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Distinct palindromic extensions of the 5'-TTC...GAA-3' motif allow STAT6 binding in vivo ¹

Department of Pharmacology and Toxicology, University of Magdeburg, Magdeburg, Germany

³Correspondence: Institute of Pharmacology and Toxicology, University of Magdeburg, 44 Leipzigerstrasse, 39120 Magdeburg, Germany. E-mail: juergen.kraus{at}medizin.uni-magdeburg.de

SPECIFIC AIMS

The seven STAT (signal transducer and activator of transcription) transcription factors have distinct, nonoverlapping functions in cytokine and growth factor signaling, and all members bind to regulatory cis-active elements that contain the core sequence 5'-TTC(N)_2–4GAA-3'. The aim of this study was to identify regulatory elements for STAT6 as a first step toward discrimination of specific target sequences for the different STATs.

PRINCIPAL FINDINGS

1. STAT6 binding sequences require palindromic extension of the 5'-TTC...GAA-3' core palindrome
A typical function of STAT6 is to mediate transcriptional stimulation evoked by interleukin-4 (IL-4). Previously, a STAT6 element had been localized at nt -997 on the human µ-opioid receptor gene promoter, whereas two additional motifs with the core sequence 5'-TTC(N)₃GAA-3' at nt -1583 and -1061 were not involved in transcriptional regulation by IL-4/STAT6. Such motifs with n = 2, n = 3, and n = 4 will be termed N2, N3, and N4, respectively. Similarly, the rat gene promoter contains one STAT6 site at nt -727 (N4) and two non-IL-4-responsive motifs (N2 and N3). In contrast to the non-IL-4-responsive elements, the -997 and -727 STAT6 sites have extensions of the core palindromes. Our hypothesis was that palindromically prolonged 5'-TTC(N)_2–4GAA-3' motifs may be specific binding sites for STAT6. To identify STAT6 sites among N3 motifs (Fig. 1 A), oligonucleotides containing modifications of the -1583 motif with systematically prolonged palindromes were cloned into a chloramphenicol acetyl transferase reporter vector in front of the Herpes simplex virus thymidine kinase promoter, transfected in SH SY5Y neuroblastoma and Raji B cells, and assayed for responsiveness to IL-4. Indeed, responsiveness to IL-4 was observed for two motifs with external palindromic extensions (-1C/+10G and -1T/+10A). Internal palindromic extensions of the -1583 motif had no effect on IL-4 responsiveness. The -1C/+10G and -1T/+10A modifications of the -1061 motif were IL-4 responsive. 5'-PyTTC...GAAPu-3' sequences, in which the external residues were nonpalindromic, did not confer IL-4 responsiveness. Next, experiments with mutations of the -727 motif were performed to examine binding of STAT6 to N4 motifs (Fig. 1B ). Besides the wild-type sequence (-1T/+11A) and the -1C/+11G mutation, two internal modifications were IL-4 responsive as well (+4A/+7T and+4C/+7G). Again, nonpalindromic 5'-PyTTC...GAAPu-3' N4 motifs were not responsive. N2 motifs with prolongations of the core palindrome were not IL-4 responsive.

View larger version (39K): [in this window] [in a new window]   Figure 1. Identification of IL-4-responsive STAT6 binding motifs. A) N3 motifs. Top: numbering of N3 motifs. Middle: modifications (underlined) of the non IL-4-responsive -1583 motif of the human µ-opioid receptor gene promoter. All oligonucleotides were cloned into the pBLCAT2 reporter plasmid and tested in transfection experiments in SH SY5Y cells. Fold induction of the IL-4-treated transfectants vs. untreated controls is shown. The oligonucleotides were cotransfected as decoys together with the IL-4-responsive reporter gene construct (-997.tk.CAT, which contains the STAT6 element of the human µ-opioid receptor gene), and were tested for their ability to abolish IL-4 induction of this reporter construct by intracellular competition for STAT6 proteins. The effect is expressed as the IL-4 inducibility of controls (cotransfected with decoy oligonucleotides carrying the STAT6 unrelated AP-2 binding motif; in front of the arrow) compared with the IL-4 inducibility of cells cotransfected with decoy oligonucleotides carrying the various modifications (behind the arrow). Below: modifications of the nonresponsive -1061 motif (Py: pyrimidine; Pu: purine). B) N4 motifs. Similar experiments performed with modifications of the -727 motif.

