CD44 alternative splicing senses intragenic DNA methylation in tumors via direct and indirect mechanisms.

Eric Batsché,Charlotte Hanmer-Lloyd,Oriane Mauger,Benjamin Hopkins,Christian Muchardt,Jia Yi,Etienne Kornobis

doi:10.1093/nar/gkab437

Abstract

DNA methylation (meDNA) is a modulator of alternative splicing, and splicing perturbations are involved in tumorigenesis nearly as frequently as DNA mutations. However, the impact of meDNA on tumorigenesis via splicing-mediated mechanisms has not been thoroughly explored. Here, we found that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1/3b undergo a partial epithelial to mesenchymal transition associated with increased CD44 variant exon skipping. These skipping events are directly mediated by the loss of intragenic meDNA and the chromatin factors MBD1/2/3 and HP1γ and are also linked to phosphorylation changes in elongating RNA polymerase II. The role of meDNA in alternative splicing was confirmed by using the dCas9/DNMT3b tool. We further tested whether the meDNA level could have predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes, mostly involved in splicing and signal transduction, are correlated with the local modulation of meDNA. Our observations suggest that, although DNA methylation has multiple avenues to affect alternative splicing, its indirect effect may also be mediated through alternative splicing isoforms of these meDNA sensors.

Highlights

DNA methylation involves the addition of a methyl group at position 5 of cytosines (5mC) by a small family of DNA cytosine-5 methyltransferase enzymes (DNMTs), which transfer methyl groups from the co-factor S-adenosyl-Lmethionine to DNA [1]
As a first approach to meDNA-guided alternative splicing and its possible impact on cancer, we examined HCT116 human colorectal cancer cells with the DNA methylases DNMT1 and DNMT3b inactivated
In this paper we sought a relationship between the dysregulation of alternative splicing and the modified meDNA frequently observed during tumorigenesis

Summary

Introduction

DNA methylation involves the addition of a methyl group at position 5 of cytosines (5mC) by a small family of DNA cytosine-5 methyltransferase enzymes (DNMTs), which transfer methyl groups from the co-factor S-adenosyl-Lmethionine to DNA [1]. This heritable epigenetic modification, which is crucial for mammalian development and cell differentiation, occurs predominantly at CpG dinucleotide sequences in mammals [2,3,4]. MeDNA has mostly been described for its role in the inhibition of transcriptional initiation, in the context of imprinting, X inactivation, retrotransposon silencing and at promoters containing CpG-rich regions. Within the body of genes, meDNA-enrichment prevents spurious RNA-polymerase II (RNAPII) entry, and cryptic transcriptional initiation [7,8,9], while regulating the activity of intragenic enhancers [10,11]

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Conclusion