Mechanism by which the IFN-beta enhanceosome activates transcription - PubMed (original) (raw)
Mechanism by which the IFN-beta enhanceosome activates transcription
J Yie et al. Proc Natl Acad Sci U S A. 1999.
Abstract
We demonstrate that in contrast to previous findings by using simple synthetic promoters or activators, the natural IFN-beta enhanceosome activates transcription by causing a dramatic increase of the rate by which preinitiation complexes assemble at the promoter. This effect totally depends on the recruitment of the CBP-PolII holoenzyme by the enhanceosome, because its depletion from the extract decelerates the rate of transcription. However, addition of the CBP-PolII holoenzyme back to these extracts fully restores the speed by which the enhanceosome activates transcription. Strikingly, preincubation of the enhanceosome with the CBP-RNA PolII holoenzyme complex results in instant assembly of preinitiation complexes. In contrast, individual IFN-beta gene activators function solely by increasing the number of functional preinitiation complexes and not the rate of their assembly. Thus, fast recruitment of the CBP-RNA PolII holoenzyme complex is critical for the rapid activation of IFN-beta gene expression by virus infection.
Figures
Figure 1
The IFN-β enhanceosome stimulates the rate of PIC assembly. (A) The IFN-β enhanceosome was assembled on the wild-type enhancer (see Materials and Methods) and was incubated with a HNE for the indicated amounts of time shown at the top part of the figure. Sarcosyl and NTPs were added, and the incubations were continued for an additional 60 min. Transcript levels were detected by primer extension, quantitated by PhosphorImager, and used as a measure of PIC formation. The right part of the figure shows the relative levels of transcription plotted as a function of time along with a best-fit curve as described in Materials and Methods. Shown is one of six independent experiments. The variability from experiment to experiment for the half-time of PIC formation was <8% for the basal level and <10% for activated transcription. (B) Same as in A but the template contains half-helical DNA insertion between PRDI and PRDII (IFN-β I/II6 CAT). Shown is one of three independent experiments. The variability from experiment to experiment was <12% for the basal and activated transcription. (C) Same as in A but the template contains four copies of the PRDII element cloned upstream of the IFN-β TATA box. The activator used was NF-κB. Shown is one of three independent experiments, and the variability was <10% and <15% for basal and activated transcription, respectively. (D) Same as in A but the template contains four copies of the PRDIII-I element cloned upstream of the IFN-β TATA box. The activator used was IRF-1. Shown is one of two independent experiments, and the variability was <7% and 9% for basal and activated transcription, respectively. (E) Same as in A but the enhanceosome used at Middle lacks IRF-1’s activation domain, whereas that used at Bottom lacks p65’s activation domain. Shown is one of two independent experiments, and the variability was <8%, <10%, and 7%, respectively.
Figure 2
Recruitment of the CBP-RNA PolII complex by the enhanceosome is the rate-limiting step that determines the speed of PIC assembly at the IFN-β promoter. (A) The IFN-β enhanceosome recruits CBP and the PolII holoenzyme. A biotinylated IFN-β enhancer oligonucleotide (−105 to −40) with or without the enhanceosome was coupled to magnetic Dynabeads and was incubated with 500 μg of HeLa nuclear extract. Western blotting by using CBP, PolII, TBP, TFIIB, TFIIEβ, and SRB7 specific antibodies detected bound proteins. Lane 1 detects the indicated proteins in the HNE, lane 2 corresponds to the precipitated proteins in the absence of the enhanceosome, whereas lane 3 depicts the proteins precipitated by the enhanceosome. (B) Antibodies against CBP coprecipitate components of the RNA PolII holoenzyme. HNE (3.6 mg) was incubated with CBP and p300 specific antibodies (10 μg each), and 1/100 of the pellet or supernatant was analyzed by Western blotting by using antibodies against the depicted. (C) CBP mediates PolII recruitment by the enhanceosome; same as in A except that a CBP-depleted HNE (lane 2) was used in parallel with a complete extract (lane 3). (D) Removal of CBP-RNA PolII holoenzyme from the extract decelerates PIC formation at the IFN-β promoter. Shown is an in vitro transcription experiment performed as detailed in Fig. 1 except that complete (lanes 5–8), CBP-depleted (lanes 9–12), or reconstituted nuclear extracts (lanes 1–4) were used. (E) Same as in D except that the CBP pellet was preincubated with the enhanceosome (lanes 7–9) before the addition of the extract for the indicated amounts of time.
Figure 3
Enhanceosome–CBP RNA PolII holoenzyme interactions are required at the early steps of PIC assembly. (A) Immobilized IFN-β enhancer–promoter DNA with (lane 3) or without (lane 2) the enhanceosome was preincubated with HNE followed by washes and Western blot analysis by using antibodies against the depicted proteins. (B) An immobilized IFN-β enhancer–promoter DNA with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) the enhanceosome was preincubated with a complete nuclear extract (lanes 1 and 2) or with the CBP/p300-depleted extract (lanes 3–8), followed by wash and addition of NTPs to initiate transcription. In lanes 5 and 6, the CBP/p300 immunoprecipitate was added before wash whereas in lanes 7 and 8, the immunoprecipitate was added after washing the beads. (C) Same as in A except a CBP-depleted HNE (lanes 2, 5, and 6) was used in parallel with the complete HNE (lanes 1, 3, and 4).
Figure 4
CBP/p300 that is not complexed with the PolII holoenzyme inhibits enhanceosome-dependent transcription. (A) Shown is an in vitro transcription experiment by using the IFN-β enhancer–promoter as template and the enhanceosome as an activator (lanes 1–3), PRDII4CAT and NF-κB (lanes 4–6), PRDI-III3CAT and IRF-1 (lanes 7–9), or G5E1BCAT and GAL4 p65 or GAL4 IRF-1 fused proteins (lanes 10–18). In lanes 3, 6, 9, 12, 14, 16, and 18, we added 200 ng of GST-CBP (1–771) protein. The top part of the figure depicts a diagrammatic illustration of the p65 protein. The two independent activation domains (TA1 and TA2) as well as the synergism-specific domain required for interaction with CBP are indicated. (B) Shown is an in vitro transcription experiment by using the IFN-β enhanceosome along with 100 ng of baculovirus-expressed and purified, full-length p300. In lanes 1–6, a complete HNE was used whereas in lanes 7–9, a CBP/p300-depleted HNE was used. Recombinant p300 was added either with the enhanceosome lanes 3 and 9 or after preincubation with the complete (lane 6) or CBP/p300-depleted (lane 10) HNE. (C) Recombinant His-tagged p300 (lane 2) was incubated with a HNE followed by the addition of Ni2+-nitrilotriacetic acid agarose beads. The precipitated complexes were washed extensively and immunoblotted by using antibodies against PolII and SRB7. Lane 1 represents nonspecific binding to the agarose beads.
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