I am a little bit confused about how Gro-seq could identify the expression of enhancer RNA.
Will the most mapped reads are actually belong to coding mRNA?
Thanks,
GRO-seq captures the pieces of _RNA_ that are being generated by active RNA polymerase. Think of it as if you were retrieving (parts of the) RNA molecule as it comes out of the copy machine that is RNA polymerase. That means that most sequences will indeed correspond to gene regions (including introns, though!), but in contrast to normal poly(A)-enriched mRNA-sequencing you will mostly have captured incomplete transcripts.
As Kevin pointed out, your mix of transcripts will include RNA molecules that were being produced outside the gene loci or from intronic regions, which are often thought to correspond to enhancers.
GRO-seq, global run-on sequencing, captures sites of active transcription where RNA polymerase is bound to DNA. The region of DNA where the polymerase is bound will be sequenced by next generation sequencing. The way that this can be used to infer the location of an enhancer RNA is based on the genomic location:
Enhancer regions, i.e., regions from which enhancer RNAs are transcribed, are found in intergenic regions up- or down-stream of the target gene's transcription start site (TSS), and expression at these regions can modify ['enhance'] the expression of these target genes. Thus, the binding of RNA polymerase at a region assumed to be an enhancer adds to evidence that it may indeed be an enhancer region.
Note that the genomic locations of enhancer regions can also be inferred by the presence of H3K27-acetylation marks.
GRO-seq is one of the methods recognised as being capable of identifying enhancer regions:
[source: http://www.nature.com/nrg/journal/v17/n4/fig_tab/nrg.2016.4_T1.html]
Further reading:
This is a difficult question to answer, it's kind of difficult to tease apart eRNA from mRNA when you are looking at intragenic enhancers. This is why a lot of studies focus on intergenic enhancers because they are easy to identify. Typically to be sure you'd have to overlap your GRO-seq to some other sequencing data such as ChIP-seq, otherwise you can never really be sure.
Some people complement the GRO-seq run with a poly(A)-mRNA sequencing experiment, which will give you some idea of the general expression pattern. In addition, you can probably classify the intragenic enhancers based on the patterns of transcription you're detecting. If you have a gene that's basically not covered by any sequences, but only one intron seems to have generated some, then that may be an indication that this is the enhancer region for a different gene.
Was indeed a difficult question - thanks for providing commentary. I imagine that there are undoubtedly many scenarios that occur in relation to eRNA and mRNA transcription.
One really intriguing example that I always use is from the study by Mansour and colleagues, where insertion variants create a 'super enhancer' region, which appear to result in transcription all the way from the eRNA site to the mRNA. See An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element
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It's quite intriguing, isn't it? The RNA polymerase appears to bind all across the entire genome, but in many cases I'm not sure that transcription even occurs. Transcription factors and other binding proteins and ncRNAs also bind at various loci.
Our genomes are very much 'alive' and fluid molecules, not fixed and rigid like we once though.
ChIP-seq of RNA Pol only tells you where it binds, but if sequences are captured by GRO-seq it means that transcription is occurring.
Yes, but I imagine that there are many situations in which the polymerase becomes blocked or loses binding efficacy due to other issues, and therefore transcription of downstream targets will not occur. One example is antisense transcripts, where a separate polymerase binds to sense and antisense strands at the same time and can actually physically block each other from transcribing further as they move along the target gene's sequence.
It would be interesting to compare GRO-seq to matched RNA-seq
I'd be surprised to see clear evidence of that in bulk GRO-seq samples. Not saying it doesn't happen. However, the point stands: if a piece of RNA was captured in GRO-seq, it was, in fact, being transcribed. Whether it produced a full-length transcript that went on to become a protein, is a different story, I completely agree.