2. STAT6 decoy oligonucleotides attenuate the effect of IL-4 on reporter gene expression
A strategy using "decoy" oligonucleotides was used to demonstrate the specificity of STAT6/DNA interactions in vivo. In this strategy, double-stranded oligonucleotides with specific binding sequences for transcription factors are used to disrupt the function of these factors in vivo, which is achieved by binding of the transcription factors to an excess of decoy oligonucleotides rather than to regulatory cis elements of a gene within cells. Thus, oligonucleotides containing the motifs that were tested in the transfection experiments were cotransfected as decoys along with a STAT6/IL-4-responsive reporter gene construct (Fig. 1) . The same motifs that were found to confer IL-4 responsiveness efficiently disrupted STAT6 function and attenuated IL-4 induction of the reporter gene construct, indicating that these sequences bind STAT6 in vivo.

3. STAT6 decoy oligonucleotides inhibit IL-4 induction of µ-opioid receptor gene transcription in Raji cells
The potency of the decoy oligonucleotides to disrupt the in vivo function of STAT6 was tested in another approach taking advantage of the physiological effect of IL-4 to induce transcription of the µ-opioid receptor gene, which is silent in unstimulated Raji cells. Raji cells were transfected with various decoy oligonucleotides, all cells were stimulated with IL-4 for 24 h, and µ-opioid receptor gene transcripts were assayed by quantitative real-time PCR (Fig. 2 ). Similar amounts of transcripts were detected in untransfected Raji cells and cells transfected with two motifs that were shown not to bind STAT6 in the above experiments. In contrast, at least 32-fold fewer µ-opioid receptor-specific transcripts were detected in cells transfected with the six STAT6 decoy oligonucleotides.

View larger version (20K): [in this window] [in a new window]   Figure 2. STAT6 decoy oligonucleotides inhibit IL-4 induction of human µ-opioid receptor gene transcription in Raji cells. Real-time PCR experiment showing amplification of µ-opioid receptor gene transcripts from Raji cells that were either untransfected (curve 1), transfected with decoy oligonucleotides that do not bind STAT6 (curves 2 and 3), or transfected with decoy oligonucleotides containing the six STAT6 binding elements (curves 4–9). Insert: amplification of a GAPDH fragment. Quantification: curve 1: untransfected cells, fractional cycle number (C) representing the threshold of amplification "T": 32.53, for the GAPDH: 27.27; curve 2: cells transfected with -1583 -1G/+10C, 32.51, 27.97; curve 3: -727–1A/+11T, 30.93, 28.77; curve 4: -1583–1C/+10G, 36.64, 28.46; curve 5: -1583–1T/+10A, NA, 27.99; curve 6: -727–1C/+11G, 38.82, 27.16; curve 7: -727–1T/+11A, 36.79, 27.41; curve 8: -727 + 4A/+7T, NA, 27.62; curve 9: -727 + 4C/+7G, NA, 27.57. NA: no amplification.

4. STAT6 binding sequences also confer responsiveness to IL-13, IL-15, and PDGF
It is known that STAT6 can be activated by cytokines like IL-13 and IL-15 and growth factors like PDGF. Transfection experiments in SH SY5Y cells revealed that the six above defined IL-4-responsive elements were significantly induced by IL-13, IL-15, and PDGF.

