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Originally published In Press as doi:10.1074/jbc.M706767200 on October 25, 2007

J. Biol. Chem., Vol. 283, Issue 3, 1317-1323, January 18, 2008
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Interplay between E-box and NF-{kappa}B in Regulation of A20 Gene by DRB Sensitivity-inducing Factor (DSIF)*

Liat Amir-Zilberstein and Rivka Dikstein1

From the Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

Received for publication, August 14, 2007 , and in revised form, October 16, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The NF-{kappa}B target gene A20 serves as a paradigm for gene-specific control of transcription elongation. This gene is regulated by the elongation factor DSIF (DRB sensitivity-inducing factor) under basal and NF-{kappa}B-activated states by two distinct mechanisms. Prior to NF-{kappa}B stimulation, the A20 gene is occupied by polymerase II, and elongation is inhibited by DSIF. This inhibition is mediated by an upstream promoter element termed ELIE (elongation inhibitory element). Upon NF-{kappa}B activation, inhibition of the A20 gene by DSIF persists, but now NF-{kappa}B and the core promoter regulate DSIF instead of ELIE. Here we investigated the regulation of DSIF by ELIE and the regulatory switch from ELIE to NF-{kappa}B following NF-{kappa}B induction. Electrophoretic mobility shift assays revealed two distinct protein complexes that specifically interact with ELIE, one of which is the E-box protein USF1. Interestingly, USF1 is displaced from the A20 promoter upon induction of NF-{kappa}B. A mutation in the E-box section of ELIE diminished the binding of USF1 and DSIF recruitment. Consistent with these findings, the E-box is crucial for DSIF inhibition in resting, but not NF-{kappa}B-stimulated, cells. These findings reveal a dynamic regulation of DSIF involving either E-box or NF-{kappa}B depending on the physiological circumstances.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription of mRNA encoding genes is a complex process divided into three discrete interconnected steps: initiation, elongation, and termination. In recent years, data have accumulated revealing the importance of the transcription elongation stage in regulation of gene expression (1, 2). DRB sensitivity-inducing factor (DSIF)2 and NELF are two negative elongation factors that inhibit RNA pol II elongation in the presence of DRB (3, 4). DSIF is composed of two subunits, p160 and p14, which are homologous to the Saccharomyces cerevisiae transcription factors Spt5 and Spt4, respectively. DSIF and NELF function cooperatively to inhibit RNA pol II elongation (4). Under certain conditions DSIF can act as a positive elongation factor (5, 6). P-TEFb, a positive elongation factor, is a DRB-sensitive cyclin-dependent protein kinase that phosphorylates the heptapeptide repeats within the pol II carboxyl-terminal domain and induces its processivity (7). P-TEFb can relieve DSIF and NELF inhibition of elongation (8, 9).

In mammalian cells, genes targeted by NF-{kappa}B are subjected to differential regulation at the elongation level by P-TEFb and DSIF (10–13). NF-{kappa}B is a family of transcription factors central to cellular response to a broad range of extracellular signals including inflammatory cytokines, tumor promoters, and chemotherapeutic agents (for review see Refs. 14 and 15). We have investigated the mechanism underlying the rapid transcriptional induction that occurs in some NF-{kappa}B target genes. We showed that the promoters of these genes are bound by the general transcription machinery prior to stimulation of NF-{kappa}B, and upon induction NF-{kappa}B acts to increase the rate of reinitiation (16). Using the NF-{kappa}B-responsive gene A20 as a model, we found that the basal A20 transcription (before stimulation) is repressed at the level of elongation in a promoter-dependent manner. Immunodepletion experiments in vitro and RNA interference (RNAi) experiments in vivo indicated that the elongation inhibition is conferred by DSIF, which in this system acts in the absence of DRB and without NELF (10). Dissection of the A20 promoter led to the identification of ELIE (elongation inhibitory element), an element that mediates DSIF inhibition under basal conditions (10). The identity of this novel type of elongation repressor and the mechanism by which it regulates the activity of DSIF are unknown.

