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Proc. Natl. Acad. Sci. USA Vol. 92, pp.

3041-3045, March 1995 Biochemistry

Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity
(response elements/interleukin 4/interleukin 6/cytokine signal transduction/gene expression)

H. MARTIN SEIDEL*t, LAWRENCE H. MILOCCO*, PETER LAMB*, JAMES E. DARNELL, JR.O, ROBERT B. STEIN*,
AND JONATHAN ROSEN*
*Ligand Pharmaceuticals Inc., 9393 Towne Centre Drive, San Diego, CA 92121; and tLaboratory of Molecular Cell Biology, Rockefeller University, New York, NY 10021

Contributed by James E. Darnell, Jr., October 21, 1994

ABSTRACT Signal transducers and activators of transcription (STAT proteins) bind to palindromic sequence elements related to interferon y (IFN-y) activation sites, which were first identified in the promoters of IFN-'yinducible genes. Although the sequences of the natural palindromic STAT-binding elements vary considerably, they conform to the general structure TT(N)5AA. We have systematically examined the effects of the spacing between the TT and AA core half sites on the binding of the STAT complexes activated by IFN-y, interleukin (IL) 6, granulocytemacrophage colony-stimulating factor, and IL-4. We show that (i) as suggested earlier, a core palindromic TT-AA motif with a 5-bp spacing displays general STAT binding, (ii) a palindromic motif with a spacing of 4 bp selectively binds to complexes containing Stat3, and (iii) a motif with a 6-bp spacing selectively binds the STAT complexes activated by IL-4. We have examined natural elements in the promoters of cytokine-responsive genes that differ in half-site spacing and found that they display binding properties predicted from the synthetic binding sites. Furthermore, the observed differential selective binding characteristics for the most part correlate with the ability to mediate transcriptional activation of transfected test genes in response to the cytokines tested. Our results thus demonstrate that the specificity of STAT-directed transcription in response to particular cytokines or cytokine families depends in part on the spacing of half sites within the conserved response element sequence. After binding to their cell surface receptors, many soluble extracellular signaling proteins are thought to exert their biological effects by inducing alterations in the pattern of gene expression (1, 2). Since many cells simultaneously express receptors for a host of extracellular signaling proteins and the phenotypic changes induced by different polypeptides vary, specificity in the signaling process must exist (2). Consequently, the mechanisms by which signals are transduced from an occupied cell surface receptor to the nucleus have been the focus of intensive investigation (3, 4). Recent experiments explaining interferon (IFN) induction of transcription have shed light on a general signal transduction mechanism used by many extracellular signaling proteins (for review, see refs. 5 and 6). IFN--y stimulates the phosphorylation on Tyr and activation of two cytoplasmic tyrosine kinases belonging to the JAK family. After JAK activation, Statl, a latent cytoplasmic transcription factor that is a member of a larger gene family termed signal transducers and activators of transcription (STAT), is phosphorylated on a specific Tyr residue. Statl then homodimerizes, translocates to
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the nucleus, binds to a distinct DNA element, the IFN-,y activation site (GAS) found in the promoters of IFN-yinduced genes, and induces transcription. In contrast, IFN-a leads to phosphorylation of both Statl and Stat2 and activates a DNA binding complex that binds a different consensus sequence, the IFN-a-stimulated response element, thus explaining the distinctive patterns of gene induction that occur after IFN-a and -,y treatments (5). It was discovered subsequently that a host of cytokines, including virtually all of those that act through the cytokine receptor superfamily (5-7), activate overlapping sets of STAT family members and/or STAT-like complexes that bind to the same GAS sequence elements (5). However, the details of specificity of binding and the functional consequences of STAT binding to these elements remain obscure. By systematically determining the affinity of various cytokine-activated proteins to a series of synthetic and natural STAT binding elements (SBEs), we have found that spacing between inverted repeats (palindromic half sites) influences the selectivity of both STAT DNA binding and transcriptional activation.

