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Previous Article | Next Article 
Molecular and Cellular Biology, August 2006, p. 6094-6104, Vol. 26, No. 16
0270-7306/06/$08.00+0 doi:10.1128/MCB.02366-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8503, Japan,1 Graduate School of Medicine, Osaka City University, Abeno-ku, Osaka 545-8585, Japan2
Received 12 December 2005/ Returned for modification 4 January 2006/ Accepted 30 May 2006
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ABSTRACT |
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INTRODUCTION |
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1-AT locus, transcription initiation is inhibited before induction, although RNA polymerase II (RNAPII) associates with the promoter (29). In contrast, at the human c-fos and c-myc loci and some Drosophila heat shock loci, elongating RNAPII pauses in the promoter-proximal region before induction (12, 23, 24). The latter example is referred to as "promoter-proximal pausing," although the mechanism and function of this regulatory mode are not well understood (13). At the Drosophila hsp70 locus, promoter-proximal pausing is thought to be mediated by two transcription elongation factors, 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) (34). DSIF is a heterodimeric protein complex consisting of the Spt4 and Spt5 subunits (31). Human Spt5 (hSpt5) has a repeat region called the CTR and multiple copies of the KOW motif, which is also found in bacterial elongation factor NusG (39). NELF is a DSIF cofactor that consists of four subunits (38). NELF-A, encoded by Wolf-Hirschhorn syndrome candidate gene 2, has an N-terminal homology to hepatitis delta antigen, which binds to RNAPII and activates elongation (36). NELF-B, encoded by the COBRA1 gene, is known to interact with the BRCA1 protein (41). NELF-C and -D are translational variants of the TH1 gene products (17). NELF-E, also known as RD, has Arg-Asp dipeptide repeats and an RNA recognition motif (38). Biochemical analysis has revealed that DSIF and NELF cooperatively bind to elongating RNAPII and induce transcriptional pausing, possibly through an interaction between NELF-E and nascent RNA (37). Transcriptional pausing is alleviated when positive elongation factor b (P-TEFb) phosphorylates the heptapeptide repeats of the C-terminal domain of RNAPII, as well as the CTR in the hSpt5 subunit of DSIF (32, 35). After pausing is reversed, DSIF instead stimulates elongation by an as-yet-unknown mechanism. Interestingly, DSIF and NELF are not evolutionarily conserved to the same extent (17). Although DSIF is highly conserved among eukaryotes and is essential for viability in yeast, some species, including yeast, lack all of the NELF subunits (10, 17). Indeed, promoter-proximal pausing has not been observed in yeast (13). Thus, promoter-proximal pausing may be involved in transcriptional regulation only in some species.
Only a limited number of studies have been reported on DSIF- and NELF-mediated transcriptional pausing. At the Drosophila hsp70 locus, both DSIF and NELF associate with RNAPII paused at positions +20 to +40 before induction (34). After heat shock, NELF dissociates from RNAPII but DSIF translocates downstream with the polymerase. Another study showed that human estrogen receptor recruits the NELF complex to target gene promoters by physically interacting with NELF-B (3). It is suggested that NELF acts as a transcriptional attenuator at these loci and is important for controlling the duration and magnitude of hormonal responses. In zebra fish, the mutant called foggy, which carries a point mutation in the Spt5 gene and lacks the repression activity of DSIF, shows specific defects in neuronal differentiation during development, suggesting that DSIF- and NELF-mediated transcriptional pausing may be involved in the expression of only a limited number of genes (9). However, its precise role in gene expression on a genome-wide basis remains unclear.