We also examined DSIF regulation of A20 transcription in NF-{kappa}B stimulated cells. Surprisingly, we found that inhibition of A20 gene transcription by DSIF persists but is now regulated by NF-{kappa}B rather than ELIE (10). Thus the A20 gene is regulated by DSIF under basal and NF-{kappa}B-activated states by two distinct mechanisms. Most recently we demonstrated that regulation of NF-{kappa}B target genes by DSIF upon NF-{kappa}B induction is not a general phenomenon but is dependent on a promoter configuration that includes an NF-{kappa}B enhancer in a context of a TATA-less core promoter (11). By contrast, DSIF is released from TATA-containing NF-{kappa}B target genes, and these are positively regulated by the elongation factor P-TEFb. Converting a TATA-less to a TATA promoter is sufficient to switch the regulation of NF-{kappa}B from DSIF to P-TEFb. Accumulation of either DSIF or P-TEFb involves NF-{kappa}B itself and the formation of distinct initiation complexes (TFIID-dependent or -independent) on each of the two types of core promoter (11).

In the present study, we investigated further the regulation of transcription elongation of the A20 gene by DSIF under basal and stimulated conditions mediated by ELIE and NF-{kappa}B, respectively. Our results suggest that an E-box-binding protein, most likely USF1, mediates DSIF inhibition and recruitment under basal conditions. Once NF-{kappa}B is induced, the E-box is no longer involved in DSIF inhibition. Consistent with that, USF1 is displaced from the promoter by NF-{kappa}B. These findings highlight a dynamic exchange between USF1 and NF-{kappa}B according to the physiological state of the cell to regulate transcription elongation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructions—A20, A20 mut-{kappa}Bs, I{kappa}B{alpha}, 2{kappa}B(A20)-{alpha}-actin, 2{kappa}B(A20)-TK, and DSIF RNAi have been described previously (10, 16). An A20 promoter E-box mutant (mutE-box) was generated by replacing the HindIII-PmlI fragment corresponding to –234 to –74 of the A20 promoter with a PCR fragment, which included a three-nucleotide substitution in the E-box.

Transient Transfection Assays and Chromatin Immunoprecipitation—293T cells (human embryonic kidney fibroblasts) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfections were performed using the standard CaPO4 method. To avoid basal NF-{kappa}B activity, cells were kept from reaching confluence and replated no more than nine times. For reporter assays, subconfluent cells were transfected in a 24-well plate using 1.1 µg of pSuper or DSIF RNAi plasmid, 20 ng of the luciferase reporter plasmids, 1 ng of RSV-Renilla, 10 ng of CMV-GFP, and, where indicated, 1 ng of p65/RelA. 48 h after transfection, cells were harvested and their luciferase activities were measured.

Chromatin immunoprecipitation (ChIP) assays of transfected and endogenous promoters were carried out as described (11). The ChIP data were quantified by densitometric analysis using Quantity One one-dimensional analysis software (Bio-Rad).

Protein Analysis—Whole-cell extract preparation and immunoblot assays were performed as described previously (11). For nuclear extracts, 293T cells were washed twice with cold phosphate-buffered saline and then lysed in buffer A containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 1 µg/ml leupeptin. The lysates were incubated for 20 min on ice and pipetted three times. Cytoplasmic proteins were removed by discarding the supernatant after centrifugation. The nuclei pellet was resuspended in buffer C containing 20 mM HEPES (pH 7.9), 250 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 1 µg/ml leupeptin and was incubated for 15 min on a rotator wheel at 4 °C followed by centrifugation and collection of the supernatant containing the nuclear proteins.

Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared as described above from 293T cells that were untreated or treated with 20 ng/ml tumor necrosis factor {alpha} (TNF{alpha}) for 1 or 4 h. Binding reactions were performed using 3–5 µg of protein in a 20-µl reaction volume consisting of 20 mM HEPES (pH 7.9), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.25 µg bovine serum albumin, and 1 µg of poly(dI·dC). Samples were incubated at room temperature for 10 min, and then the appropriate 32P-end-labeled double-stranded oligonucleotide was added for an additional 10 min. DNA-protein complexes were resolved using nondenaturing polyacrylamide gels. For competition, a 100-fold excess of unlabeled oligo was added. For antibody supershift experiments, 1 µl of specific antibody was added to the binding reaction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of ELIE-binding Proteins—Under basal conditions, transcription directed by the A20 promoter is inhibited at the level of elongation by DSIF. The DSIF inhibition was shown to be mediated by an upstream promoter element, termed ELIE, located at –74 to –67 relative to the transcription start site (Fig. 1A) (10). To characterize the transcription factors that bind ELIE we employed the electrophoretic mobility shift assay. For a probe we used an oligonucleotide corresponding to –83 to –48 of the A20 promoter. This region contains the ELIE and an overlapping E-box as well as an adjacent NF-{kappa}B site (Fig. 1B, A20 WT). This probe was reacted with a nuclear extract prepared from unstimulated 293T cells. The results show that under basal conditions two protein-DNA complexes, designated A and B, are formed (Fig. 1C, lane 2). To characterize these complexes, competition experiments were carried out. As can be seen in Fig. 1C, lane 3, both complexes were competed with by an excess of cold DNA that was used as a probe. Neither complex was competed with by an oligo containing only the two NF-{kappa}B binding sites of the A20 promoter (Fig. 1C, lane 4) suggesting that they do not comprise NF-{kappa}B, a result consistent with the extract being prepared from unstimulated cells. We next used an oligo containing mutations in ELIE (Fig. 1B, mutELIE) in competition experiments. This mutant failed to compete for either complex (Fig. 1D, lane 3). Because the mutELIE also destroys the overlapping E-box, we synthesized another oligo in which the E-box is mutated but ELIE remains intact (Fig. 1B, mutE-box). This oligo effectively competed with the faster migrating complex B but not with the slow migrating complex A (Fig. 1D, lane 4). These results suggest that complex A is an E-box-binding protein and complex B is a different protein that binds ELIE. To test this possibility, we added to the EMSA reactions antibodies against the E-box-binding proteins, USF1 and USF2, or the major NF-{kappa}B protein p65/RelA. Consistent with the competition results, complex A, but not complex B, was supershifted by USF1 antibodies, whereas p65 and USF2 antibodies had no effect on either complex (Fig. 1E, lanes 2–4). Thus USF1 appears to be the major E-box-binding protein of the A20 promoter.

To analyze further complex B, an EMSA was performed with a labeled mutE-box oligo. As expected, only one complex is formed with this oligo, and it is competed with by the cold mutE-box that was used as a probe but not by an oligo in which ELIE is mutated (Fig. 1F, lanes 3 and 4). USF1 antibody had no effect on this complex (Fig. 1F, lane 5), confirming that complex B contains proteins distinct from USF1. These results suggest that the DNA element that we designated ELIE is actually composed of two overlapping binding sites, one of which is bound by the E-box protein USF1.

USF1 Associates with the A20 Promoter and Is Displaced upon NF-{kappa}B Induction—To examine the interplay between ELIE-binding proteins and NF-{kappa}B we performed EMSA experiments with nuclear extracts prepared from control and TNF{alpha}-stimulated cells (TNF{alpha} is a potent inducer of NF-{kappa}B). The probe we used was, as before, the –83 to –48 oligonucleotide of the A20 promoter that contains both the ELIE region and the NF-{kappa}B site (Fig. 1B). Like the results shown in Fig. 1, under basal conditions two protein-DNA complexes are formed, both subject to competition by excess cold DNA probe (Fig. 2, lane 2). The slower migrating complex A was competed with by the oligo in which the NF-{kappa}B site is mutated (Fig. 2, lane 3, mut-{kappa}B) but not by the oligo in which E-box is mutated (Fig. 2, lane 4, mutE-box), and it was supershifted by USF1 antibody confirming that it is USF1 (Fig. 2, lane 5). One h after induction with TNF{alpha}, the complexes (Fig. 2, lane 6) were competed with by the DNA probe (Fig. 2, lane 7), but surprisingly, complex A was not competed with by the mut-{kappa}B oligo as had occurred before stimulation (Fig. 2, compare lane 8 with lane 3). The complexes were now efficiently competed with by the mutE-box oligo (Fig. 2, lane 9), and no supershift was observed with USF1 antibodies (Fig. 2, lane 10). Similar results were observed after a 4-h induction with TNF{alpha} (Fig. 2, lanes 11–14). The TNF{alpha}-induced complex A reacted with the NF-{kappa}B p65 antibody (Fig. 2, lanes 17 and 20), suggesting that the composition of complex A changed from USF1 to NF-{kappa}B. Thus upon NF-{kappa}B induction by TNF{alpha}, USF1 is displaced from the promoter.