MATERIALS AND METHODS Reagents. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 4, IL-6, and oncostatin M (OSM) were obtained from R & D Systems. Human IFN-,y was from Amgen Biologicals. Cells and Cell Culture. HepG2 (human hepatoma) cells were obtained from the American Type Culture Collection (ATCC) and grown in Eagle's minimum essential medium/ 10% (vol/vol) fetal bovine serum (FBS). U-937 (human promonocytic) cells (ATCC) were grown in RPMI 1640 medium/10% FBS. ME-180 cells (human cervical carcinoma) were obtained from the ATCC and grown in McCoy's 5A/10% FBS. Cytokines were used at the following concentrations: IFN-,y, 5 ng/ml; IL-4, 10-30 ng/ml; IL-6, 10 ng/ml; GM-CSF, 5 ng/ml.
Preparation of Nuclear Extracts and Gel Retardation Assays. Nuclear extracts were prepared as described (7). Nuclear extracts were prepared from untreated HepG2 cells, HepG2 cells treated for 15 min with IFN-y or 15 min with IL-6, untreated U-937 cells, and U-937 cells treated for 30 min with GM-CSF or 30 min with IL-4. The double-stranded oligonucleotide probes were formed by annealing oligonucleotides with the sequences (the antisense strand, except where indicated, was constructed such that, when annealed, both strands
Abbreviations: IL, interleukin; IFN, interferon; OSM, oncostatin M; GM-CSF, granulocyte-macrophage colony-stimulating factor; STAT, signal transducers and activators of transcription; GAS, IFN-,y activation site; SBE, STAT binding element; EMSA, electrophoretic mobility shift assay; SIE, sis-inducible element. tTo whom reprint requests should be addressed.

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Biochemistry: Seidel et aL

Proc. NatL Acad Sci USA 92 (1995)


Table 1. STAT binding elements in the promoters of cytokine-responsive genes Gene Nucleotide sequence GAS elements GBP TTACTCTAA IRF-1 TTCCCCGAA TTCCTGTAA Ly-6E

would have 5'-GATC overhangs; core elements are underlined): 5'-GATCTGCTTCC(T)nGGAACGT-3', where n = 0 (4spA/4spB), 1 (5spA), 2 (6spA), and 3 (7spA); 5'GATCTGCTTCC(C)nGGAACGT-3', where n = 1 (5spB), 2 (6spB), 3 (7spB), and 4 (8spB); 5'-GATCTGCTTC(C)nGAACGT-3', where n = 1 (3spC), 2 (4spC), 3 (5spC), 4 (6spC), 5 (7spC), and 6 (8spC); 5'-GATCTGCTTC(T) GAACGT-3', where n = 2 (4spD), 3 (5spD), 4 (6spD), and 5 (7spD); 5'-GATCTGCTTCT(C)nAGAACGT-3', where n = 0 (4spE), 1 (5spE), 2 (6spE), and 3 (7spE); 5'-GATCTCACTGTCAGGAAGCGT-3' (junB); 5'-GATCCATTTCTGGAAATG-3' (SAA3); 5'-GATCGATTTCCCCGAAATG-3' (IRF-1); 5'-GATCCACTTCCCAAGAACAGA-3' (Ce); 5'-GATCCATTCCCCGTAAATC-3' (SIE-6sp); 5'-GATCCACTTTCCAAGAACAGA-3' (CE-Ssp); 5'-GATCCATTTCCCGTAAATC-3' (m67/SIE-sense); 5'-CTAGGATTTACGGGAAATG-3' (m67/SIE-antisense). Electrophoretic mobility shift assays (EMSAs) were performed as described (7). Transient Transfection Assays. The luciferase reporter plasmids, based on the parent tk-luc plasmid (7), were constructed by linking the appropriate oligonucleotide sequences in multiple copies to the promoter of the herpes simplex virus thymidine kinase gene at position -35 with respect to the cap site. HepG2 and ME-180 cells were transfected by calcium phosphate coprecipitation with the reporter and a transfection control plasmid expressing f-galactosidase as described (7). Recombinant cytokines were then added and the cells were harvested after 5 h. Cells were lysed and luciferase and 13-galactosidase activities were determined. For each sample, the normalized response was determined by dividing relative light units measured in a luciferase assay with the f3-galactosidase activity in the same lysate. Fold induction (induced/ untreated) was calculated by using the averaged normalized responses from three experiments.

