Inhibition of Transcription by the Trimeric Cyclin-dependent Kinase 7 Complex*

Daniel A. BocharDagger , Zhen-Qiang Pan§, Ronald Knights, Robert P. Fisher, Ali Shilatifardparallel , and Ramin ShiekhattarDagger Dagger Dagger

From the § Derald H. Ruttenberg Cancer Center, The Mount Sinai Medical Center, New York, New York 10029-6574, the  Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, the parallel  Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, and the Dagger  Wistar Institute, Philadelphia, Pennsylvania 19104

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cyclin-dependent kinase 7 (CDK7) can be isolated as a subunit of a trimeric kinase complex functional in activation of the mitotic promoting factor. In this study, we demonstrate that the trimeric cdk-activating kinase (CAK) acts as a transcriptional repressor of class II promoters and show that repression results from CAK impeding the entry of RNA polymerase II and basal transcription factor IIF into a competent preinitiation complex. This repression is independent of CDK7 kinase activity. We find that the p36/MAT1 subunit of CAK is required for transcriptional repression and the repression is independent of the promoter used. Our results demonstrate a central role for CAK in regulation of messenger RNA synthesis by either inhibition of RNA polymerase II-catalyzed transcription or stimulation of transcription through association with basal transcription repair factor IIH.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cyclin-dependent kinase 7 (CDK7)1 was originally isolated as the catalytic subunit of the trimeric cdk-activating kinase (CAK) complex. This complex, consisting of CDK7, cyclin H, and MAT1, is responsible for activation of the mitotic promoting factor in vitro (1-3). The discovery that CDK7 was also a component of the basal transcription repair factor IIH (TFIIH) implicated a dual role for CDK7 in transcription as part of TFIIH and in the control of the cell cycle as the trimeric CAK complex (4-7). TFIIH is a multisubunit protein complex identified as a factor required for RNA polymerase II (RNAPII)-catalyzed transcription (8-11), and subsequently this complex was found to play a key role in nucleotide excision repair (12-14). At least nine polypeptides with molecular masses of 89, 80, 62, 52, 44, 40, 37, 36, and 34 kDa co-purify with mammalian TFIIH. The cDNAs encoding all of these subunits have now been cloned. p89 and p80 are the gene products of ERCC3 (XPB) and ERCC2 (XPD), respectively (13-15). p62 and p44 are the mammalian counterparts of the yeast TFB1 and SSL1 gene products that are required for DNA nucleotide excision repair (16). p34 exhibits partial sequence homology to p44 and also contains zinc-finger motifs (17). The p40, p37, and p36 subunits of TFIIH are identical to the vertebrate CAK complexes, CDK7, cyclin H, and p36/MAT1, respectively (5-7). Two subcomplexes containing some TFIIH polypeptides can also be isolated from extracts of HeLa cells (18, 19): (i) a five-subunit core TFIIH complex that includes ERCC3 (XPB), p62, p52, p44, and p34 but is devoid of detectable levels of ERCC2 (XPD) or CAK; (ii) an XPD·CAK complex that includes XPD and all three CAK components (CDK7, cyclin H, and p36/MAT1). The addition of XPD·CAK to the core TFIIH potently stimulates the TFIIH transcriptional activity (18, 19). These observations suggest that core TFIIH and XPD·CAK interact to form a complex that constitutes the TFIIH holoenzyme (holo-TFIIH). Biochemical analysis has therefore revealed that CDK7 is a component of at least three complexes, the trimeric CAK complex (20-22), the quaternary complex with the XPD, and the nine-subunit TFIIH complex (18, 19). In addition, a number of studies have suggested that the trimeric CAK complex is the CDK7-containing complex involved in cell cycle control (21, 23). These studies were based on initial reports that identified the trimeric CAK complex as the kinase responsible for activating a number of cdks (1-3). However, in Saccharomyces cerevisiae, Kin28 and Ccl1, the counterparts of CDK7 and cyclin H, associate with S. cerevisiae TFIIH, although they do not exhibit detectable CAK activity (4, 24). The CAK activity of S. cerevisiae has recently been identified as the gene product of Cak1/Civ1 (25, 26). However, a recent report indicates that CDK7 is essential for mitosis and cdk-activating kinase activity in Drosophila melanogaster (27).

