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. Author manuscript; available in PMC: 2016 Jan 29.
Published in final edited form as: Cell. 2015 Jan 22;160(3):367–380. doi: 10.1016/j.cell.2014.12.023

Figure 3. TOP1 Recruits toAR -Regulated Enhancers and Nicks the DNA.

Figure 3

(A) UCSC browser screenshot displaying the KLK3 enhancer. Arrows indicate the PRO-caps representing (putative) eRNA TSS flank the NKX3.1 peak. (B) eRNA readout assay showing that Tyr723 of TOP1 is required for eRNA induction. LNCaP cells were hormone-starved for 24 h, transfected with siTOP1 to deplete the endogenous protein and then electroporated with either empty expression vector (Veh), wild-type TOP1 (WT), or the Y723F-TOP1 mutant (Mut) before treatment with either ethanol or DHT for 1h. eRNA for KLK2, KLK3 and TMPRSS2 gene enhancers was quantified by RT-PCR. TOP1 mRNA and protein levels are also shown. qPCR data show mean ± s.d.; (n=3). **P<0.01. (C) Knockdown of endogenous TOP1 affects nick/break formation as measured by incorporation of Biotin 11-dUTP at selected AR-enhancers after 10 min DHT treatment. Data represent mean ± s.d.; (n=3). **P<0.01.

See also Figure S3 and Table S2.