The C-Terminal Transactivation Domain of STAT1 Has a Gene-Specific Role in Transactivation and Cofactor Recruitment

Abstract

STAT1 has a key role in the regulation of innate and adaptive immunity by inducing transcriptional changes in response to cytokines, such as all types of interferons (IFN). STAT1 exist as two splice isoforms, which differ in regard to the C-terminal transactivation domain (TAD). STAT1β lacks the C-terminal TAD and has been previously reported to be a weaker transcriptional activator than STAT1α, although this was strongly dependent on the target gene. The mechanism of this context-dependent effects remained unclear. By using macrophages from mice that only express STAT1β, we investigated the role of the C-terminal TAD during the distinct steps of transcriptional activation of selected target genes in response to IFNγ. We show that the STAT1 C-terminal TAD is absolutely required for the recruitment of RNA polymerase II (Pol II) and for the establishment of active histone marks at the class II major histocompatibility complex transactivator (CIIta) promoter IV, whereas it is dispensable for histone acetylation at the guanylate binding protein 2 (Gbp2) promoter but required for an efficient recruitment of Pol II, which correlated with a strongly reduced, but not absent, transcriptional activity. IFNγ-induced expression of Irf7, which is mediated by STAT1 in complex with STAT2 and IRF9, did not rely on the presence of the C-terminal TAD of STAT1. Moreover, we show for the first time that the STAT1 C-terminal TAD is required for an efficient recruitment of components of the core Mediator complex to the IFN regulatory factor (Irf) 1 and Irf8 promoters, which both harbor an open chromatin state under basal conditions. Our study identified novel functions of the STAT1 C-terminal TAD in transcriptional activation and provides mechanistic explanations for the gene-specific transcriptional activity of STAT1β.

Keywords: IFNγ; IRF8; RNA polymerase II; interferon regulatory factor 1 (IRF1); macrophage; mediator; signal transducer and activator of transcription; transcriptional coactivator.

Figure 1

Transcriptional activity of STAT1β at the Irf1, Irf8, CIIta, Gbp2, and Irf7 genes. BMDMs from WT and Stat1^β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h, -). Total RNA (A–D, G) or protein extracts (E) were isolated. Irf1 (A), Irf8 (B), CIIta (C), Gbp2 (D), and Irf7 (G) pre-mRNA expression was determined by RT-qPCR. Data were normalized to the housekeeping gene Ube2d2. Mean values ± SE from three independent experiments are shown. (E) IRF1 protein levels were determined by Western blotting. ERK p42 was used as loading control. Data are as representative of two independent experiments. Original uncropped blots are shown in Supplementary Figure 1. (F) BMDMs from WT and Stat1^β/β mice were stimulated with IFNγ for 24 h. MHC class II surface levels were determined by flow cytometry. Median fluorescence intensities (MdFI) ±standard error (SE) from two independent experiments are shown. (A–D, F–G) ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001. Significances are only indicated for the comparisons between genotypes.

Figure 2

STAT1 and IRF1 binding to the CIIta promoter IV (pIV) and the Gbp2 promoters and IFNγ-induced histone modifications. (A,B) Schematic representation of the murine CIIta and Gbp2 promoter regions. STAT1 and IRF1 binding sites, the TSS, and the position of the primers used for the ChIP analyses are depicted.(C–M) BMDMs from WT and Stat1^β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). STAT1 and IRF1 binding to the CIIta (C, D) and the Gbp2 (E–G) promoter binding sites was analyzed by ChIP. H3 pan-acetylation (H3ac), H4 pan-acetylation (H4ac), and H3 lysine 4 trimethylation (H3K4me3) around the CIIta (H–J) and the Gbp2 (K–M) TSS was determined by ChIP. Data were normalized to the input control (C–G) and the total levels of H3 (H–M). Mean values ± SE from three (C–G, H,J,K,M) or four (I,L) independent experiments are shown; ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001. Significances are only indicated for the comparisons between genotypes.