The goal of this study was to understand the physiological role of DSIF- and NELF-mediated transcriptional pausing. As a model, we used junB, an immediate-early gene (IEG) that is activated transiently and rapidly in response to a wide variety of extracellular stimuli, such as interleukin-6 (IL-6). junB encodes a basic leucine zipper protein, which functions as a component of the AP-1 transcriptional activator. Several studies indicate that the regulation of junB gene expression is important for cell growth and differentiation (11, 15, 20). Here we report that DSIF- and NELF-mediated transcriptional pausing has a dual role in the regulation of junB expression in human hepatoma HepG2 cells. Our data indicate that pausing contributes to the negative regulation of junB expression not only by inducing transcriptional pausing before induction but also by attenuating the mRNA expression level after induction.
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MATERIALS AND METHODS |
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Cell culture and IL-6 stimulation. HepG2 cells were grown in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). The medium was replaced with 0.1% FBS-DMEM 24 h prior to IL-6 stimulation. IL-6 (0.5-mg/ml stock in 10 mM acetic acid) was added to the culture medium to a final concentration of 100 ng/ml.
Reverse transcription (RT)-PCR analysis. Total RNA was prepared by a guanidine thiocyanate extraction method using Sepasol I Super (Nacalai Tesque), followed by treatment with RQ1 RNase-free DNase (Promega). The following primers were used: junB, 5'-CACCAAGTGCCGGAAGCGGA-3' and 5'-AGGGGCAGGGGACGTTCAGA-3'; GAPDH, 5'-ATCCTGGGCTACACTGAGCA-3' and 5'-GGTGGTCCAGGGGTCTTACT-3'; c-fos, 5'-CACTCCAAGCGGAGACAGAC-3' and 5'-GAGCTGCCAGGATGAACTCT-3'; tis-11, 5'-GGGACTTGGGGGACAGTAAT-3' and 5'-GAACCTCGGAAGACACTCCA-3'; 18S rRNA, 5'-TAGAGGGACAAGTGGCGTTC-3' and 5'-TCCTCGTTCATGGGGAATAA-3'. RT-PCR was performed with the Titan One Tube RT-PCR system (Roche) as follows: 50°C for 30 min; 95°C for 15 min; and 20 cycles of 94°C for 15 s, 57°C for 30 s, and 72°C for 30 s. Real-time RT-PCR was performed with the iCycler iQ Detection System (Bio-Rad) and the QuantiTect SYBR Green RT-PCR kit (QIAGEN) as follows: 50°C for 30 min; 95°C 15 min; and 40 cycles of 94°C for 15 s, 57°C for 30 s, and 72°C for 30 s. The mRNA levels of junB shown in Fig. 5, as well as those of c-fos and tis-11 in Fig. 6, were normalized to the mRNA levels of GAPDH to allow comparisons among different experimental groups. The GAPDH expression level was measured and normalized to the level of 18S rRNA.
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Modified ChIP with ligation-mediated PCR (LM-PCR). ChIP assays were performed with some modifications. Cross-linked chromatin was suspended in modified sonication buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 0.5 mM EGTA). After a brief sonication, HaeIII (40 U) and PvuII (80 U) were added per 100 µl of cross-linked lysate. Partial digestion of chromatin was performed at 37°C for 30 min and terminated by the addition of sodium dodecyl sulfate to 1%. Following rotation for 1 h, samples were centrifuged and supernatants were used for immunoprecipitation. Coprecipitated DNAs and 1% of input DNAs in 5 µl of TE were subjected to LM-PCR as previously described (5). Briefly, DNA was blunt end ligated with the unidirectional linker consisting of two partially complementary oligonucleotides, LM-PCR.1 (5'-GCGGTGACCCGGGAGATCTGAATTC-3') and LM-PCR.2 (5'-GAATTCAGATC-3'). Next, ligated DNA was subjected to a brief PCR amplification with LM-PCR.1 and the gene-specific primer ChIP.LM-PCR.GSP.2.upst (5'-AGCGCACTTCCGTGGCTGAC-3'). Lastly, the 32P-end-labeled primer ChIP.LM-PCR.GSP.3.upst (5'-TTCCGTGGCTGACTAGCGCGGTA-3') was used for primer extension analysis of the PCR products.