Here we show that the trimeric CAK complex exhibits an inhibitory activity in RNAPII-dependent transcription. This inhibition results from the preclusion of TFIIF and RNAPII from the preinitiation complex. These studies reveal a novel role for the trimeric CAK in regulation of transcription.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Purification of CAK from HeLa Nuclear Extract-- Trimeric CAK was purified from 3 g of HeLa nuclear extract (Fig. 1A). Nuclear extract was loaded on a 1-liter column of phosphocellulose (Whatman) and fractionated stepwise by the indicated KCl concentrations in buffer A (20 mM Tris-HCl, pH 7.9, 0.2 mM EDTA, 10 mM 2-mercaptothanol, 20% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. The phosphocellulose 0.3 M KCl fraction (400 mg) was dialyzed to 0.1 M KCl in buffer A and loaded on a 100-ml DEAE-Sephacel column (Amersham Pharmacia Biotech). The column was eluted with 0.5 M KCl in buffer A. The 0.5 M KCl elution (260 mg) was dialyzed to 100 mM KCl in buffer A and loaded on a 100-ml Q-Sepharose column (Sigma). The column was resolved using a linear 10-column volume gradient of 100-600 mM KCl. Fractions containing CDK7 (~200 mM KCl, 37 mg) were dialyzed to 10 mM potassium phosphate in buffer B (5 mM Hepes, pH 7.5, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 10 µM CaCl2, 10% glycerol, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) and loaded on a 20-ml hydroxyapatite column (American International Chemical). The column was resolved using a linear 10-column volume gradient of 10-600 mM potassium phosphate in buffer B. CDK7-containing fractions (100 mM potassium phosphate, 8.6 mg) were dialyzed to 1 M ammonium sulfate in buffer C (20 mM Hepes, pH 7.9, 4 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM EDTA, 10% glycerol) and fractionated on a phenyl-Superose HR 5/5 column (Amersham Pharmacia Biotech). The phenyl-Superose column was resolved using a linear 10-column volume gradient of 1 M to 0 mM ammonium sulfate in buffer A. CDK7-containing fractions (~0.4 M ammonium sulfate, 0.5 mg) were precipitated with 60% ammonium sulfate and fractionated on a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated in 1 M KCl in buffer A.

Generation of Recombinant CAK Polypeptides in SF9 Cells-- We generated and purified CAK using a detailed protocol for the production of CAK in insect SF9 cells as described previously (21).

CTD Kinase Assay-- CTD kinase assays for phosphorylation of the carboxyl-terminal domain of RNAPII were described previously (7).

In Vitro Transcription-- Transcription assays were reconstituted as described (7) using recombinant TBP (10 ng), TFIIB (10 ng), TFIIE (10 ng), TFIIF (10 ng), highly purified HeLa holo-TFIIH (7) or highly purified rat TFIIH (6), and highly purified HeLa core RNAPII (28). The rat somatostatin promoter (29), human T-cell leukemia virus, type 1 (HTLV-1) promoter (30), or Ad-MLP leading to a 390-nucleotide G-less cassette was used as the template (100 ng).

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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The Trimeric CAK Complex Purified from HeLa Cells Is a Transcriptional Repressor-- The trimeric CAK complex from HeLa nuclear extract was purified as described under "Experimental Procedures" (Fig. 1A) using the CTD-kinase activity and Western blot analysis with antibodies against CDK7 and cyclin H. Nuclear extract was initially fractionated by phosphocellulose chromatography. The 0.3 M KCl step elution contained approximately 40% of CDK7 immunoreactivity and was devoid of TFIIH activity or ERCC3 immunoreactivity, which fractionated into the 0.5 M KCl step elution (data not shown). The 0.3 M KCl fraction was therefore further purified through five additional steps (Fig. 1A). Analysis of the CTD kinase activity of CAK on the sizing column (Superose 6) revealed the peak of activity eluting at ~200 kDa, consistent with the previously reported size for the trimeric CAK complex (21). We estimate that our purification resulted in ~2000-fold enrichment in CAK. Analysis of column fractions in a transcription system reconstituted with recombinant TBP, TFIIB, TFIIE, TFIIF, highly purified HeLa or rat holo-TFIIH, and highly purified HeLa core RNAPII revealed an inhibitory activity copurifing with the CDK7 complex throughout the purification (Fig. 1B). The transcriptional inhibitory activity was also observed in the last two steps of the purification (phenyl-Superose and Superose 6) (Fig. 1, C and D). Highest concentrations of the CAK complex tested resulted in about 95% inhibition in basal transcription.


