Dr. Shalini Oberdoerffer covers the role of splicing in epigenetics, how splicing and DNA methylation affect exon definition and the crosstalk between nucleosome and splicing mechanisms.
Splicing Up Epigenetics
In the splicing field several years ago, it was a somewhat surprising study coming out of whole genome sequencing studies of ChIP-seq data wherein it was found that exons presented a differential DNA landscape relative to introns. For instance, exons have shown higher levels of nucleosome occupancy, higher levels of DNA methylation, specific histone modifications relative to introns. This was a pretty exciting finding in the RNA field, because for years, RNA biologists have tried to identify the mechanisms of exon definition.
So exon definition is a process in which the spliceosome can distinguish exonic sequence from intronic sequence co-transcriptionally. In higher eukaryotes, the majority of introns are moved co-transcriptionally. This is somewhat different than what you find in lower eukaryotes, and it presents the spliceosome with a relatively difficult task because sometimes there can be mega bases of intronic sequences prior to presentation of the exonic DNA.
So for the last couple of years in the RNA field, there’s been a major effort to identify whether these histone modifications – in particular, that’s where the majority of focus has been – may be related to splicing. For instance, there have been studies in which histone modifications have been altered by the use of drugs, by the use of siRNA against methyltransferases and there have been global changes in alternative splicing.
“I think that the extensive coupling between splicing and Pol II elongation probably could be applied one step further to actual changes in chromatin dynamics.”
In the case of DNA methylation which is gaining interest in the epigenomics field, there has also been an account from my own laboratory wherein we found that the reciprocal effects of CTCF binding – CTCF is a methyl sensitive, zinc finger binding protein that can act when bound to non-methylated DNA – can act as a barrier to Pol II elongation, and that during the process of development, methylation may act to evict CTCF such that there is no longer Pol II pausing and exons can be excluded.
In the epigenomics field, it’s been found that there’s been quite a bit of effort looking at DNA methylation overlapping exons. And there seems to be some correlation between the level of methylation and exon inclusion such that exons that appear to have higher methylation are more likely to be included. So what I think is really exciting in the last few years is the synergy that we’re finding between the splicing field, which was my background and the epigenomics field, where we’re finding quite a bit of regulation in terms of the impact that these modifications may have on co-transcriptional splicing.
DNA Methylation in Exon Definition
The DNA methylation field has identified a link between methylation and gene silencing. These studies have largely focused on promoter DNA wherein variations in methylation can either act to in the case of hypermethylation can act to silence genes, or in the case of hypomethylation may act to activate genes.
The intergenic landscape is completely different, wherein we find that DNA methylation is highly linked to exonic sequence and we know that it’s not acting as a barrier to Pol II elongation. There was a Genome Biology paper from the Troy lab showing this quite effectively, that there doesn’t seem to be a correlation between gene body methylation and overall gene silencing.
My feeling is that DNA methylation probably does play an important role in the process of exon definition. However, I think it’s actually quite complicated. In a seminar from KG Zhou, where he was looking at the role of methylation in exon definition, he found that variations in methylation can either lead to exon inclusion or exclusion, which I think probably is a hint at how complex this process is.
So, in our case we see that methylation acts to evict CTCF. But it’s certainly not the case that all methylation sensitive exons are binding a methyl sensitive DNA binding protein. I think an area that has been largely unexplored is methyl-sensitive DNA binding proteins, and ones that will go and bind to methyl DNA.
So, I think we could find that for instance maybe in cases where methylation is linked to inclusion, perhaps a methyl binding protein is going to bind, and thereby mediating changes in elongation.
Crosstalk Between Epigenetics and Splicing
Going forward, a lot of focus in my own laboratory and probably many laboratories all over the world is going to be to examine in greater depth how many of the aspects of the DNA landscape influence Pol II elongation and spliceosome assembly. There are several recent studies showing that – one from the Bentley lab and the Garcia-Blanco lab suggesting that specific modifications to chromatin can occur, actually, co-transcriptionally such that they’re transcription dependent. They’ve shown this for trimethylation of H3K36.
I think that the extensive coupling between splicing and Pol II elongation probably could be applied one step further to actual changes in chromatin dynamics. And I think that this will be a major area that we’ll be interested in looking forward. For instance, how do variant nucleosomes incorporating variant histones affect Pol II elongation? How do they affect spliceosome assembly? We’d be interested to see how changes in histone chaperones – obviously nucleosome turnover will be at some point implicated in RNA processing by the mere fact that if you have an open chromatin, you can have cryptic transcription. So nucleosomes have to be remodeled and nucleosomes do pose a barrier to Pol II elongation. So, I think there’s going to be quite a bit of cross talk between these pathways.