Ref(s).
5
23

Fc-yR1
ICSBP MIG

aL2-macro Core spacing 5 bp


IL-6 responsive genes

TTCTGGGAA TTCTCGGAA TTACTATAA TTCTGGGAA TTNNNNNAA

5 5
32

5 8
9 10
11, 12 12, 13 14 12, 15

junB SAA3 Core spacing 4 bp IL-4 responsive genes


Cs

GTCAGGAA TTCTGGAA TTNNNNAA TTCCCAAGAA TTCCTAGGAA TTCCTAGGAA TACCTGAGAA

C-yl Csy4

FcsRIIa Core spacing 6 bp TTNNNNNNAA Ly-6E, murine surface antigen; ICSBP, interferon consensus sequence binding protein; MIG, monokine induced by IFN-,y; a2-macro, rat a2-macroglobulin; Cyl, Cyl sterile transcript; Cy4, Cy4 sterile transcript; FcsRIIa, low-affinity IgE receptor. The GAS elements listed include elements known to be responsive to IFN-,y or IL-6.

RESULTS STAT Protein-DNA Complexes: Effect of Variations in Core Half-Site Spacing on DNA Binding. The observation that multiple cytokines appear to activate genes through STAT binding elements with similar DNA sequences raises the question of how cytokine-specific gene regulation is achieved. A partial list of previously characterized GAS-like elements is shown in Table 1. The sequences of these palindromic SBEs vary considerably, the only strongly conserved hallmark being a core half site composed of a TT sequence separated from its palindromic counterpart, AA, by 5 bp. This core half site (boldface type) is embedded in the observed consensus half sites, which have the sequences TTCC, TTAC, and TTCT (Table 1). We noted that the promoters of some previously reported cytokine-induced genes contain possible SBEs (9, 10, 12) that differ in nucleotide spacing between their half sites. These observations led us to test the role of half-site spacing in cytokine selectivity of SBEs. We designed sets of DNA sequence elements that contain fixed core STAT binding motifs (TT-AA) separated by a defined number of base pairs, keeping the flanking sequences identical in each case. The ability of these elements to bind to STAT complexes activated by different cytokines was tested by EMSA. Treatment of HepG2 (human hepatoma) cells with high doses of IL-6 activated three STAT-containing complexes called SIF-A, -B, and -C (refs. 16 and 17; these three complexes can be seen in Fig. 1A, lane 5spA/IL-6). Antibody supershift experiments showed that SIF-C and SIF-A were likely to be homodimers of Statl and Stat3, respectively, while SIF-B reacted with both antisera and is, therefore, thought to be a heterodimer. The relative positions of SIF-A, -B, and -C in an EMSA are indicated on Fig. 1A. IFN-,y induced a complex that is a homodimer of Statl and migrated identically to SIF-C. Treatment of the human promonocytic cell line U-937 with

GM-CSF (7) and IL-4 (7, 18) also activated distinct GAS binding complexes that did not react with antisera to Statl, -2, -3, or -4 and, therefore, contained different STAT proteins. These complexes are likely the same as those described for these ligands in other cell lines (12, 19, 20). A STAT activated by IL-4 in THP-1 cells has been cloned recently and could be a constituent of all reported IL-4-induced complexes (21). Having established the migration pattern of extracts from IFN--y- and IL-6-treated HepG2 cells and GM-CSF- and IL-4-treated U-937 cells, we tested the effect of changing the spacing of the TT-AA conserved sequences on the binding of the STAT complexes activated by these four cytokines (Fig. 1 A-F). The individual members of the set of oligonucleotides designated spA [TTCC(T),GGAA] exhibited clear STAT binding preferences (Fig. 1 A and B). The oligonucleotide with the 5-bp spacing (5spA, n = 1), which corresponds to the spacing associated with most of the SBEs characterized to date, bound strongly to the complexes activated by all four of the tested cytokines (Fig. 1A) and specifically showed the IFN--y induction of a complex identical to SIF-C and the IL-6 induction of a complex that migrated like SIF-A [these bands were confirmed to be reactive to Statl- or Stat3-specific antisera (16) and will be referred to as Statl and Stat3 homodimers]. The slightly different migrations of the IL-4and GM-CSF-induced bands can also be seen. The oligonucleotide with the 4-bp spacing between the TT-AA half sites (4spA, n = 0) showed selective binding to the Stat3 homodimer that is preferentially induced by IL-6 (Fig. 1A). This spacing corresponds to the spacing of the STAT binding sites found in the IL-6-responsive junB and SAA3 promoters (Table 1). In contrast, oligonucleotides with the 6-bp spacing (6spA, n = 2), which coincides with the elements found in the regulatory regions of IL-4-responsive genes, selectively bound the STAT complexes activated by IL-4 (Fig. 1B). The binding specificities observed for the spA spacing set were mostly recapitulated by using two additional sets of spacing elements, spB [TTCC(C)nGGAA, Fig. 1 C and D] and spC [TTC(C)nGAA, Fig. 1 E and F], which differ from the spA series in the sequences between the TT-AA conserved sequence. One notable difference was that the 4spC element