Figure 1
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  		FIGURE 1.
Characterization of ELIE-binding proteins. A, a schematic representation of the A20 promoter and the DNA sequence of the region that is relevant to DSIF regulation is shown. ELIE, E-box, the two NF-{kappa}B binding sites, and the core promoter are indicated. Two additional putative E-boxes (CANNTG) are indicated by dashed underlines. B, the sequence of the oligonucleotides used for the EMSA. C–F, EMSA using either the A20 WT (–83 to –48) region (C, D, and E) or mutE-box (F) as probes with nuclear extracts from unstimulated 293T cells. Lane 1 in C, D, and F shows the DNA probe without nuclear extract. Lane 2 in C, D, and F, and lane 1 in E shows the DNA probe with the nuclear extract. The competitor DNAs or the antibodies added to the reactions are indicated on top.

 
To examine further the association of USF1 with the A20 promoter under basal and NF-{kappa}B-activated states, 293T cells were transfected with an A20 promoter-reporter plasmid treated with TNF{alpha} for 1 h to induce NF-{kappa}B and then subjected to ChIP assay using antibodies against pol II, USF1, p65/RelA, the p160 subunit of DSIF, and a nonrelevant antibody as a control. Under basal conditions, pol II and DSIF constitutively bind the A20 promoter (Fig. 3A) consistent with previous findings (10, 11). As predicted from the EMSA experiments, USF1 also occupies the A20 promoter in vivo. Upon TNF{alpha} treatment, NF-{kappa}B p65/RelA associates with the A20 promoter with a concomitant increase in pol II and DSIF occupancy. By contrast, USF1 occupancy is sharply decreased upon NF-{kappa}B induction, suggesting that USF1 is displaced from the A20 promoter in vivo once NF-{kappa}B is induced.

To test the relevance of these findings to the endogenous A20 gene, Jurkat cells were treated with TNF{alpha} for 1 h and analyzed by ChIP. The results (Fig. 3B) confirm USF1 occupancy of this promoter along with pol II and DSIF under basal conditions. As with the transfected promoter, USF1 occupancy is significantly reduced upon TNF{alpha} induction. In contrast, pol II and DSIF occupancies are induced together with NF-{kappa}B.

Regulation of DSIF Is Switched from E-box to NF-{kappa}B—The EMSA experiments distinguish between two distinct factors that bind to the ELIE region of the A20 promoter. Previously we reported that mutations in ELIE abolished DSIF inhibition of A20 under basal conditions (10). As these mutations disrupt the binding of both E-box- and complex B-binding proteins, we wished to determine whether there is a connection between USF1 and DSIF inhibition under basal and NF-{kappa}B-activated states. For this purpose we constructed a luciferase reporter driven by an A20 promoter in which the E-box is mutated but the complex B binding site remains intact (Fig. 4A, mutE-box). We used DSIF RNAi to down-regulate DSIF p160 subunit levels. 293T cells were transfected with A20 reporter gene derivatives: wild type (WT), mutE-box, and mut-{kappa}Bs in which NF-{kappa}B sites were mutated (Fig. 4A) together with DSIF RNAi or pSuper (parental expression vector) for 48 h. DSIF depletion by the RNAi was confirmed by immunoblotting (Fig. 4E). Consistent with previous results (10), down-regulation of DSIF expression under basal conditions enhanced A20 WT promoter activity, a result also seen when there are mutations in the NF-{kappa}B sites (Fig. 4B). Remarkably, the mutation in the E-box alone was sufficient to diminish DSIF inhibitory activity (Fig. 4B). These results suggest that it is the overlapping E-box section of the ELIE that is most significant for the regulation of DSIF activity under basal conditions.


Figure 2
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  		FIGURE 2.
USF1 is displaced upon NF-{kappa}B induction. EMSA using the A20 WT (–83 to –48) region as a DNA probe (A20 WT) in the presence of unstimulated 293T (lanes 1–5) or TNF{alpha}-stimulated (lanes 6–20) nuclear extract. The sequences of the DNA fragments used are shown at the top of the figure. Competitor DNA and antibodies that were used are indicated above the lanes. The arrows indicate the positions of complexes A and B.

