Efficient repression of endogenous major histocompatibility complex class II expression through dominant negative CIITA mutants isolated by a functional selection strategy.
Major histocompatibility complex class II (MHC-II) molecules present peptide antigens to CD4-positive T cells and are of critical importance for the immune response. The MHC-II transactivator CIITA is essential for all aspects of MHC-II gene expression examined so far and thus constitutes a master regulator of MHC-II expression. In this study, we generated and analyzed mutant CIITA molecules which are able to suppress endogenous MHC-II expression in a dominant negative manner for both constitutive and inducible MHC-II expression. Dominant negative CIITA mutants were generated via specific restriction sites and by functional selection from a library of random N-terminal CIITA deletions. This functional selection strategy was very effective, leading to strong dominant negative CIITA mutants in which the N-terminal acidic and proline/serine/threonine-rich regions were completely deleted. Dominant negative activity is dependent on an intact C terminus. Efficient repression of endogenous MHC-II mRNA levels was quantified by RNase protection analysis. The quantitative effects of various dominant negative CIITA mutants on mRNA expression levels of the different MHC-II isotypes are very similar. The optimized dominant negative CIITA mutants isolated by functional selection should be useful for in vivo repression of MHC-II expression.
Bontron
S
S
Department of Genetics and Microbiology, University of Geneva Medical School, Switzerland.
Ucla
C
C
Mach
B
B
Steimle
V
V
D002051:Burkitt Lymphoma; D002469:Cell Separation; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005434:Flow Cytometry; D005799:Genes, Dominant; D005802:Genes, MHC Class II / Q000235:genetics*; D006684:HLA-DR Antigens / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D009687:Nuclear Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D017384:Sequence Deletion; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D006684:HLA-DR Antigens; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D007371:Interferon-gamma