Biochemistry: Seidel et aL
A
4spA Probe: Cytokine: , _ , 9 STAT _A 5spA c: . X 9 . 9

Proc. Natl. Acad. Sci. USA 92 (1995)

3043

Complexes[
a':. :,,,' ..............,5

Probe:

6spA
c _

7spA
z
-

Cytokine:

CD

Probe:

4sp. 4spA)

5spB

STAT , C ~~~~ Complexes


Probe:

In
spC

-'

4spB
C

u_ X Cytokine: ccD
STAT
Complexes
~

Probe: 5spC STAT [::

7spC

ComplexesL

Cytokine:
Probe:

u v

, c

CD

5spC

4spC
,=Zt.

Cytokine: 'u,c-

(n = 2) bound to both the Stat3 homodimer and the presumed Statl-Stat3 heterodimer (lane IL-6). Thus, the 4-bp spacing can accommodate Stat3-containing complexes other than Stat3 homodimers but does not bind the Statl homodimer. A second difference was that the 5spC element (n = 3) did not bind to the Stat3 homodimer and bound only weakly to the Statl-Stat3 heterodimer but still bound the Statl homodimer (lanes IFN-,y and IL-6). Additional sets of spacing elements (spD and spE) were tested in the same fashion by EMSA, and the results are summarized in Table 2. These data confirm the spacing preferences of the GM-CSF- and IL-4-activated STAT complexes noted in Fig. 1 A and B. No specific binding was observed for oligonucleotides with a spacing of 3 bp or 7 bp or more (Fig. 1 B, D, and E and data not shown). The composition of the spacing nucleotides also has an impact on the ability of oligonucleotides to bind. Data with 5spC (described above) and other oligonucleotide sets point to the contribution of the extended half sites and spacing nucleotides themselves to STAT recognition, as the spD [TTC(T)nGAA] and spE [TTCT(C)nAGAA] sets do not bind to Statl or Stat3 complexes regardless of spacing (Table 2). Binding of STAT Complexes to Natural Elements. We next wished to correlate specific binding ability of various synthetic spacing elements with that of naturally occurring elements that differ in half-site spacing, such as the junB and SAA3 (4-bp spacing), high-affinity SIE (22) and IRF-1 (5-bp spacing), and human CE (6-bp spacing) elements. The junB element behaved much like the 4spA and 4spB elements and selectively bound the Stat3 homodimer (Fig. 1 G, IL-6 lane). The SAA3 element showed no binding discrimination between the STAT complexes tested (Fig. 1 G) and behaved much like the synthetic elements with a 5-bp spacing [however, this element has the sequence TTT(N)4AAA so that it could be considered to have a 4-, 5-, or 6-bp spacing between TT and AA]. The highaffinity SIE, a variant of the element in the c-fos promoter that confers responsiveness to platelet-derived growth factor (22), bound all STAT complexes tested (Fig. 1H) as does the IRF-1 GAS (refs. 23-26 and data not shown). The human Cs element behaved in the same manner as the synthetic 6-bp-spacing
HepG2 cells either untreated (un) or treated with IFN-y or IL-6 and from U-937 cells either untreated (un) or treated with GM-CSF (GM) or IL-4 were incubated with the indicated labeled probes and analyzed by EMSA. The STAT complexes, if present, are indicated by brackets. (A and B) EMSA analysis of the spA series [TTCC(T),GGAA]. The 4spA element has a 4-bp spacing between the TT-AA half sites, the 5spA element has a 5-bp spacing, the 6spA element has a 6-bp spacing, and the 7spA element has a 7-bp spacing. Bands A, B, and C in A denote the relative migrations of the SIF-A, SIF-B, and SIF-C complexes in lane 5spA/IL-6. (C and D) EMSA analysis of the spB series [TTCC(C)nGGAAJ. The 4spB element has a 4-bp spacing between the TT-AA half sites, 5spB has a 5-bp spacing, 6spB has a 6-bp spacing, and 7spB has a 7-bp spacing. (E and F) EMSA analysis of the spC series [TTC(C)nGAA]. The 3spC element has a 3-bp spacing between the TT-AA half sites, 4spC has a 4-bp spacing, 5spC has a 5-bp spacing, and 6spC has a 6-bp spacing. (G) Binding of STAT complexes to elements with a 4-bp spacing from the promoters of IL-6-responsive genes. JunB is from the murine junB promoter, and SAA3 is from the murine serum amyloid A3 promoter [because of its TTT(N)4AAA structure, the SAA3 element can be viewed as having ambiguous spacing between the TT-AA half sites]. (H and I) Base changes that alter core half-site spacing in natural STAT binding elements alter their STAT binding specificity. SIE is the high-affinity variant of the sis-inducible element (SIE) from the human c-fos promoter, and Cs is the STAT binding element from the human Cs control region. The SIE element has a 5-bp spacing between the TT-AA half sites. The SIE-6sp element differs from the SIE by a single base substitution and has a 6-bp spacing. The Cs element has a 6-bp spacing, and the Cs-5sp element differs from Cs by a single base substitution and has a 5-bp spacing.