5. Binding of other STAT factors to STAT6 binding sites cannot be excluded generally
Decoy oligonucleotides containing STAT6 binding motifs had no effect on reporter gene induction of a construct containing a STAT5 element of the human Bcl-x cell death regulator gene after stimulation with granulocyte-macrophage colony-stimulating factor, indicating that STAT5 does not bind to the STAT6 sequences. A similar approach was used to test whether STAT6 sites would interfere with STAT1/STAT3 binding to a regulatory sequence of the mouse metallothionein-I gene promoter after stimulation by IL-6. Decoy oligonucleotides with the wild-type -1583 element and all modifications of it attenuated IL-6 induction of the reporter gene, indicating that the -1583 element binds STAT1 and/or STAT3. STAT6 binding sequences derived from modifications of the -1061 and the -727 elements and the natural -997 STAT6 element did not interfere with IL-6 inducibility of the mMT-I reporter gene system.

CONCLUSIONS AND SIGNIFICANCE

Summarizing our results, STAT6 binding sites are characterized by eight palindromic base pairs. Thus, the 5'-TTC...GAA-3' core palindrome must have a specific palindromic extension (Fig. 3 ). This may be either a C or T residue 5' outside the core sequence with the respective G or A residue at the 3' position. These motifs may have three or four random nucleotides in the middle. Motifs that have a nonpalindromic extension of a pyrimidine 5' and a purine residue 3' do not bind STAT6. N4 motifs, in which the core palindromes are extended internally with either 5'-C..G-3' or 5'-A..T-3', are binding sites for STAT6. Binding of other STATs to STAT6 elements cannot be excluded because the -1583 sequences bound STAT1 and/or STAT3. The systematic identification of functional and specific binding sites for STAT6 is a first step toward discrimination of cis-active DNA elements as target sequences for the seven different STAT factors. In earlier investigations this complex was addressed by the EMSA technique, but studying DNA/STAT6 interactions using this technique revealed rather confusing results as almost any N3 or N4 sequence was shown to bind STAT6. Thus, it was shown in different laboratories (including ours) that in vitro STAT6 binding to randomly chosen N3 and N4 motifs resulted in the same EMSA patterns as STAT6 binding to natural IL-4-responsive elements. However, functional tests with such random sequences that bound STAT6 in EMSAs, monitoring responsiveness to IL-4 in reporter gene assays, were negative, indicating that in vivo no interactions with such sequences occur. These data lead to the conclusion that EMSAs are unsuitable for delineation of regulatory STAT6 sequences. Binding of this transcription factor to DNA sequences in vitro is probably much less specific than in vivo, due to different conditions in vitro and within a cell. Among the functional approaches used in this study, the decoy strategy could become the method of choice for characterizing STAT/DNA interactions. Since decoy oligonucleotides bind transcription factors within living cells, the approach represents the current most physiological and specific assay with which to investigate transcription factor/DNA interactions. The high specificity is clearly demonstrated in experiments with sequences that differ from one another in as few as a single nucleotide. The strategy using decoy oligonucleotides to disrupt the function of transcription factors in vivo has been discussed and applied for gene therapy. We thus provide another example of inhibiting a physiological effect by the decoy strategy by blocking transcription of the µ-opioid receptor gene in Raji cells. µ-Opioid receptors are up-regulated by IL-4 in various immune cells. The presence of these receptors on immune cells could account for some of the adverse immunosuppressive effects of morphine analgetics, since most of these substances act via the µ receptor subtype. Thus, suppression of their expression in immune cells could be advantageous. Considering potential therapeutic use of decoy oligonucleotides, detailed knowledge of the binding sequences for the different STATs is of more than academic interest in order to obtain specific effects that are due to disruption of only the desired STAT factor.

View larger version (36K): [in this window] [in a new window]   Figure 3. Above: STAT6 transcription factors bind to regulatory DNA sequences and activate transcription of target genes. Below: binding motifs for STAT6.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0482fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0482fje

² Both authors contributed equally to this manuscript.

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