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Fig. 1.   Isolation of the trimeric CAK. A, HeLa nuclear extract was fractionated by chromatography as described under "Experimental Procedures." The horizontal and diagonal lines indicate stepwise and gradient elution, respectively. Salt concentrations for protein elutions are given in millimolars. Also shown is a Western blot analysis of fractions derived from the phenyl-Superose with the antibodies indicated on the left and a CTD kinase analysis of fractions derived from the Superose 6 column. B, hydroxyapatite column fractions (3 µl) were analyzed using transcription reactions reconstituted with the full set of basal factors using linear Ad-MLP (100 ng) and Western blot analysis (20 µl of column fractions) using anti-CDK7 antibodies (Oncogene Research Products). C, transcription reactions were reconstituted with the full set of factors using a supercoiled HTLV-1 promoter as described under "Experimental Procedures," 1X represents 2 µl of the pool from the CDK7 containing fractions from a phenyl-Superose column. D is the same as B except 6 µl of the pool from the phenyl-Superose or Superose 6 columns were used. nt, nucleotide.

Recombinant Trimeric CAK Displays Transcriptional Inhibitory Activity-- To determine if the inhibitory activity associated with the HeLa fractions is the CAK complex, we expressed either the trimeric (CDK7-cyclin H-MAT1) or the dimeric CAK complex (CDK7-cyclin H) in insect SF9 cells. These complexes were purified as previously reported (21) (Fig. 2A). Moreover, as the highly purified holo-TFIIH complex also contains CAK, we sought to determine the action of the trimeric CAK complex in an assay free of TFIIH. Similar to a previous report (31), reconstituted transcription using the rat supercoiled somatostatin promoter is independent of TFIIH or TFIIE but is highly stimulated by these factors (Fig. 2B). Analysis of recombinant trimeric CAK complex in reconstituted transcription assay either in the presence (Fig. 2C, lanes 1 and 2) or the absence (lanes 3-5) of TFIIH revealed that this complex is a potent inhibitor of transcription. The inhibitory effect of CAK is achieved at roughly a 1:1 stoichiometry (0.25 pmol) with other basal factors.


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Fig. 2.   Recombinant CAK inhibits transcription. A, SDS-polyacrylamide gel electrophoresis followed by either staining with Coomassie Brilliant Blue R-250 for recombinant dimeric (lane 1) or silver for trimeric (lane 2) CAK complexes. Note that in the trimeric CAK complex the p36/MAT1 subunit contained a six-histidine tag and therefore displayed a larger molecular mass than that of cyclin H. The asterisk depicts the position of a contaminating polypeptide. B, transcription reactions were reconstituted using a supercoiled rat somatostatin promoter and the full set of factors. Lane 1 represents the complete reaction. The other reactions were identical except for the omission of the indicated protein. C, transcription reactions in the presence or absence of CAK(3) were carried out with a complete set of factors as in panel B (lanes 1 and 2) or in the absence of holo-TFIIH (lanes 3-5). 1X represents approximately 50 ng of recombinant CAK. Approximately 30 ng of holo-TFIIH was added in lanes 1 and 2. Lanes 1 and 2 employed a supercoiled HTLV-1 promoter, and lanes 3-5 employed a supercoiled rat somatostatin promoter. nt, nucleotide.

The p36/MAT1 Subunit of the Trimeric CAK Complex Is Required for the Inhibitory Activity-- To further ascertain the role of the p36/MAT1 subunit of the CDK7 complex in transcriptional repression, we compared the recombinant dimeric and trimeric CAK complexes. Although the two CAK complexes displayed similar kinase activity as ascertained by phosphorylation of a CTD peptide (Fig. 3A), analysis of the dimeric CAK in transcription revealed that p36/MAT1 is required for the inhibitory activity of the CAK complex (Fig. 3B). In contrast to the trimeric CAK, which inhibited transcription driven from either the supercoiled somatostatin (lanes 2 and 3) or supercoiled HTLV-1 (lanes 8 and 9) promoters, the dimeric CAK was devoid of any inhibitory activity with either promoter (lanes 4-6 or 10 and 11). These data indicate that the p36/MAT1 subunit of the CAK complex is required for inhibition of transcription and that the CAK-mediated inhibition is independent of the promoter used.


