Figure 1. Trans-Bound RARs on ERα Active Enhancers Regulate E₂-Liganded Transcription Activation.

(A) Heatmaps of GRO-seq and ChIP-seq data (±E₂) data for 1,333 ERα active enhancers showing strong E₂-induced eRNA transcription and E₂-enhanced binding of both RARα and RARγ, respectively.

(B) Heatmaps of GRO-seq and ChIP-seq for a control group of ERα non-active enhancers exhibiting no RARα/γ binding and no significant E₂-induced eRNA transcription.

(C) For the 1,333 ERα active enhancers, heatmaps of ChIP-seq data for the wild-type and two DNA-binding mutants of RARα/γ (+RA and E₂) show that their association with these enhancers is DNA binding independent.

(D) Knockdown of either RARα or RARγ by shRNA inhibits ERα target gene induction by E₂, as demonstrated by qPCR analysis.

(E) Knockdown of either RARα or RARγ using shRNA inhibits expression of the RAR cis-binding target HoxA1 gene in response to RA, as shown by qPCR analysis.

(F) RARs are required for the E₂-liganded activation of ERα active enhancers and their targets, as shown by GRO-seq boxplots. No significant effects were found for either ERα non-active enhancers or non-ERα enhancers.

(G) The pBox mutant RARγ fails to rescue expression of its cis-binding target HoxA1 after knockdown of endogenous RARγ. In contrast, both wild-type and pBox mutant RARγ can rescue expression of the trans-binding target GREB1. For details regarding rescue experiments see Extended Experimental Procedures.

(H) Heatmap showing that knockdown of RARs does not affect ERα binding at the 1,333 active enhancers.

Data are represented as mean ± SEM (NS not significant, **P<0.01, ***P<0.001). See also Figure S1 and S2.