Abstract

This is a continuing study on the molecular mechanisms of human Beta-IFN gene regulation. These studies involve the characterization of the properties of cis-acting DNA regulatory sequences and the identification and purification of trans-acting regulatory factors. The positive and negative regulatory DNA sequences will be precisely delineated by constructing and analyzing large numbers of single base mutations within the region already defined as being essential for Beta-IFN gene control. The properties of these regulatory sequences will be further investigated by studying their affects on heterologous promoters and enhancers. Interactions between cellular factors and specific Beta-IFN regulatory sequences will be studied using in vitro and in vivo DNAase footprinting methods. The in vitro footprinting method or other specific DNA binding assays will be used as a means of purifying the putative regulatory factors. Amino acid sequence information or antibodies raised against these purified proteins will be used to isolate the corresponding genes. These genes will then be characterized to derive the primary structure of the encoded proteins, and they will be used to produce large amounts of the proteins for use in DNA binding and in vitro transcription studies. Attempts will be made to isolate the genes encoding activating factors using differential hybridization methods, and the genes encoding Beta-IFN repressors will be isolated using a combination of genetic selection and gene rescue procedures. In order to study the mechanisms by which proteins can activate Beta-IFN gene expression, efforts will be made to establish transcription assays that are capable of supporting enhancer-dependent gene transcription. This problem will be approached by attempting to assemble transcriptionally active minichromosomes in Xenopus oocytes or in mammalian cells on extrachromosomal plasmids. The ultimate objective is to establish a fully reconstituted transcription system using purified cellular components that will mimic the in vivo regulation of the Beta-IFN gene.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI020642-12
Application #
2061294
Study Section
Special Emphasis Panel (NSS)
Project Start
1983-12-01
Project End
1996-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
12
Fiscal Year
1995
Total Cost
Indirect Cost

  Institution

Name
Harvard University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138

  Publications

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
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
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
Yang, Hongmei; Lin, Charles H; Ma, Gang et al. (2002) Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein-protein interactions. Eur J Biochem 269:6142-51
Lin, C H; Hare, B J; Wagner, G et al. (2001) A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies. Mol Cell 8:581-90
Falvo, J V; Parekh, B S; Lin, C H et al. (2000) Assembly of a functional beta interferon enhanceosome is dependent on ATF-2-c-jun heterodimer orientation. Mol Cell Biol 20:4814-25
Peters, R T; Liao, S M; Maniatis, T (2000) IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex. Mol Cell 5:513-22
Falvo, J V; Uglialoro, A M; Brinkman, B M et al. (2000) Stimulus-specific assembly of enhancer complexes on the tumor necrosis factor alpha gene promoter. Mol Cell Biol 20:2239-47
Sears, C; Olesen, J; Rubin, D et al. (1998) NF-kappa B p105 processing via the ubiquitin-proteasome pathway. J Biol Chem 273:1409-19

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