Complexes
G

STAT

I_ :

'
junB
SAA3

, G Cytokine: C

Probe:

_z C IL

2 T
n

Complexes
Constit.

Probe:

SIE
e9

SIE-6sp
z

Cytokine: c
STAT Complexes

E9

Probe:

CE

CE-5sp
0

Cytokine: c9
STAT

>

Complexes

FIG. 1. Selective binding of STAT complexes to DNA sequence elements that differ in core half-site spacing. Nuclear extracts from

3044

Biochemistry: Seidel et al

Proc. Natl. Acad Sci. USA 92 (1995) activation of the same STAT complexes induced by these cytokines in HepG2 cells. OSM, whose receptor shares the gpl3O signaling component with the IL-6 receptor (27), activated the same DNA binding complexes as does IL-6 in both cell lines (refs. 17 and 28 and data not shown). The transfection results for the reporter constructs in HepG2 cells are shown in Fig. 2A (GM-CSF and IL-4 were not tested, as HepG2 cells do not express their receptors). The reporter containing the 4spC element (4-bp spacing) was specifically induced by IL-6 and OSM, the two cytokines that predominantly activated Stat3, but not by IFN-y, which predominantly activated Statl. The reporter containing the native junB element (4-bp spacing) also showed a selective, though modest, induction by IL-6 and OSM. These results correlate directly with the binding data shown above (Fig. 1). Interestingly, the native SAA3 element [the element with the TTT(N)4AAA composition], which did not discriminate in binding between the STAT complexes tested, mediated preferential induction by IL-6 and OSM compared to IFN-,y in HepG2 cells. The reporters containing the 5spA and IRF-1

