of low SF3B1 expression in both cancer types. We identified genes co-regulated with SF3B1 expression in UVM and breast cancer and observed a significant overlap of 40% of upregulated genes (p &lt; 0.0001) and 41.1% of down regulated genes (p &lt; 0.0001) in patients with low SF3B1 expression in UVM and breast cancer. This similarity in the co-regulated gene sets suggests a possible direct regulation of gene expression by SF3B1. Next, we analyzed DNA methylation patterns in UVM samples stratified by SF3B1 expression and found that in patients with high SF3B1 expression relative to low SF3B1 expression, there was a significant increase of methylation at 755 CpG sites (Δ β-value&gt; 0.1, p&lt;0.001) and decrease of methylation at 42 CpGs (Δ β-value&lt; -0.1, p&lt;0.001) of all (&gt;450,000) CpG sites tested. At promoter CpG sites, 197 CpG sites had significantly increased methylation (Δ β-value&gt; 0.1, p&lt;0.001) while only 10 CpG sites had decreased methylation (Δ β-value&lt; -0.1, p&lt;0.001). Thus, high levels of SF3B1 are associated with increased DNA methylation, and inhibition of SF3B1 is associated with reactivation of gene expression from methylated promoters. Finally, we performed ChIP-Seq analyses for SF3B1 in YB5 colon cancer cells. In addition to SF3B1 enrichment at exons, we observed that SF3B1 was depleted at transcription start sites (TSSs) overall, but stratifying genes by expression reveled SF3B1 depletion at TSSs of expressed genes but not in unexpressed genes. This was similar to the pattern seen for histone H3 ChIP-Seq, suggesting SF3B1 association with nucleosomes. Taken together, our data suggest that SF3B1 plays a role in DNA methylation associated epigenetic silencing. In cancers, these silenced genes are potentially tumor suppressor genes which upon knockdown or inhibition of SF3B1, their expression could be reactivated leading to antitumor effects.

Citation Format: Sandra Deliard, Yasuyuki Okamoto, Jozef Madzo, Jaroslav Jelinek, Jean-Pierre Issa. Potential role of the splicing factor SF3B1 in epigenetic regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-100. doi:10.1158/1538-7445.AM2017-LB-100