HUMAN BETA INTERFERON GENE REGULATION

Harvard University, Cambridge MA

Investigators

Linked publications & trials

Abstract

DESCRIPTION: 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.