Note that the first-strand synthesis step before linker ligation was omitted because HaeIII and PvuII both generate blunt-ended DNA fragments.
Potassium permanganate in vivo footprinting analysis. Serum-starved HepG2 cells on a 10-cm dish were treated with 10 ml of 7.5 mM KMnO4 in phosphate-buffered saline for 45 s. After quick aspiration of the solution, reactions were quenched by adding 1 ml of TNESK (10 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM EDTA, 1% sodium dodecyl sulfate, 0.1 mg/ml proteinase K) containing 0.4 M ß-mercaptoethanol and incubated at 50°C for 1 h and then at 37°C overnight. Genomic DNA was purified by phenol-chloroform extraction and ethanol precipitation, and DNA pellets were suspended in 200 µl of 1 M piperidine. After vigorous mixing for 15 min to suspend the pellets completely, samples were incubated at 90°C for 30 min, frozen quickly with liquid N2, and then dried for 90 min with a vacuum evaporator. Dried DNA pellets were suspended in 30 µl of distilled H2O and lyophilized again. This step was repeated. These steps were intended to remove residual piperidine completely. The samples were resuspended in TE to a final DNA concentration of 400 ng/µl and subjected to LM-PCR. Two primer sets were used to detect the promoter-proximal region of junB. The following gene-specific primers were used as primer set A for first-strand synthesis, PCR amplification, and primer extension labeling, respectively: LM-PCR.GSP.1, 5'-GTGCGCAAAAGCCCTGT-3'; LM-PCR.GSP.2, 5'-AAGCCCTGTCAGGCTTCCCGAG-3'; LM-PCR.GSP.3, 5'-TGTCAGGCTTCCCGAGCCCCCGT-3'. Likewise, the following primers were used as primer set B: LM-PCR.GSP.1.revB, 5'-CTGCGGTGACCGGACTG-3'; LM-PCR.GSP2.revB, 5'-TGGGTGCCTGGTCGCGCGT-3'; LM-PCR.GSP3.revB, TGGGTGCCTGGTCGCGCGTTCTC.
RNA interference (RNAi) analysis with lentiviral vectors. For expression of short hairpin RNA against NELF-E, a double-stranded oligonucleotide was inserted downstream of the mouse U6 promoter (–315 to +1; GenBank accession number X06980) such that the following RNA sequence was expressed: 5'-GAUGGAGUCAGCAGAUCAGuucaagagaCUGAUCUGCUGACUCCAUCuu-3' (the sequence in uppercase corresponds to positions 1020 to 1038 of the human NELF-E mRNA [accession no. NM_002904]). The expression cassette and a control U6 promoter sequence were subcloned into lentiviral vector pLenti6 (Invitrogen), and recombinant lentiviruses were produced according the manufacturer's instructions. For infection, HepG2 cells (9 x 105) were plated in 10-cm tissue culture dishes. On the following day (day 1), 1.5-ml aliquots of the lentiviral stock and Polybrene (final concentration, 6 µg/ml; Sigma) were added to dishes containing 4.5 ml of 10% FBS-DMEM, and on day 2, the medium was replaced with fresh medium. On day 3, the cells were passaged and divided into three 10-cm dishes, and on day 5, the cells were passaged again and divided into two six-well plates (5 x 105 cells in each well) and two 10-cm dishes (2 x 106 cells each). On day 6, the medium was replaced with 0.1% FBS-DMEM, and on day 7, the cells were stimulated with IL-6. The cells in six-well plates were used for real-time RT-PCR analysis or Western blot analysis, and the cells in 10-cm dishes were used for in vivo footprinting analysis. For ChIP assays, cells were plated on two 15-cm dishes (5 x 106 cells each) on day 5.