Table 2. STAT complex binding to elements differing in core half-site spacing

Spacing,
bp spA series
4

Binding
Sequence
TTCCGGAA TTCCTGGAA TTCCTTGGAA TTCCTTTGGAA

IFN--y
++ ++
-

IL-6
+ ++ + +++
-

GM-CSF
+++ +++
-

IL-4
++ +++ ++ +++
-

5
6 7

spB series
4

5 6 7 8

TTCCGGAA TTCCCGGAA TTCCCCGGAA TTCCCCCGGAA TTCCCCCCGGAA


TTCCGAA TTCCCGAA TTCCCCGAA TTCCCCCGAA TTCCCCCCGAA TTCCCCCCCGAA

spC series
3 4 5 6 7 8
-

++
-

+++ -

+++
-

++ +++ -

A
c 0

spD series
4 5 6 7
TTCTTGAA TTCTTTGAA TTCTTTTGAA TTCTTTTTGAA
TTCTAGAA TTCTCAGAA TTCTCCAGAA TTCTCCCAGAA
-

+ ++ -

+ + ++ ++ -

~0
U-

spE series
4

5 6
7

Binding affinities as estimated by band intensity from EMSA analysis were rated as follows: -, not detectable or faintly visible; +, weak intensity; + +, moderate intensity; + + +, strong intensity. The flanking sequences were identical for all of the elements. Spacing nucleotides are in boldface type. Entries in the IFN--y column indicate binding by the Statl homodimer. Entries in the IL-6 column indicate binding by Stat3-containing complexes.

t x

"t
x

C\
x m

t x

t x

t
x

C)
cn

nc
.:

<
0

CO
:C
0

4bp Spacing

5bp 6bp Spacing Spacing


ME-180

B
C

,* IFNg

elements and bound to the IL-4-activated STAT complexes

1o B IL-6
0

selectively (Fig. 1I). The dichotomy between binding specificity conferred by a spacing of 5 bp and 6 bp was examined in more detail. A single base change was made in the high-affinity SIE that converted it from a 5-bp spacing to a 6-bp spacing (SIE-6sp, TTCCCCGTAA), and a single base change was made in the Cs element that converted it from a 6-bp spacing to a 5-bp spacing (Cs-5sp, TTCCAAGAA). These elements were then examined for their binding to the STAT complexes in the test extracts by EMSA (Fig. 1 H and I). The SIE-6sp mutation converted the SIE sequence from a nonselective STAT binding sequence to a selective IL-4-activated STAT binding sequence. Conversely, the Cs-5sp mutation converted the Cs element from an exclusive IL-4-activated STAT binding sequence to a sequence that displays a more general STAT binding pattern. These results further support the concept that spacing of the TT-AA half sites can play a pivotal role in determining the specificity of STAT binding. Transcriptional Activities of STAT Complexes on Spacing Elements. We next examined the functional consequences of varying the core half-site spacing on STAT transcriptional activation in transient transfections. Several of the synthetic
and natural SBEs were placed in multiple tandem copies upstream of the herpes simplex virus thymidine kinase minimal promoter directing the expression of the firefly luciferase gene. The reporter constructs were then transfected into HepG2 cells or IL-4-responsive ME-180 (cervical carcinoma) cells. IFN-y and IL-6 treatment of ME-180 cells resulted in

8 642-

~0

OSm

[3 IL-4

0u
_

t
x

t
x

t
x

c
x

It
x x x

C
0. C (0)

m C 2

cj

< <

fin

cn

Co CO

4 bp 5 bp 6 bp Spacing Spacing Spacing FIG. 2. Selective transcriptional activation by cytokines of reporters containing response elements that differ in their core half-site spacing. The 4spC, junB, and SAA3 elements have a 4-bp spacing between the TT-AA half sites, the 5spB and IRF-1 elements have a 5-bp spacing, and the 6spB and Cs elements have a 6-bp spacing. Multimers of each element were cloned upstream of the herpes simplex virus tk minimal promoter driving the expression of luciferase (parent vector, tk-luc). HepG2 or ME-180 cells were transfected with these constructs (including a 13-galactosidase-expressing control plasmid), treated with cytokines for 5 h, then lysed, and assayed for luciferase and ,B-galactosidase activities. The fold induction was calculated from the ratio of the normalized response in treated cells to the normalized response in untreated cells. (A) Selective transcriptional activation in HepG2 cells by IFN- y (IFNg), IL-6, or OSM. (B) Selective transcriptional activation in ME-180 cells by IFN-y (IFNg), IL-6, OSM, or IL-4.

