The current proposal is a broad investigation with seven specific aims which are directed towards two general goals: 1) understanding the structure and function of a multi-component transcriptional control element; 2) the signaling pathways the converge to activate expression of the IFNbeta gene in response to viral infection.
Aim 1 is to determine the structure of the multi-protein complex that assembles on the IFNbeta enhancer upon activation. This """"""""enhanceosome"""""""" contains binding sites for 5 factors: ATF-2/cJun, IRF-1, NFkB and HMG I(Y) within a 50 bp stretch of DNA. A combination of molecular biology and biochemical approaches to DNA-protein interactions are proposed for the Maniatis lab, including chemical protection and DNA-protein cross-linking analyses and mutagenesis of either DNA or protein. These techniques would address specific issues such as the coupling of DNA bending to protein binding, the orientation of bZIP heterodimer complexes and the configuration of the HMG: NFkB ternary complex. Collaborations that provide biophysical approaches are described including the use of x-ray crystallography for the ATF/cJun : DNA complex or the NFkB/HMG: DNA complex plus atomic force microscopy for studies of the whole complex.
A second aim will identify the """"""""correct"""""""" transcription factor that functions at region III and I since the current assignment of IRF-1 is suspect.
Aim 3 studies the putative DSP1 repressor, cloning the protein from humans, then testing its role in repression of the IFNbeta enhancer in uninduced cells by interactions with NFkB.
Aim 4 establishes a fully reconstituted in vitro transcription system to study activation of transcription via the enhanceosome. The role of transcription activators in regulating the function of the pre-initiation complex and the RNA polymerase holoenzyme will be studied.
Aim 5 focuses on the signal transduction pathways that affect the function of ATF/cJun and NFkB. SAPK is being tested for ATF while dsRNA-dependent kinase is being tested for inducing the degradation of IkB, the inhibitor of NFkB. The proteolytic processing of the NFkB protein p105 to p50 is the focus of aim 6. Two major goals are purification of the protease; testing the role of phosphorylation in the ubiquitination of p105 which is reported to be necessary for degradation.
The final aim extends recent work that identified a multi-protein complex as a new IkB kinase. Phosphorylation of IkB precedes its ubiquitination and subsequent degradation. The goals are to purify the components of the kinase, study the regulation of the kinase by a putative upstream kinase and find other targets for the kinase.
| Zhao, Mingwei; Zhang, Jiangwen; Phatnani, Hemali et al. (2012) Stochastic expression of the interferon-? gene. PLoS Biol 10:e1001249 |
| Ng, Sze-Ling; Friedman, Brad A; Schmid, Sonja et al. (2011) I?B kinase epsilon (IKK(epsilon)) regulates the balance between type I and type II interferon responses. Proc Natl Acad Sci U S A 108:21170-5 |
| Ye, Junqiang; Chen, Shuibing; Maniatis, Tom (2011) Cardiac glycosides are potent inhibitors of interferon-? gene expression. Nat Chem Biol 7:25-33 |
| Ye, Junqiang; Maniatis, Tom (2011) A prion-like trigger of antiviral signaling. Cell 146:348-50 |
| Ye, Junqiang; Maniatis, Tom (2011) Negative regulation of interferon-ýý gene expression during acute and persistent virus infections. PLoS One 6:e20681 |
| Huang, Hon-Ren; Chen, Zhijian J; Kunes, Sam et al. (2010) Endocytic pathway is required for Drosophila Toll innate immune signaling. Proc Natl Acad Sci U S A 107:8322-7 |
| Falvo, James V; Lin, Charles H; Tsytsykova, Alla V et al. (2008) A dimer-specific function of the transcription factor NFATp. Proc Natl Acad Sci U S A 105:19637-42 |
| Panne, Daniel; Maniatis, Tom; Harrison, Stephen C (2007) An atomic model of the interferon-beta enhanceosome. Cell 129:1111-23 |
| McWhirter, Sarah M; Fitzgerald, Katherine A; Rosains, Jacqueline et al. (2004) IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc Natl Acad Sci U S A 101:233-8 |
| Yang, Hongmei; Lin, Charles H; Ma, Gang et al. (2003) Interferon regulatory factor-7 synergizes with other transcription factors through multiple interactions with p300/CBP coactivators. J Biol Chem 278:15495-504 |
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