The glucocorticoid receptor blocks P-TEFb recruitment by NFkappaB to effect promoter-specific transcriptional repression

Abstract

To investigate the determinants of promoter-specific gene regulation by the glucocorticoid receptor (GR), we compared the composition and function of regulatory complexes at two NFkappaB-responsive genes that are differentially regulated by GR. Transcription of the IL-8 and IkappaBalpha genes is stimulated by TNFalpha in A549 cells, but GR selectively represses IL-8 mRNA synthesis by inhibiting Ser2 phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). The proximal kappaB elements at these genes differ in sequence by a single base pair, and both recruited RelA and p50. Surprisingly, GR was recruited to both of these elements, despite the fact that GR failed to repress the IkappaBalpha promoter. Rather, the regulatory complexes formed at IL-8 and IkappaBalpha were distinguished by differential recruitment of the Ser2 CTD kinase, P-TEFb. Disruption of P-TEFb function by the Cdk-inhibitor, DRB, or by small interfering RNA selectively blocked TNFalpha stimulation of IL-8 mRNA production. GR competed with P-TEFb recruitment to the IL-8 promoter. Strikingly, IL-8 mRNA synthesis was repressed by GR at a post-initiation step, demonstrating that promoter proximal regulatory sequences assemble complexes that impact early and late stages of mRNA synthesis. Thus, GR accomplishes selective repression by targeting promoter-specific components of NFkappaB regulatory complexes.

Figure 1.

GR differentially regulates NFκB at the IL-8 and IκBα genes in A549 cells. (A) Steady-state IL-8 and IκBα mRNA levels are stimulated in A549 cells treated for 2 h with TNFα (2.5 ng/mL). Cotreatment with TNFα and dex (100 nM) selectively repressed IL-8 mRNA accumulation. (B) A549 cells were transfected with IL-8-Luc, IκB-Luc, IL-8mut-Luc, or IκBmut-Luc (50 ng) reporter genes and RSV-βgal (50 ng) followed by treatment with combinations of TNFα and dex as indicated for 5 h. Luciferase units were normalized to β-galactosidase activity. (C) Diagram of the human IL-8 and IκBα genes. The promoter proximal κB site sequences are boxed.

Figure 2.

NFκB and GR factor occupancy does not distinguish the IL-8 and IκBα gene regulatory regions. In A, B, and D, ChIP assays were performed on A549 cells treated for 2 h as indicated using polyclonal RelA (A), p50 (B), or GR(N499) (D) antibodies. (A) RelA immunoprecipitates were probed using IκBα promoter primers (–168 to +21) and normalized to U6 snRNA genomic fragment. (B) p50 immunoprecipitates were analyzed for the presence of sequences from the IL-8 and IκBα promoter and downstream regions as indicated; fold enrichment values were normalized to a control region of the HSP70 gene. (C) qRT–PCR analysis of RNA isolated from A549 cells treated with combinations of TNFα and Kamebakaurin for 2 h as indicated. (D) GR immunoprecipitates were probed using primers specific for the IL-8 and IκBα promoter regions and normalized to the U6 control region.

Figure 3.

IL-8 transcription is repressed by dex at a post-initiation step. (A) Nuclear run-on assays were performed on nuclei isolated from A549 cells treated for 2 h with combinations of TNF and dex as indicated. RNA was hybridized to cDNA fragments of the human IL-8, IκBα, and GAPDH genes. (B) ChIP assays were performed on A549 cells treated for 2 h with combinations of TNFα and dex as indicated using a Pol II polyclonal antibody and probed for sequences located in the IL-8 promoter and 3′-UTR. Fold enrichment values for the IL-8 regions were normalized to a control region from the HSP70 gene.

Figure 4.

The P-TEFb kinase complex is differentially recruited to and utilized by the IL-8 and IκBα genes in A549 cells. ChIP assays using polyclonal antibodies to Cdk9 (A) or Cyclin T1 (B) were performed on A549 cells treated with combinations of TNF and dex as indicated for 2 h. The immunoprecipitates were probed using qRT–PCR for regions of the IL-8 and IκBα promoter and downstream regions as indicated. The fold enrichment data were normalized to a HSP70 control genomic region.

Figure 5.

IκBα mRNA synthesis is independent of P-TEFb function. (A–C) qRT–PCR analysis of RNA isolated from A549 cells treated with combinations of TNF, dex, and DRB as indicated for 2 h. The abundance of IL-8 and IκBα mRNA were normalized to a RPL19 control. (B,C) A549 cells were transfected with siRNA oligos directed against the Cdk9 or Cyclin T1 subunits of P-TEFb or siRNA control for 26 h. The cells were untreated or treated with TNF (2.5 ng/mL) for 2 h and total RNA was isolated and analyzed by qRT–PCR using primers specific to the IL-8 (B) and IκBα (C) transcripts. These values were normalized to the RPL19 gene.

Figure 6.

Interaction between RelA and Cyclin T1 is diminished by GR in vitro. Purified GST-Cyclin T1 (1–272) or GST alone was incubated with purified recombinant RelA. The resulting complexes were isolated and washed on glutathione agarose beads and then were incubated in the presence or absence of 100 nM GR. The amount of RelA bound to the beads was determined by Western blot using sc-109.

Figure 7.

A model for differential regulation of the IL-8 and IκBα genes by the glucocorticoid receptor. Heterodimeric complexes of RelA and p50 occupy both the promoter proximal κB elements of these genes; however, P-TEFb is selectively recruited to the IL-8 gene. Dex treatment results in GR recruitment to both genes, which results in loss of P-TEFb recruitment to the IL-8 promoter, suggesting a direct competition, but has no effect at IκBα.