Figure 3

Promoter occupancy of Pol II, S5 phosphorylated Pol II (S5pPol II), and S2 phosphorylated Pol II (S2pPol II) around the CIIta and Gbp2 TSS and of S2pPol II at the Gbp2 gene body. BMDMs from WT and Stat1^β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). The association of Pol II, S5pPol II, and S2pPol II with the TSS of (A–C) CIIta pIV and (D–F) Gbp2 TSS, and (G) association of S2pPol II with the Gbp2 gene body was determined by ChIP. Data were normalized to the input control. Mean values ± SE from three (D,G), four (A,B) or five (C,E,F) independent experiments are shown; ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001. Significances are only indicated for the comparisons between genotypes.

Figure 4

IFNγ induced expression of Irf1 and Irf7 in Stat1^β/β cells in the absence of STAT2 or IRF9. BMDMs derived from Stat1^β/β, Stat1^β/βStat2^−/− and Stat1^β/βIrf9^−/− mice were stimulated with IFNγ for the times indicated or left untreated (0 h, -). (A) Protein was isolated and Tyr701-phosphorylated STAT1 (pSTAT1) and STAT1 protein levels determined by Western blotting. ERK p42 was used as loading control. One representative out of three independent experiments is shown. Original uncropped blots are shown in Supplementary Figure 2. (B,C) Total RNA was isolated and Irf1 (B) and Irf7 (C) mRNA expression was determined by RT-qPCR. Data were normalized to Ube2d2. Mean values ± SE from three (C) or four (B) independent experiments are shown. ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001. Significances are only indicated for the comparisons between genotypes.

Figure 5

STAT1 and IRF1 binding to the Irf7 and Irf8 promoters and H3ac and H4ac and H3K4me3 around the Irf1, Irf8, and Irf7 TSS before and after IFNγ treatment. (A–C) Schematic representation of the murine Irf7 (A), Irf1 (B), and Irf8 (C) promoter regions. GAS and ISRE sites, the TSS and the position of the primers used for the ChIP analyses are depicted. (D–O) BMDMs from WT and Stat1^β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). STAT1 and STAT2 binding at the Irf7 ISRE (D,E) and STAT1 binding at the Irf8 (F) GAS element was analyzed by ChIP. H3 pan-acetylation (H3ac), H4 pan-acetylation (H4ac), and H3 lysine 4 trimethylation (H3K4me3) around the Irf1 (G–I), the Irf8 (J–L), and the Irf7 (M–O) TSS was determined by ChIP. Data were normalized to the input control (D–F) or the total levels of H3 (G–O). Mean values ± SE from three to four independent experiments are shown; ^**P ≤ 0.01, ^***P ≤ 0.001. Significances are only indicated for the comparisons between genotypes.

Figure 6

Recruitment and phosphorylation of Pol II at Irf1gene and occupancy of TFIIH, p-TEFb, and Mediator components at the Irf1 TSS. (A) Schematic representation of Pol II, GTFs, and the Mediator complex at a GAS-driven gene promoter. Components of the Mediator, TFIIH, and p-TEFb complexes analyzed by ChIP are indicated. (B–L) BMDMs from WT and Stat1^β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). Association of Pol II, S5pPol II, and S2pPol II with the Irf1 promoter around the TSS (B–D) and of S2pPol II within the Irf1 gene body (E). Association of ERCC3 (F) and CDK9 (G) at the Irf1 TSS and of MED18 (H), MED4 (I), MED24 (J), MED26 (K), and MED1 (L) at the Irf1 GAS. Data were normalized to the input control. Mean values ± SE from two (H) or three (all others) independent experiments are shown; ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001.

Figure 7

Recruitment and phosphorylation of Pol II at the Irf8 and Irf7 genes and occupancy of Mediator components at the Irf8 promoter. (A–K) BMDMs from WT and Stat1^β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). Association of Pol II, S5pPol II, and S2pPol II with the Irf8 (A–C) and the Irf7 promoters (H–J) around the TSS and of S2pPol II within the respective gene bodies (D,K) was determined by ChIP. The promoter occupancy of MED26 (E), MED1 (F), and MED24 (G) at the Irf8 GAS. Data were normalized to the input control. Mean values ± SE from two (K), three (A–I) or four (J) independent experiments are shown; ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001.