 
The A20 gene is activated by NF-{kappa}B. DSIF inhibition in the presence of NF-{kappa}B persists, but the regulation and occupancy by DSIF becomes dependent on NF-{kappa}B and the TATA-less core promoter (11). We now determined the effect of the E-box mutation on DSIF inhibition when A20 is activated by NF-{kappa}B. 293T cells were transfected with the wild-type, mut-{kappa}B, or mutE-box A20 reporter genes together with NF-{kappa}B protein p65/RelA in the presence or absence of the DSIF RNAi plasmid. Down-regulation of DSIF by RNAi enhanced the NF-{kappa}B-induced transcription of the WT promoter (compare Fig. 4, B and C). In contrast to the basal state, this inhibition is abolished in the NF-{kappa}B-mutated A20 promoter as shown previously (10, 11) but is unaffected by the E-box mutation (Fig. 4C). This E-box mutation did not affect the level of activation by NF-{kappa}B, whereas mutations in NF-{kappa}B site diminished it (Fig. 4D). These findings suggest that the elongation-inhibitory activity of DSIF is regulated by the promoter via two specific elements, E-box and NF-{kappa}B, which switch according to the physiological circumstances.


Figure 3
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  		FIGURE 3.
USF1 is associated with the A20 promoter and is displaced upon NF-{kappa}B induction in vivo. A, A20 reporter gene was transfected to 293T, and 24 h later cells were treated with TNF{alpha} for 1 h and then used for ChIP assay using the indicated antibodies and a nonrelevant control antibody (Con). Promoter occupancy was analyzed by PCR with primers from the A20 promoter and the beginning of the luciferase gene. Representative ChIP results and analysis of 0.1% of the input DNA (Input) of four independent experiments with similar results are shown. B, analysis of the endogenous A20 promoter is shown. Jurkat T cells were treated with TNF{alpha} for 1 h and then subjected to ChIP assay with the indicated antibodies and a nonrelevant control antibody. The immunoprecipitated DNAs were analyzed by PCR with primers specific to the promoter and beginning of the A20 gene. Representative ChIP and input results of two independent experiments are shown. C, the ChIP results were quantified by densitometry and normalized to the input. The left and right graphs represent data from panels A and B, respectively.

 
USF1 Mediates DSIF Recruitment to the A20 Promoter under Basal Conditions—To investigate the mechanism of E-box-dependent regulation of DSIF under basal conditions, we tested whether this element mediates DSIF recruitment to the A20 promoter. 293T cells were transfected with the A20 WT or E-box mutant and then were subjected to ChIP assay using antibodies against USF1, the p160 subunit of DSIF, and a nonrelevant control antibody. To ensure that equivalent amounts of extract were used for immunoprecipitation, 0.1% of each input extract was examined by increasing PCR cycles with the same primer set (Fig. 5A, Input panel). As with the EMSA experiments, USF1 occupied the A20 promoter, and the E-box mutation diminished USF1 binding (Fig. 5A). The residual binding of USF1 to the promoter could be attributed to two additional E-boxes present in the proximal promoter (see Fig. 1A). Interestingly, reduced USF1 binding is accompanied by a significant drop (3-fold) in DSIF occupancy (Fig. 5A). These results suggest that the binding of an E-box protein to the ELIE region mediates DSIF recruitment to the A20 promoter under basal conditions.


Figure 4
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  		FIGURE 4.
Regulation of DSIF is switched from E-box to NF-{kappa}B. A, the sequence of wild-type (A20 WT) and mutant A20 promoter reporters. B, 293T cells were transfected with luciferase reporter plasmids directed by the A20 promoter derivatives with either DSIF p160 RNAi or pSuper (parental vector) and Rous sarcoma virus promoter-driven Renilla luciferase reporter plasmids that served to normalize the transfection efficiency. Cells were harvested 48 h after transfection, and luciferase activity was measured. The effect of DSIF p160 depletion on the various reporter genes is presented as the ratio of the relative luciferase activity in the presence of DSIF RNAi to its activity in the presence of pSuper. The data represent the means ± S.D. of three independent experiments each with independent duplicates. The asterisks indicate statistically significant difference (p < 0.01). C, 293T cells were cotransfected with wild-type and mutant A20 promoter reporters (shown in A) and the NF-{kappa}B protein p65 together with either pSuper or DSIF p160 RNAi. 48 h later the inhibitory effect of DSIF p160 was determined as in B. The data represent the means and standard deviations of three transfection experiments, each performed twice. The asterisks indicate statistically significant difference (p < 0.01). D, the responsiveness of the A20 reporter gene derivatives to p65/RelA NF-{kappa}B protein. E, a representative immunoblot showing down-regulation of DSIF p160 by the RNAi.

