Abstract
In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.
Highlights
The removal of introns and ligation of exons in the process of pre-mRNA splicing takes place predominantly co-transcriptionally[1,2,3]
In recent years there has been mounting evidence that pre-mRNA splicing is closely coupled with RNA synthesis and the chromatin environment of template DNA4,36
The original findings suggested that chromatin modifications modulate alternative splicing[11,13,14,21,23,37,38] and further studies have shown that pre-mRNA splicing reciprocally feeds back on histone modifications[7,8,9]
Summary
The removal of introns and ligation of exons in the process of pre-mRNA splicing takes place predominantly co-transcriptionally[1,2,3]. The first evidence implying chromatin affects alternative splicing came from genome-wide ChIP-Seq studies that correlated the presence of certain histone modifications with exon inclusion rates[5,6] Determining whether these histone marks are causative for splicing changes or a consequence of splicing cannot be addressed by this correlative approach. In order to experimentally determine the role of chromatin in alternative splicing regulation, most studies have used a global approach to perturb histone modifications genome-wide, such as small molecule inhibitors or knockdown/overexpression of histone modifying enzymes (HMEs)[10,11,12,13,14,15] As this method globally affects the transcriptional program of the cell, secondary effects cannot be fully excluded. We further studied the global distribution of H3K9me[3] within genes and showed this histone mark plays an unanticipated role in splicing of constitutive exons
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