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. 2009 Mar;29(5):1123-33.
doi: 10.1128/MCB.00841-08. Epub 2008 Dec 22.

Postrecruitment regulation of RNA polymerase II directs rapid signaling responses at the promoters of estrogen target genes

Miltiadis Kininis  1 , Gary D Isaacs, Leighton J Core, Nasun Hah, W Lee Kraus
Affiliations

Postrecruitment regulation of RNA polymerase II directs rapid signaling responses at the promoters of estrogen target genes

Miltiadis Kininis et al. Mol Cell Biol. 2009 Mar.

Abstract

Under classical models for signal-dependent transcription in eukaryotes, DNA-binding activator proteins regulate the recruitment of RNA polymerase II (Pol II) to a set of target promoters. However, recent studies, as well as our results herein, show that Pol II is widely distributed (i.e., "preloaded") at the promoters of many genes prior to specific signaling events. How Pol II recruitment and Pol II preloading fit within a unified model of gene regulation is unclear. In addition, the mechanisms through which cellular signals activate preloaded Pol II across mammalian genomes remain largely unknown. We show here that the predominant genomic outcome of estrogen signaling is the postrecruitment regulation of Pol II activity at target gene promoters, likely through specific changes in Pol II phosphorylation rather than through recruitment of Pol II to the promoters. Furthermore, we show that negative elongation factor binds to estrogen target promoters in conjunction with preloaded Pol II and represses gene expression until the appropriate signal is received. Finally, our studies reveal that the estrogen-dependent activation of preloaded Pol II facilitates rapid gene regulatory responses which play important physiological roles in regulating estrogen signaling itself. Our results reveal a broad use of postrecruitment Pol II regulation by the estrogen signaling pathway, a mode of regulation that is likely to apply to a wide variety of signal-regulated pathways.

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Figures

FIG. 1.
FIG. 1.
The promoters of many unexpressed genes are preloaded with Pol II, which in many cases is prephosphorylated at Ser5 of the CTD. (A) Heat map of ChIP-chip binding analyses for Pol II at the promoters of expressed and unexpressed genes in MCF-7 cells. (B) Averaging of Pol II (blue) and Ser5P (green) promoter ChIP-chip signals for expressed, unexpressed Pol−, and unexpressed Pol+ genes. (C) Flowchart outlining the identification of promoters where Pol II is regulated at a postrecruitment level. (D) Averaging of Pol II (blue) and Ser5P (green) promoter ChIP-chip signals for unexpressed Pol+ genes with classification based on Ser5P status. (E) Gene-specific ChIP analyses of Pol II, TFIIB, and Ser5P binding at TSS (black) and downstream regions (+1 kb) (yellow) for a set of representative genes. Each bar represents the mean plus the standard error of the mean (SEM) (n ≥ 3).
FIG. 2.
FIG. 2.
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).
FIG. 3.
FIG. 3.
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).
FIG. 4.
FIG. 4.
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).
FIG. 5.
FIG. 5.
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.
FIG. 6.
FIG. 6.
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).
FIG. 7.
FIG. 7.
Pol II preloaded genes are induced by E2 more rapidly than Pol II recruited genes. (A and B) Gene-specific analysis of mRNA expression for representative Pol II preloaded (A) or Pol II recruited (B) genes in MCF-7 cells before or after E2 treatment for 1, 3, and 6 h. The E2 response is shown as the percentage of the maximum E2 response for each individual gene. Each bar represents the mean plus the SEM (n ≥ 3). (C) Averaging of E2 responses for all Pol II preloaded and Pol II recruited genes shown in panels A and B, respectively. Error bars represent the SEM. The associated t test P value for differences between the two points at 1 h is shown. (D) Scatterplot of absolute E2 responses for all Pol II preloaded and Pol II recruited genes, as determined by expression microarray analysis of MCF-7 cells treated with or without E2 for 3 h. The horizontal lines represent the mean for each class of genes. (E) Biological functions of E2-stimulated genes containing preloaded Pol II. Pol II preloaded genes, which are induced earlier than Pol II recruited genes, play key roles in propagating or attenuating estrogen signaling. See the text for details.
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