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RESULTS |
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Whereas STAT3 was detected negligibly on the junB gene before induction, 15 min after IL-6 stimulation, STAT3 was specifically cross-linked to both the 5'- and 3'-flanking regions (Fig. 1C). The 3' region was previously reported to be a putative downstream enhancer (7). STAT3 remained bound to the promoter and downstream enhancer regions for up to 45 min after induction and disappeared by 90 min. Concomitantly after induction, acetylation of histone H4 increased significantly in more distal 5' and 3' regions, while the transcribed region was modestly and constitutively acetylated (Fig. 1D). Interestingly, the levels of histone H4 acetylation, as well as those of histone H3, were quite low in the promoter and downstream enhancer regions (Fig. 1D and E). These findings, together with the previous report that these regions are hypersensitive to DNase I (21), suggest that the density of nucleosomes in these regions is constitutively low.
To investigate transcription elongation along the junB gene, we next mapped the distributions of RNAPII, DSIF, and NELF. ChIP with anti-RNAPII antibody (Fig. 1F) showed that RNAPII is efficiently recruited to the promoter region before induction. Fifteen minutes after IL-6 addition, RNAPII became cross-linked to the entire region of the gene, and maximal cross-linking was observed 45 min after induction. At 90 min, the distribution of RNAPII reverted to that of the uninduced state. These results are consistent with the idea that junB expression is controlled both before and after the PIC assembly step. We obtained very similar results with anti-hSpt5 antibody (Fig. 1G). A significant amount of hSpt5 was found associated with the promoter region before induction, and its cross-linking to the downstream regions was observed 15 min after induction. In contrast, the cross-linking pattern for NELF was different (Fig. 1H). Although NELF-E was cross-linked to the promoter region throughout the time course, it did not associate with the downstream regions appreciably, even after induction.
The above experiments revealed three important aspects of transcriptional regulation on the junB locus. First, RNAPII, DSIF, and NELF associate with the promoter region, but not with the downstream regions, before induction. Second, RNAPII, DSIF, and NELF accumulate in the promoter region after induction. Third, RNAPII and DSIF, but not NELF, associate with the downstream regions after induction. The first finding suggests that junB transcription is negatively regulated at the post-PIC assembly step before induction, and the second finding suggests that this negative regulation persists even after induction.
Association of RNAPII, DSIF, and NELF with the promoter-proximal transcribed region of junB. Conventional ChIP analysis cannot distinguish between polymerases in the PIC and those paused in the promoter-proximal region. To obtain more precise information on the location of RNAPII, DSIF, and NELF, we performed a modified ChIP assay as previously described (4). Briefly, soluble chromatin was partially digested with HaeIII and PvuII, which cleave at positions +16 and +21 and at position +133 of the junB gene, respectively (Fig. 2A). After immunoprecipitation of the digested chromatin, coprecipitated DNAs and 1% of the input DNAs were subjected to LM-PCR analysis such that three digested genomic DNA fragments (fragments I, II, and III) were amplified. The three DNA fragments should be coimmunoprecipitated at different efficiencies, depending on the binding sites of the proteins of interest. For example, if RNAPII exclusively associates with the transcription initiation site, immunoprecipitation with anti-RNAPII antibody should lead to recovery of the three DNA fragments with equal efficiency. If RNAPII exclusively associates with the region between positions +21 and +133, only fragment III should be detected. Thus, a higher recovery of fragment III indicates the presence of RNAPII within the region between positions +21 and +133.
