Characterization of a palindromic enhancer element in the promoters of IL4, IL5, and IL13 cytokine genes.
BACKGROUND
The genes encoding the cytokines IL-4, IL-5, IL-13, and GM-CSF are located in close proximity on human chromosome 5. We have previously described a motif in the promoters of these genes with a common palindromic sequence: CCAAG em leader CTTGG. These half sites are variably spaced and, within GMCSF, flank a second internal palindromic site. The GMCSF palindrome was shown to act as a strong enhancer of transcription when linked to a heterologous promoter.


OBJECTIVE
We sought to determine whether the related palindromic elements from IL4, IL5, and IL13 also act as enhancers of gene transcription.


METHODS
Reporter plasmids driven by palindromic elements were transfected into Jurkat T and HeLa cells to determine enhancer activity, and T-cell extracts were used in electrophoretic mobility shift and methylation interference assays to determine the DNA-binding characteristics of palindrome-binding proteins.


RESULTS
Enhancer activity was observed in unactivated T cells and HeLa cells, whereas in T cells the IL4 palindrome mediated an activation-specific response. Mutational analysis of this element revealed that both halves of the CCAAG em leader CTTGG palindrome and part of the intervening sequence were essential for mediating interaction with protein complexes. Electrophoretic mobility shift assays suggested that the 4 palindromes bound similar factors because complexes formed between Jurkat nuclear extracts and each palindromic element showed identical mobility, and these elements were able to cross-compete for binding.


CONCLUSIONS
These data suggest that constitutively expressed factors are involved in mediating the enhancer function of these elements in T cells and that these factors might either be present or have closely related homologues in other cell types. Also, an activation-dependent factor might be recruited to modulate the function of the IL4 element.
Codlin
Sandra
S
Department of Asthma, Allergy and Respiratory Science, King's College London, Guy's Hospital, London.
Soh
Cecilia
C
Lee
Tak
T
Lavender
Paul
P
D001483:Base Sequence; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D006801:Humans; D018793:Interleukin-13 / Q000235:genetics*; D015847:Interleukin-4 / Q000235:genetics*; D015848:Interleukin-5 / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic
D018793:Interleukin-13; D015848:Interleukin-5; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor