Silencer activity of NFATc2 in the interleukin-12 receptor beta 2 proximal promoter in human T helper cells.
Interleukin 12 (IL-12) is a potent enhancer of interferon gamma production by activated T cells. The high-affinity IL-12 receptor (IL-12R) is a heterodimer of a beta1 and a beta2 subunit. Expression of the signaling IL-12Rbeta2 chain is usually low, as compared with the more abundant beta1 chain, and may be rate-limiting for IL-12 sensitivity. Little is known about the mechanisms controlling IL-12Rbeta2 gene expression. Reporter gene assays in IL-12Rbeta2-expressing Jurkat cells showed that truncation of the region from -151 to -61 abrogated promoter activity. The proximal promoter region does not contain a typical TATA box, suggesting a role for SP-1. Indeed, mutagenesis of the -63 SP-1 consensus site decreased transcription by 50%. Electrophoretic mobility shift experiments confirmed the binding of SP-1 and SP-3 at this site. In contrast, truncation of -252 to -192 increased promoter activity. Likewise, mutagenesis of the consensus nuclear factor of activated T cells site at -206 increased promoter activity by 70%, suggesting silencer activity of this element. Electrophoretic mobility shift experiments with primary Th (T helper) cells showed the formation of a specific, T-cell receptor-inducible complex at this site that is sensitive to cyclosporin A and supershifted with anti-NFATc2 in both Th1 and Th2 cells. Accordingly, cyclosporin A dose-dependently increased IL-12Rbeta2 mRNA expression. These first data on IL-12Rbeta2 gene regulation indicate a TATA-less promoter, depending on SP-1/SP-3 transcription factors, and a negative regulatory NFAT element at -206. This element may contribute to the overall low level of IL-12Rbeta2 expression on Th cells.
van Rietschoten
J G
JG
Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology, P. O. Box 22700, 1100 DE Amsterdam, The Netherlands.
Smits
H H
HH
van de Wetering
D
D
Westland
R
R
Verweij
C L
CL
den Hartog
M T
MT
Wierenga
E A
EA
D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005809:Genes, Regulator; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011033:Podophyllin / Q000031:analogs & derivatives* / Q000378:metabolism; D011034:Podophyllotoxin / Q000031:analogs & derivatives; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D012333:RNA, Messenger / Q000032:analysis; D018123:Receptors, Interleukin / Q000235:genetics*; D053707:Receptors, Interleukin-12; D006377:T-Lymphocytes, Helper-Inducer / Q000378:metabolism*; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; C508438:IL12RB1 protein, human; C508439:IL12RB2 protein, human; D050778:NFATC Transcription Factors; C494006:NFATC2 protein, human; D009687:Nuclear Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12; D014157:Transcription Factors; C003170:mitopodozide; D011033:Podophyllin; D011034:Podophyllotoxin