The molecular events that mediate Ig class switching, a process critical for generating a functionally diverse humoral immune response, are poorly understood. A role for NF-kB/Rel transcription factors in switching is suggested by multiple binding sites for these proteins within the Ig heavy chain locus, including promoters, enhances, and switch regions. Thus, NF-kB/Rel proteins might regulate transcriptional events that determine the accessibility of constant heavy (CH) genes for switching, as well as the switching process itself. Indeed, B cells from mice made genetically deficient in p50/NFkB (p50-/-), the transactivation domain of c-Rel (dc-Rel), or RelA/p65 show distinct defects in germline CH RNA expression and/or switch recombination itself. This proposal will determine the 1) functional roles, 2) expression patterns, and 3) mechanism(s) of action of NFkB/Rel proteins in Ig class switching in normal murine B cells in vitro in response to distinct combinations of activators and cytokines. The ability of NF-kB/Rel proteins to regulate Ig isotype selection through modulation of an enhancer complex 3' to Cha (3'aE) and/or I exon promoters, as well as through changes in the cell cycle will be determined. The functions and expression patterns of NF-kB/Rel knockout (KO) and KO/replacement mice activated with anti-lg-dextran, CD40-ligand, and/or LPS +/- multiple cytokines. The locus of action of NF-kB/Rel proteins in germline CH gene expression will be determined using B cells from 3'aE and/or IXSXCX transgenic mice. We will determine the relationships between 1) Ig class switching by flow cytometry and digestion-circularization PCR assays, 2) germline CH RNA levels by RT-PCR, 3) expression of NF-kB/Rel proteins by EMSA immunoblotting, and in vivo footprinting, 4) transgene activation, and 5) DNA synthesis, cell cycle progression, and apoptosis; CFSE labeling will relate division number with Ig isotype expression. These studies will elucidate key parameters that underlie the molecular basis of Ig switching.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032560-09
Application #
6373265
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Deckhut Augustine, Alison M
Project Start
1992-09-01
Project End
2003-05-31
Budget Start
2001-06-01
Budget End
2003-05-31
Support Year
9
Fiscal Year
2001
Total Cost
$249,602
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
City
Rockville
State
MD
Country
United States
Zip Code
20817
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