Biochemistry: Seidel et at
elements (5-bp spacing), which bound to all of the STAT complexes tested, showed no selectivity and mediated transcriptional induction by all three cytokines. The reporters containing the expanded 6-bp spacing did not respond to IFN-,y, IL-6, or OSM. Thus, the observed in vitro STAT binding specificity for the various binding sites is in most instances a reliable indicator of their transcriptional activity in HepG2 cells. The transfection results for the reporter constructs in ME180 cells are shown in Fig. 2B. The elements with a 4-bp spacing again selectively mediated transcriptional induction by IL-6 and OSM, activators of Stat3. The reporters containing the 5-bp spacing elements again responded to IFN-,y, IL-6, and OSM; however, somewhat surprisingly, they did not respond to IL-4 though they bound to the IL-4-activated STAT complex. The reporter containing the 6-bp synthetic spacing element 6spB did not respond to any of the cytokines tested (though it bound specifically to the IL-4-activated STAT complex), but the natural Cs element with a 6-bp spacing mediated strong and extremely selective transcriptional induction by IL-4 and is, therefore, a functional activation site.

Proc. Natl. Acad. Sci. USA 92 (1995)

3045

caveats, we feel that the experiments clearly establish a difference between the ability of GAS-like elements containing the core motif TT-AA spaced 4, 5, and 6 bp apart to direct gene expression in response to different cytokines. The spacing effects observed with STAT binding to SBEs are reminiscent of results obtained for intracellular receptors such as the retinoic acid and vitamin D3 receptors binding to their response elements (29-31). Thus, by analogy, STAT response elements composed of variously spaced direct repeats and inverted palindromes of the SBE core half site may exist. Intriguingly, the consensus IFN-a-stimulated response element is made up of a direct repeat of the SBE half-site sequence GAA separated by either 2 or 3 bp (5). In conclusion, our results demonstrate that the specificity of STAT-directed transcription in response to particular cytokines or cytokine families depends in part on the spacing of half sites within the conserved response element sequence.
We thank D. E. Levy, P. Tapley, S.-S. Tian, L. Kessler, J. Haslam, and J. Miner for helpful discussions. J.E.D. is a consultant with Ligand Pharmaceuticals.
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DISCUSSION The recent discoveries that the STAT complexes activated by a variety of extracellular signaling proteins bind to similar DNA elements raises the question of whether and how transcriptional specificity in endogenous genes occurs (2, 5, 6). Our results provide evidence that at least a part of any specificity that exists in STAT-mediated transcriptional induction depends on the spacing between the palindromic core half sites of SBEs. In addition, it is clear that sequences between the TT and AA palindromic boundaries also affect specificity. The spacing of 5 bp between the TT-AA core sequence is found in many previously characterized natural sites, and several of the 5-bp-spaced synthetic sequences bound complexes induced by all four test cytokines. These 5-bp-spacing elements mediated transcriptional induction by cytokine activators of either Statl or Stat3 (IFN-,y, IL-6, and OSM), but they did not direct transcriptional activation by the IL-4-induced STATs. It has been reported that the FceRIIb element (15), which has a 5-bp spacing (TTCTAAGAA), binds to the IL-4-activated STAT complex (12, 18) and is necessary for IL-4-induced expression of the FcsRIIb gene (18). We found that this 5-bp spacing element also does not confer IL-4 responsiveness in the context of a minimal promoter even when multimerized (H.M.S. and L.H.M., unpublished data). The clearest results with respect to specific STAT binding and selective transcriptional activation were with changes in spacing of the TT-AA core to 4 or 6 bp. The 4-bp spacing (particularly with CCCG as the bases) allowed binding only by complexes containing Stat3. Accordingly, the natural (junB and SAA3) and synthetic 4-bp-spacing elements mediated a selective response to activators of Stat3 (IL-6 and OSM). Elements with a 6-bp spacing, which corresponds to the SBEs in several IL-4-responsive genes, strongly and selectively bind to the STAT proteins activated by IL-4. Though the construct containing the synthetic 6-bp-spacing element was not induced by IL-4, the natural site from the human Cs control region, which contains a site with a 6-bp spacing, was active. These results show that while binding of STAT complexes to SBEs may be a guide to effectiveness of a promoter element in transcription, there is no assurance that binding will ensure induction. In addition to the importance of sequences within the core, we have also noted some effects of sequences outside the 9- to 10-bp boundary of the conserved element (H.M.S., L.H.M., P.L., and J.E.D., unpublished data). In spite of these

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