Multiple roles for acetylation in the interaction of p300 HAT with ATF-2

Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Biochemistry (Impact Factor: 3.19). 08/2007; 46(28):8207-16. DOI: 10.1021/bi7000054
Source: PubMed

ABSTRACT The transcriptional coactivator paralogues p300 and CBP contain acetyltransferase domains (HAT) and catalyze the lysine acetylation of histones and other proteins as an important aspect of their functions. Prior studies revealed that the basic leucine zipper domain (b-ZIP) of transcription factor ATF-2 (also called CRE-BP1) can interact with the CBP HAT domain. In this study, we have examined the ATF-2 b-ZIP interaction with the p300 HAT domain and shown that p300 HAT autoacetylation can enhance the binding affinity. Pull-down assays revealed that hyperacetylated p300 HAT is more efficiently retained by immobilized ATF-2 b-ZIP than hypoacetylated p300 HAT. Loop deleted p300 HAT lacking autoacetylation was retained about as well as hyperacetylated p300 HAT, suggesting that the loop and ATF-2 compete for p300 HAT binding. While ATF-2 b-ZIP is a weak inhibitor of hypoacetylated p300 HAT acetylation of a histone H4 peptide, hyperacetylated p300 HAT is much more potently inhibited by ATF-2 b-ZIP. Moreover, we showed that ATF-2 b-ZIP could serve as an acetyltransferase substrate for p300 HAT. Using mass spectrometry, two p300 HAT lysine acetylation sites were mapped in ATF-2 b-ZIP. Immunoprecipitation-Western blot analysis with anti-acetyl-lysine antibody revealed that ATF-2 can undergo reversible acetylation in vivo. Mutational analysis of the two ATF-2 b-ZIP acetylation sites revealed their potential contributions to ATF-2-mediated transcriptional activation. Taken together, these studies suggest multiple roles for protein acetylation in the regulation of transcription by p300/CBP and ATF-2.

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    • "A cell could specify different transcriptional programs by controlling the dynamic ratio of ac-p300/p300 through SIRT2. Consistent with this idea, p300 isolated from cells treated with HDAC inhibitors interacts more tightly with p53 (Stiehl et al., 2007) and ATF-2 preferentially interacts with ac-p300 in vitro (Karanam et al., 2007). The dynamics of p300 autoacetylation and deacetylation by SIRT2 provide another level of precision in the fine-tuning of gene regulation programs. "
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    ABSTRACT: Autoacetylation of the p300 histone acetyltransferase controls the transition between VP16-mediated chromatin acetylation and preinitiation complex (PIC) assembly. Currently, it is unknown if and how autoacetylated p300 is deacetylated. We found that the NAD(+)-dependent histone deacetylase SIRT2 deacetylates p300 in vitro and in cells. SIRT2 deacetylates lysine residues in the catalytic domain of p300 and restores binding of p300 to the PIC. RNAi-mediated depletion or chemical inhibition of SIRT2 in cells results in accumulation of acetylated p300. The altered ac-p300/p300 ratio in SIRT2-depleted cells results in decreased p300 recruitment to an integrated VP16-responsive gene and inhibition of transcription. We conclude that p300 undergoes a dynamic cycle of autoacetylation and deacetylation.
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    ABSTRACT: Induction of type I interferons by the transcription factors IRF3 and IRF7 is essential in the initiation of antiviral innate immunity. Activation of IRF3/7 requires C-terminal phosphorylation by the upstream kinases TBK1/IKKi, where IRF3/7 phosphorylation promotes dimerization, and subsequent nuclear translocation to the IFN-beta promoter. Recent studies have described the ubiquitin-editing enzyme A20 as a negative regulator of IRF3 signaling by associating with TBK1/IKKi, however the regulatory mechanism of A20 inhibition remains unclear. Here we describe the adaptor protein, TAX1BP1, as a key regulator of A20 function in terminating signaling to IRF3. Murine embryonic fibroblasts (MEFs) deficient in TAX1BP1 displayed increased amounts of IFN-beta production upon viral challenge compared to WT MEFs. TAX1BP1 inhibited virus-mediated activation of IRF3 at the level of TBK1/IKKi. TAX1BP1 and A20 blocked antiviral signaling by disrupting K63-linked polyubiquitination of TBK1/IKKi independently of the A20 deubiquitination (DUB) domain. Furthermore, TAX1BP1 was required for A20 effector function as A20 was defective for the targeting and inactivation of TBK1 and IKKi in Tax1bp1–/– MEFs. Additionally, we found the E3 ubiquitin ligase TRAF3 to play a critical role in promoting TBK1/IKKi ubiquitination. Collectively, our results demonstrate TBK1/IKKi to be novel substrates for A20 and further identifies a novel mechanism whereby A20 and TAX1BP1 restrict antiviral signaling by disrupting a TRAF3/TBK1/IKKi signaling complex. Several viruses utilize a number of strategies to evade the host innate immune response by inhibiting the production of type I interferons. The Human T-cell leukemia virus type 1 (HTLV-1) has been shown to block interferon signaling, however the mechanism of inhibition is poorly understood. We show here that the HTLV-1 encoded protein, Tax plays a critical role in blunting the activation of type I interferons. Tax expression rendered MEFs hyper-permissive in supporting virus replication. Correspondingly, Tax blocked the production of IFN-beta. Interestingly, Tax did not require NEMO interaction to inhibit antiviral signaling to IRF3/7. Instead, Tax targeted RIP1 and further blocked IRF7 K63-linked polyubiquitination. Altogether, we show that Tax inhibits IFN activation by disrupting the ubiquitin dependent activation of IRF7 mediated by RIP1.


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