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RNAPII strongly accumulates at around position +50 of the junB gene. To determine the distribution of RNAPII over the junB promoter-proximal region at nucleotide resolution, we performed potassium permanganate (KMnO4) in vivo footprinting analysis. Because KMnO4 preferentially oxidizes thymidine residues in single-stranded DNA, transcription bubbles within elongating polymerases are detected by this analysis. Genomic DNA was prepared from KMnO4-treated cells and cleaved at oxidized sites with piperidine, and these sites were mapped by LM-PCR with two primer sets that are specific to the junB promoter-proximal region (Fig. 3). As a control, purified genomic DNA was treated with KMnO4 in vitro before and after heat denaturation in order to estimate the KMnO4 sensitivity of double-stranded and single-stranded DNAs, respectively (lanes 4, 5, 9, and 10). Consistent with the data in Fig. 1 and 2, transcription bubbles detected in the junB promoter-proximal region were similar in pattern before and after induction (lanes 2, 3, 7, and 8). Before induction, there were strong KMnO4-sensitive sites at positions +47 and +54 and minor sensitive sites at positions +29, +78, +80, and +105, the intensities of which were slightly increased by short-term (15 min) IL-6 treatment. T residues at positions +8, +9, and +124 to +127 were not sensitive to KMnO4 in unstimulated cells but became modestly sensitive after IL-6 stimulation. Similar results were obtained when IL-6 treatment was prolonged to 45 min (data not shown). These results indicate that a strong pause occurs at about position +50 of the junB gene before and after IL-6 induction.
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Next, we performed KMnO4 in vivo footprinting analysis with NELF-E-RNAi and U6 cells (Fig. 4C). NELF-E knockdown resulted in a two- to threefold decrease in transcription bubbles at positions +29 and +47 both before and after induction, indicating that pausing was reduced at these sites. The signals at site +54 seemed to be less affected by the knockdown, possibly because of residual NELF (see Discussion). These results are consistent with the findings in Fig. 4B, and together they indicate that NELF activity is important for transcriptional pausing in the junB promoter-proximal region both before and after induction.
DSIF- and NELF-mediated pausing attenuates expression of junB and other IEGs. We then investigated the role of NELF in junB expression by quantifying the mRNA level by real-time RT-PCR (Fig. 5A, left). Although the junB mRNA level reached its peak at 60 min after induction in both NELF-E-RNAi and U6 cells, NELF-E knockdown resulted in a reproducible twofold increase in the junB mRNA level both before and after induction. In contrast, the GAPDH mRNA level was not affected by NELF-E knockdown (Fig. 5A, right). The increase in junB expression was also observed at the protein level. Western blot analysis showed that NELF-E knockdown resulted in a twofold increase in the JunB protein level both before and after induction (Fig. 5B). Next, we examined whether or not NELF-E knockdown affects the IL-6-STAT3 signaling pathway. STAT3 is known to be activated by phosphorylation on Tyr705 and Ser727 (1, 43). As shown in Fig. 5C, STAT3 phosphorylation increased transiently after IL-6 treatment and was not appreciably affected by NELF-E knockdown. These results indicate that NELF directly downregulates both basal and activated levels of transcription of the junB gene. Moreover, the similar effects of NELF-E knockdown on both the mRNA and protein levels of junB suggest that NELF is not involved in processing, export, and translation of the mRNA.
Like junB, c-fos and tis-11 are IEGs that are controlled by STAT3 and are rapidly induced by IL-6 (16, 40). Moreover, c-fos is thought to be regulated in the transcription elongation phase (23). We therefore examined whether NELF is also involved in the regulation of these genes. As shown in Fig. 6, NELF-E knockdown resulted in a two- to threefold enhancement of the mRNA levels of c-fos and tis-11 both before and after IL-6 induction. These results indicate that c-fos and tis-11 are subject to negative regulation by NELF as well.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Paused polymerases have been identified at several loci, of which Drosophila hsp70 is the best studied. Pausing at hsp70 occurs between positions +20 and +40 of the transcribed region and is thought to involve DSIF and NELF (33, 34). In addition, the promoter region of hsp70 is known to be decondensed in the absence of heat shock (8). Several lines of evidence suggest that the junB promoter region has a similar chromatin structure. We have shown that the promoter and downstream enhancer regions are not immunoprecipitated efficiently by anti-acetylated histone H4 and anti-histone H3 antibodies (Fig. 1D and E). Moreover, both regions are hypersensitive to DNase I (21, 22). Such a decondensed chromatin structure probably makes the promoter region accessible to the basal transcription machinery and facilitates PIC assembly and transcription initiation, and then transcription is subject to negative regulation by DSIF and NELF.
