AGBT 2010 - Jesse Gray - Harvard Medical School

Widespread RNA Polymerase II Recruitment and Transcription at Enhancers During Stimulus-Dependent Gene Expression

Mamalian brain is [paraphrased] Awesome technology
* Sensory experience shapes brain wiring via neuronal activation
* Whiskers compete for real estate in meta-sensory cortex.
* Brain can re-wire to adapt to environment
* Transcriptional changes in nucleous as brain cells reprogram
* (Discussion in terms of real-estate for rat whisker areas of brain.)

Neuronal activation affects circuit function by altering gene expression
* Activity dependent gene expression

Cascade
* Ca++ influx
* kinases & phosphatases
* CREB + SRF TFs
* recruit Creb binding protein
* Induce about 50-100x expression in genes (eg, fos)
* Can we do genome wide approaches to understand what's being expressed?

An experimental system for genome-wide analysis of activity-regulatee gene expression
* grow in dish
* depolarize with KCl
* do ChIP-seq and RNA-seq

CBP and transcription factor binding at fos locus
* see CBP binding at conserved region up stream, as well as promotor for fos gene
* also see NPAS4 CREB and SRF with similar (but not identical) binding sites

Is the activity dependent binding CBP restricted to the locus or genome wide?
* compare CBP peaks in both conditions
* binding appears limited to KCL stimulated only.

Are CBP-bound sites enhancers or promoters or both?
* Promoters don't necessarily drive transcription
* Promoters have H3K4Me3 histone modifications (enhancers dont)
* 3d configuration to bring enhancers together with promoters.

Most CBP peaks are not at TSSs and do not show H3K4Me3
* 5079 at TSSSs
* 36,069 not at TSSs

Align all seq that are enhancers
* there is much M3K4Me1 (clear pattern)
* there is not much M3K4Me3

Use known site
* upstream from Arc - used to build a construct

CBP and HK4Me1-marked loci function as activity-dependent transcriptional enhancers.
* Found 8 enhancers

Summarize:
* about 20,000 CBP sites that are activity-regulated enhancers
* do not correspond to annotated start sites
* H3K4Me1 modified
* lack H3K4Me3 mark
* do not initiate long RNAs
* confer activity-regulation on the arc promotor

Questions about activity-regulated enhancers
* do they play a role in binding RNA Polymerase II?
* Evidence is tending towards saying that most enhancers do not seem to have RNAPII binding.

fos enhancers bind RNAPII
* use chip for RNAPII and CBP
* 10-20% of sites have RNAPII at enhancer
* potential artifact - crosslinking conditions may exaggerate this by tying promotors and enhancers.

Does RNAPII at enhancers synthesize RNA?
* Enhancers at the fos locus produce enhancer RNAs
* non-polyadenylated RNA? Yes.
* you do get some transcription at enhancers... [doesn't this start to describe lincRNA?]

Enhancer transcription is correlated with promoter transcription.

The Arc enhancer can be activated without the presence of the Arc promoter
* increases in polymerase binding at enhancer even when promoter is gone.
* preliminary - but may not be transcription when the promoter is gone.
* what is the function of eRNA transcription? (don't know the answer yet)
* Could be that it helps to lay down epigenetic marks.