Postrecruitment Regulation of RNA Polymerase II Directs Rapid Signaling Responses at the Promoters of Estrogen Target Genes

The majority of estrogen-regulated promoters are preloaded with Pol II prior to E2 treatment. (A) Gene expression microarray analysis in MCF-7 cells with (+) or without (−) E2 (3 h). (B) ChIP-chip analyses for Pol II at the promoters of all E2-regulated genes in MCF-7 cells ± E2 (45 min). (C) Heat maps of ChIP-chip for Pol II at the promoters of E2-stimulated and E2-repressed genes with (+) or without (−) E2 (45 min). (D) Averaging of Pol II promoter ChIP-chip signals for three classes of E2-stimulated genes with (+) or without (−) E2 (45 min).

Preloaded Pol II localizes to TSS prior to E2 treatment and moves into the body of the gene upon activation. (A) Comparison of Pol II occupancy at the TSS (−200 to +100 bp) and downstream regions (DS; +300 to +500 bp) for all Pol II preloaded genes (n = 171), as determined by ChIP-chip with (+) or without (−) E2 (45 min). The associated t test P value is shown. (B and C) Gene-specific ChIP analyses of Pol II binding at the TSS and +1-kb regions of Pol II recruited (B) and Pol II preloaded (C) E2-stimulated genes with (+) or without (−) E2 (45 min). Each bar represents the mean plus the SEM (n ≥ 3).

Preloaded Pol II is partially phosphorylated at Ser5, but not Ser2, prior to E2 treatment, and Pol II CTD phosphorylation increases in conjunction with Cdk9 recruitment upon E2 treatment. (A to C) Gene-specific ChIP analyses of Pol II Ser5P (A) and Pol II Ser2 (B), as well as Cdk9 occupancy (C), at the TSS and +1-kb regions of representative E2-stimulated genes with (+) or without (−) E2 (45 min). Each bar represents the mean plus the SEM (n ≥ 3).

NELF binds to promoters containing preloaded Pol II and prevents transcription through the gene prior to E2 treatment. (A) Averaging of Pol II and NELF-A ChIP-chip signals at 221 E2-stimulated promoters for the following conditions: Pol II -E2 (blue), Pol II +E2 (red), and NELF-A −E2 (green). (B) Gene-specific ChIP analysis of NELF-A binding at promoters of representative E2-stimulated genes with (+) or without (−) E2 (45 min). Each bar represents the mean plus the SEM (n ≥ 3). (C) Western blot showing the shRNA-mediated depletion of NELF-B (shNELF-B) in MCF-7 cells versus a GFP knockdown control (shGFP). NELF-B, as well as NELF-A and NELF-E, protein levels decrease in the shNELF-B cells. ACTB, loading control. (D and E) Gene-specific analysis of mRNA expression for representative Pol II recruited (D) and Pol II preloaded (E) genes in MCF-7 cells with or without NELF knockdown with (+) or without (−) E2 (3 h). Each bar represents the mean plus the SEM (n ≥ 3). (F) Summary of observations regarding the E2-dependent regulation of Pol II binding and activity. See the text for details.

Pol II recruited and Pol II preloaded genes are enriched in different ontological categories. GO analysis was performed by using Genecodis and filtered for the universe of applicable GO terms (see Materials and Methods). Each gene set in each category was expressed as the percentage of genes in the class and assigned its own P value (chi-square analysis).