Distinct palindromic extensions of the 5′-TTC...GAA-3′ motif allow STAT6 binding in vivo

STATs (signal transducers and activators of transcription) are transcription factors downstream of cytokine and growth factor signals. All of the seven different STATs bind to regulatory promoter elements with the common core motif 5'-TTC(N)(2-4)GAA-3'. A key question is how the different STAT factors recognize "their" response elements, that is, what distinguishes for example STAT1 from STAT6 binding sites. In vivo, binding of the different STATs to DNA elements is highly specific and disruption of the genes for the different STAT factors is accompanied with distinct, non-overlaping phenotypical effects. As a first step towards discrimination of target sequences for the various STATs, we determined requirements for binding sites for STAT6. In functional assays, six sequences were identified. These have palindromic extensions of the core motif in common (underlined): 5'-TTTCNNNGAAA-3', 5'-CTTCNNNGAAG-3', 5'-TTTCNNNNGAAA-3', 5'-CTTCNNNNGAAG-3', 5'-TTCCNNGGAA3' and 5'-TTCANNTGAA-3'. Different approaches and mutational analysis demonstrated the functionality of these sequences and high specific binding to STAT6. (I) These elements mediate transcriptional induction by interleukin-(IL)-4, IL-13, IL-15, and platelet-derived growth factor. (II) When used as "decoy" oligonucleotides, they bind STAT6 and disrupt its function in vivo, attenuating (a) STAT6/IL-4-mediated reporter gene transcription and (b) STAT6/IL-4-mediated induction of mu-opioid receptor mRNA of Raji cells.