 


Figure 5
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  		FIGURE 5.
USF1 mediates DSIF recruitment under basal conditions. A, the top panel shows the sequence of the A20 wild type and the E-box derivatives that are linked to the luciferase reporter gene that was transfected into 293T cells. 24 h after transfection, ChIP assays were performed using the USF1 and DSIF p160 antibodies and a nonrelevant antibody as control (Con). The precipitated DNAs were then analyzed by PCR as in Fig. 3A. Representative ChIP results of three independent experiments are shown. 0.1% of the input DNA (Input) was subjected to an increasing number of PCR cycles. Quantified results, normalized to the input, are shown on the right. B, USF1 and DSIF expression plasmids were cotransfected into 293T cells for 48 h. Whole-cell lysate was then prepared and used for immunoprecipitation with anti-USF1, anti-DSIF, and a nonrelevant antibody as a control as indicated above the lanes (IP). The input (5%) and the immune complexes were separated by SDS-PAGE and subjected to immunoblotting (IB) with the indicated antibodies.

 
To gain insight into the mechanism by which USF1 recruits DSIF, we examined by co-immunoprecipitation assays whether these proteins interact with each other. 293T cells were cotransfected with expression plasmids of USF1 and DSIF and then subjected to immunoprecipitation with anti-USF1, anti-DSIF, and a nonrelevant antibody as a control. The input and the immunoprecipitated proteins were then analyzed by immunoblotting with the USF1 and DSIF antibodies. The results (Fig. 5B) show that a fraction of DSIF is specifically coprecipitated with USF1, and similarly, a fraction of USF1 is coprecipitated with DSIF. The association of just a small portion of USF1 with DSIF was expected because USF1 generally functions as a positive transcriptional regulator, a context that would mask interaction with a negative regulator such as DSIF. Thus recruitment of DSIF is mediated, at least in part, through interaction with USF1.

DSIF Inhibition Is Promoter-specific—To investigate the specificity of DSIF inhibitory activity under basal conditions, we examined the effect of DSIF p160 RNAi on other promoters that contain NF-{kappa}B sites but no E-box. These include I{kappa}B{alpha}, HIV-LTR (without the TAR), and the artificial NF-{kappa}B reporters 2{kappa}B-{alpha}-actin and 2{kappa}B-TK. The promoter-reporter constructs were cotransfected with either DSIF RNAi or parental expression vector, and 48 h later luciferase activity was measured (Fig. 6). The results show that a reduced level of DSIF in the cell enhanced A20 promoter activity but had no effect on transcription directed by the I{kappa}B{alpha}, HIV-LTR, 2{kappa}B-{alpha}-actin, and 2{kappa}B-TK promoters, confirming the promoter-specific nature of DSIF inhibitory activity. These findings are consistent with DSIF presence on the endogenous A20 promoter but its absence from the I{kappa}B{alpha} gene under basal conditions (11). In stimulated cells, however, transcription of the I{kappa}B{alpha} gene is attenuated by DSIF as it bears a TATA-less core promoter (11).


Figure 6
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  		FIGURE 6.
DSIF inhibition is specific to the A20 promoter. 293T cells were transfected with a plasmid expressing DSIF RNAi or the empty expression plasmid (pSuper) together with a luciferase reporter gene driven by different promoters as indicated and Rous sarcoma virus promoter-driven Renilla luciferase that served to normalize the transfection efficiency. DSIF effect is presented as in Fig. 4. The data represent the means and standard deviations of three transfection experiments, each performed twice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The NF-{kappa}B response gene A20 serves as an excellent model for understanding gene-specific aspects of elongation control. A20 is inhibited by DSIF both under basal and NF-{kappa}B-activated states, but in each of these physiological states an alternative mechanism is utilized to regulate DSIF. Different promoter elements including ELIE, NF-{kappa}B, and the core promoter modulate DSIF inhibition of the A20 gene according to the extracellular signals (10, 11). The present study extends those observations by characterizing the proteins that bind ELIE and the regulatory switch between ELIE and NF-{kappa}B with respect to DSIF inhibition of elongation. Our results revealed that ELIE activity is mediated primarily by an E-box and USF1 protein, which is displaced by NF-{kappa}B upon its induction.