Although DSIF and NELF were biochemically identified on the basis of the ability to stall RNAPII movement along a DNA template, it is not well understood when and where these proteins function on the genome of living cells. At the human junB locus, DSIF and NELF likely induce transcriptional pausing in the promoter-proximal region. It appears that DSIF also travels downstream with RNAPII following induction (Fig. 1), an observation that is consistent with another biochemical activity of DSIF, the promotion of elongation (32). We assume that pausing before induction acts to reduce unwanted "leaky" expression of junB. Our results showed, however, that depletion of NELF does not lead to full activation of junB in an uninduced state. Other processes occurring after induction, such as activator-dependent recruitment of more RNAPII to the promoter, may also be required to fully activate the gene. Alternatively, residual NELF activity after the knockdown may obscure true phenotypic effects. In this regard, it is noteworthy that a substantial amount of NELF-E remained associated with the junB promoter-proximal region after its knockdown in spite of a large overall decrease in the level of its protein (Fig. 4). This apparent discrepancy may be explained by possible preferential depletion of a free pool of NELF.
Importantly, our study shows that DSIF- and NELF-mediated pausing on junB persists and attenuates junB expression even after induction. This finding contrasts with the observation that on Drosophila hsp70, heat shock causes dissociation of NELF and alleviation of promoter-proximal pausing (34). In the case of some human estrogen-responsive genes, it is reported that NELF attenuates the activated level of transcription (3), similar to what we observed for junB and other IEGs. Before induction, however, RNAPII and NELF are not appreciably associated with the estrogen-responsive genes (3). Thus, DSIF- and NELF-mediated pausing at the junB locus is partly similar but not identical to those found on Drosophila hsp70 and human estrogen-responsive genes.
On the basis of these results, we propose the following model for transcriptional regulation of junB by DSIF- and NELF-mediated pausing (Fig. 7). In an uninduced state, DSIF and NELF cause RNAPII pausing at around position +50 of the promoter-proximal region, where chromatin structure is decondensed and permissive for PIC assembly and transcription initiation. Immediately after IL-6 stimulation, the transcriptional activator STAT3 binds to both the promoter and 3' enhancer regions and increases the efficiency of transcription elongation, allowing the polymerase to reach the 3' end of the gene. STAT3 also recruits more RNAPII to the promoter region. The enhancement of transcription elongation by STAT3 may be mediated by P-TEFb or other proteins having elongation activation activity. From a number of in vitro and/or in vivo studies, it has been shown that P-TEFb, FACT, TFIIS, TFIIF, and the capping enzyme are capable of reversing transcriptional repression imposed by DSIF and NELF (2, 14, 19, 25, 30). However, DSIF- and NELF-mediated pausing persists and continues to attenuate junB expression. We provide two possible explanations as to how transcriptional pausing attenuates the level of mRNA expression. First, RNAPII paused in the promoter-proximal region may prevent transcription reinitiation by posing an obstacle to progression of the second polymerase, which we detected in Fig. 2. Second, pausing may eventually lead to premature termination of transcription. DSIF and NELF induce slowdown or pausing but not termination of polymerases in vitro (25, 38). When DSIF and NELF cause a prolonged pausing of polymerases in living cells, however, other proteins, such as the transcription termination factor Pcf11 (42), may cause premature termination. In either case, pausing acts as a rate-limiting step to reduce the maximal level of target gene expression.
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ACKNOWLEDGMENTS |
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This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.W. and H.H.) and a grant from NEDO (to H.H.). This study was also supported in part by the Tokyo Tech. Award for Challenging Research to T.W. and a grant from the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. M.A. is a research fellow of the Japan Society for the Promotion of Science.
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FOOTNOTES |
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