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Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling - PubMed

  • ️Mon Jan 01 1996

Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling

J J Zhang et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Interferon gamma (IFN-gamma) induces rapid tyrosine phosphorylation of the latent cytoplasmic transcription factor, Stat1, which then forms homodimers, translocates to the nucleus and participates in IFN-gamma-induced transcription. However, little is known of the interactions between Stat1 and the general transcription machinery during transcriptional activation. We show here that Stat1 can directly interact with the CREB-binding protein (CBP)/p300 family of transcriptional coactivators. Specifically, two interaction regions were identified: the amino-terminal region of Stat1 interacts with the CREB-binding domain of CBP/p300 and the carboxyl-terminal region of Stat1 interacts with the domain of CBP/p300 that binds adenovirus E1A protein. Transfection experiments suggest a role for these interactions in IFN-gamma-induced transcription. Because CBP/p300-binding is required for the adenovirus E1A protein to regulate transcription of many genes during viral replication and cellular transformation, it is possible that the anti-viral effect of IFN-gamma is based at least in part on direct competition by nuclear Stat1 with E1A for CBP/p300 binding.

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Figures

Figure 1
Figure 1

Stat1 interacts with CBP/p300 at two distinct regions. (A) A schematic diagram of CBP and variants of Stat1. The fragments of CBP used as GST-fusion proteins were residues: 1-170, 1-356, 451-682, 571-687, 1-116 plus 720-1459, 1680-1891, and 2164-2325. The fragment of p300 used was residues 566-664. NR, nuclear hormone receptor; C/H, cysteine/histidine-rich region; Stat1tc, a truncated version of Stat1 (residues 132-713); ID, interaction domain; Y, Tyr-701; S, Ser-727. (B) Stat1α interacts with two different regions of CBP/p300. Purified Stat1α protein was incubated with different regions of CBP/p300 as GST-fusion proteins bound to glutathione-Sepharose-beads. The bound proteins were analyzed by Western blotting using an anti-Stat1 antibody. Equivalent amounts of various GST-fusion proteins were used in the assay, as shown by Coomassie staining. (C) Two different interaction regions between Stat1 and CBP/p300. Purified Stat1α, Stat1β, or Stat1tc proteins were incubated with Sepharose beads-bound GST-fusion proteins containing fragments of CBP/p300 that interacted with Stat1α in B and analyzed by Western blotting.

Figure 2
Figure 2

Endogenous Stat1 homodimers interact with CBP/p300. (A) Both unphosphorylated and phosphorylated cellular Stat1α and β interact with the CREB-binding domain of CBP/p300. GST-fusion proteins of CBP/p300 bound on glutathione-Sepharose beads were incubated with nuclear extracts from untreated or IFN-γ-treated U3A cells deficient for endogenous Stat1 and stably expressing FLAG-tagged Stat1α or β. The specifically bound Stat1 proteins were analyzed by Western blotting using an anti-FLAG antibody. 84F, extracts from cells expressing FLAG-tagged Stat1β; 91F, extracts from cells expressing FLAG-tagged Stat1α. (B) Interaction between cellular Stat1α and the E1A-binding domain of CBP requires the carboxyl terminus of Stat1α. Whole-cell extracts from treated cells stably expressing FLAG-tagged Stat1α or β as in A were incubated with GST-fusion proteins and the resulting bound Stat1 analyzed as above. (C) The CREB-binding domain of CBP/p300 interacts with Stat1α dimer bound to DNA. Nuclear extracts from IFN-γ-treated U3A cells reconstituted with FLAG-tagged Stat1α were incubated with a 32P-labeled Stat1-DNA-binding site before being exposed to various purified GST-fusion proteins, and the resulting DNA–protein complexes were analyzed by EMSA.

Figure 3
Figure 3

IFN-γ-induced transcriptional activation by Stat1α is enhanced by CBP and repressed by E1A. (A) IFN-γ induces reporter activity in cells expressing endogenous Stat1α. (B) Overexpression of CBP enhances IFN-γ-induced transcription. (C) E1A inhibits IFN-γ-induced Stat1α transactivation by competing for CBP/p300-binding. U2-OS cells were transfected with 0.5 μg of luciferase reporter, 0.3 μg of CMVβgal, and various amounts of RSVCBP, CMVE1A12S, and CMVE1A(Δ64-68) as indicated. Twenty-four hours after transfection, cells were treated with 5 ng of IFN-γ per ml for 6 hr and harvested for luciferase assay and β-gal assay. All transfections were done in triplicates. Results shown are the mean ± SD of two to four experiments. Results from only the IFN-γ-treated samples are presented in B and C as reporter activities from untreated cells did not change significantly.

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