Under basal conditions, the A20 gene is bound constitutively by the general transcription machinery, and transcription is inhibited at the level of reinitiation (16) and is attenuated at the level of elongation by DSIF through ELIE (10). Dissection of the ELIE region revealed that it is a composite element consisting of two partially overlapping sites: an E-box motif that binds USF1 in vitro and in vivo and an unknown protein that we called complex B. Although two distinct factors were observed to bind the ELIE region of the A20 promoter, the ChIP and functional assays point to the E-box section of ELIE as the major element responsible for the recruitment of DSIF and its inhibitory effect under basal conditions. When the E-box section of ELIE was mutated in a manner that left the complex B binding site intact, USF1 and DSIF association with the promoter was reduced, and the DSIF inhibitory effect of the basal activity was abolished. A mutation that disrupted the complex B, but not the E-box, binding site also somewhat reduced DSIF inhibition (m2, Ref. 10), suggesting that the E-box may be assisted by the adjacent overlapping element in recruiting DSIF. Presently, the nature of the proteins that bind to complex B is unknown.

USF1 is mainly known for its positive role in transcription. However, numerous recent studies suggest that it may be also involved in transcriptional repression. In Sertoli cells, USF1 binds to the sex hormone-binding globulin gene and represses its activity (17), and USF1 and USF2 serve as negative regulators for human telomerase reverse transcriptase gene expression (18, 19). It is possible that the inhibitory effect of USF1 in these genes is, as in the A20 gene, at the level of elongation.

How does the E-box-binding factor USF1 regulate the activity of DSIF? Co-immunoprecipitation experiments revealed specific interaction between USF1 with DSIF that is likely to mediate DSIF recruitment to the A20 promoter. Their association was observed only upon overexpression of the two factors, suggesting that this interaction is transient or unstable.

The A20 gene is activated by NF-{kappa}B following TNF{alpha} induction. It was therefore unexpected to find that DSIF also attenuates the TNF{alpha}- or p65/RelA-induced transcription directed by the A20 promoter (10, 11). Interestingly, under these conditions mutations in the NF-{kappa}B sites, but not E-box, eliminated the DSIF inhibition. EMSA and ChIP assays revealed that NF-{kappa}B acts directly or indirectly inhibits USF1 binding to the A20 promoter as USF1 is displaced from the A20 promoter upon NF-{kappa}B induction by TNF{alpha}. Displacement of USF1 from the promoter may be because of steric hindrance as the 5' NF-{kappa}B site is only five nucleotides distant from the E-box. However, this possibility seems unlikely because in stimulated cells DSIF inhibition is also diminished when the A20 promoter bears mutations that abolish NF-{kappa}B binding, and the E-box is intact (Fig. 4). A more likely possibility is that NF-{kappa}B activation leads to a modification of USF1 that impairs its DNA binding activity.

Transcriptional repression at the elongation stage has been described for several rapidly induced stress genes such as Drosophila hsp70, c-myc, c-fos, A20, and others (10, 20–22). Recent studies suggest that this form of repression also occurs during Drosophila development in the process of cell fate specification (23). It will be interesting to see whether gene-specific inhibition of elongation by E-box or other promoter elements is a widespread mechanism in mammalian gene expression programs and what its impact is on the transcription process in general.


   FOOTNOTES
 
* This work was supported by grants from the Israel Cancer Research Foundation and the Israel Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 972-8-9342117; Fax: 972-8-9344118; E-mail: rivka.dikstein{at}weizmann.ac.il.

2 The abbreviations used are: DSIF, DRB sensitivity-inducing factor; DRB, 5,6-dichloro-1-D-ribofuranosylbenzamidazole; NELF, negative elongation factor; WT, wild type; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; TNF, tumor necrosis factor; RNAi, RNA interference; pol II, polymerase II; ELIE, elongation inhibitory element. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Sandra Moshonov for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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