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Friday, February 6, 2009

Jesse Gray, Harvard Medical School - “Neuronal Activity-Induced Changes in Gene Expression as Detected by ChIP-Seq and RNA-Seq”

Now “widespread overlapping sense/antisense transcription surrounding mRNA transcriptional start sites.”

Thousands of promoters exhibit divergent transcriptional initiation. Annotated TSS come from NCBI. There are 25,000 genes. There is an additional anti-sense TSS (TSSa) 200 bp upstream. [Nifty, I hadn't heard about that.]

Do RNA-Seq and ChIP-Seq. Using SOLiD. SOLiD or Ambion [not sure which] plans to sell the method as a kit for WTSS/WT-Seq.

Using RNA Pol II ChIP-Seq.

Anti-sense transcription peaks about ~400 bases upstream of TSS. When looking at the genome browser, you see overlapping TSS-associated transcription. (you see negative strand reads on the other direction, upstream from TSS, and on the forward strand at the TSS, with a small overlap in the middle.)

It is a small amount of RNA being produced.

Did a binomial statistical test, fit to 4 models:
1.sense only initiation
2.divergent initiation (overlap)
3.anti-sense only initiation
4.divergent (no overlap)

The vast majority are TSSs with divergent overlap, 380 with divergent (no overlap), 900 sense only, 140 anti-sense only. Many other sites were discarded because it was unclear what was happening. This is apparently a wide-spread phenomenon.

Might this be important? Went back to ChIP-Seq to classify the peaks into these categories from RNA Pol II expt. (Same categories.) Is this a meaningful way to classify sites, and what does it tell us?

How many of those peaks have a solid PhastCons score, which should tell something about the read. No initiation has the lowest scores... the ones with the antisense models have the highest conservation at the location of antisense initiation.

Where do the peaks fall, when they have anti-sense? Anti-sense are bimodal, sense only and bi-direcitional are just before the TSS, and non-bi-modal.

Tentatively, yes, it seems like this anti-sense is functionally important.

Does TSSo change efficiency of initiation?

Break into two categories. Non-overlap TSSs, and overlap TSSs. It appears that overlap TSSs produce more than twice the RNA than non-overlap. This could be a bias... could be selecting for highly expressed genes. Plot the RNA Pol II occupancy at the star sites, there is a big difference at the overlapping TSS. Non-overlap has higher occupancy at non-overlap, but lower up or down stream than overlap. Thus the transition to elongation may be less efficient.

Does TSSo change efficiency of initiation? Tentatively, yes.

Comment from audience: this was discovered a year ago in a paper a year ago by Kaplan (Kaparov?). Apparently this was lately described that these are cleaved into 31nt capped reads. THus, the fate of the small RNA should be of interst. 50% of genes had this phenomenon.

Question from audience: what aligner was used, and how were repetitive sections handled. Only uniquely mapping read, using SOLID pipeline. (Audience member thinks that you can't do this analysis with that data set.) Apparently, someone else claims it doesn't matter.

My Comment: This is pretty cool. I wasn't aware of the anti-sense transcription in the reverse direction from the TSS. It will be interesting to see where this goes.



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