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Fig. 3.   p36/MAT1 is required for the repressing activity of CAK. A, CTD kinase activity of dimeric (50 ng, lane 1), trimeric (50 ng, lane 2), and a trimeric kinase-deficient mutant of CAK (50 ng, lane 3) was measured using a heptapeptide repeat of the RNAPII-CTD. B, transcription was reconstituted using either a supercoiled rat somatostatin promoter (lanes 1-6) or a supercoiled HTLV-1 promoter (lanes 7-11). Reactions were carried out in the absence of holo-TFIIH with the addition of the indicated proteins. 1X represents 50 ng for each protein preparation. C, transcription was reconstituted using a supercoiled HTLV-1 promoter and a full set of basal factors in the absence of holo-TFIIH (lane 1). The other reactions were identical to lane 1 except for the addition of proteins indicated (lanes 3-5). 1X represents 50 ng of p36/MAT1 or 50 ng of CAK. D, transcription was reconstituted as in C. 50 ng of each factor was added to each transcription reaction. nt, nucleotide.

p36/MAT1 Is Not Sufficient for Transcriptional Inhibition-- To determine whether the p36/MAT1 subunit of the CAK complex is sufficient for transcriptional repression, the p36/MAT1 subunit was expressed in Escherichia coli, and the purified p36/MAT1 was analyzed for its activity in transcription. In contrast to trimeric CAK (Fig. 3B, lane 2), the addition of p36/MAT1 not only failed to inhibit transcription (lanes 3-5) but also displayed a small stimulatory activity (compare lanes 1 and 3). These results demonstrate that although p36/MAT1 is required for the inhibitory activity of the CAK complex, it is not sufficient for inhibition.

Addition of p36/MAT1 to Dimeric CAK Can Reconstitute Transcriptional Repression-- The p36/MAT1 subunit of CAK was produced in SF9 cells, and purified protein was tested for its ability to confer repression when added to the dimeric CAK complex. As Fig. 3C indicates, neither the dimeric CAK (lane 2) nor the p36/MAT1 protein alone were sufficient to mediate repression. However, the addition of the p36/MAT1 subunit to dimeric CAK reconstitutes the transcriptional repression observed with trimeric CAK (lane 4).

The Trimeric CAK Inhibits Transcription by Precluding RNAPII and TFIIF from the Preinitiation Complex-- To analyze which step during the formation of the preinitiation complex the trimeric CAK may target to repress transcription, we incubated the basal transcription factors in a stepwise fashion with the DNA for 30 min before adding the trimeric CAK complex (Fig. 4A). Preincubation of DNA with either TBP (lane 2) or TBP and TFIIB (lane 3) could not overcome the inhibitory effect of CAK, indicating that the TBP·TFIIB complex (TB) formation is not the target of the CAK complex. However, the addition of TFIIF to the preinitiation complex, which results in the formation of the TBF complex, could partially relieve the CAK repression (lane 4). Formation of the TBPolF or TBPolFE complex by further preincubation with RNAPII or RNAPII and TFIIE resulted in a complete recovery of transcription (lanes 5 and 6). These results indicate that trimeric CAK precludes the entry of RNAPII and TFIIF into a competent preinitiation complex, and a preformed preinitiation complex is refractory to the action of trimeric CAK. This contention is further substantiated when we analyzed whether the addition of excess TFIIF or RNAPII can overcome the inhibitory activity of the trimeric CAK complex. As shown in Fig. 4B, the addition of increasing amounts of TFIIF partially overcomes the CAK-mediated repression (lanes 3 and 4), whereas the addition of excess RNAPII could completely restore transcription (lane 5). We conclude that trimeric CAKs repress transcription by precluding RNAPII and TFIIF entry into the preinitiation complex.


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Fig. 4.   CAK represses transcription by precluding TFIIF and RNAPII entry into the pre-initiation complex. A, transcription reactions were initiated by incubating the factors shown on the right panel of the figure with a supercoiled rat somatostatin DNA template for 30 min. The remainder of the transcription factors, CAK, and the nucleotide mixture were then added to the reaction for an additional 60 min. The numbers to the right of the right panel correspond to the lane numbers shown in the bottom of the left panel. B, transcription reactions were reconstituted with the full set of factors using a supercoiled HTLV-1 promoter in the absence of holo-TFIIH (lane 1). Other reactions are as in lane 1 except for the addition of proteins indicated. 1X represents 25 ng for TFIIF and 100 ng for RNAPII. CAK (50 ng) was added to lanes 2-5. C, transcription reaction was reconstituted as in B. 1X represents 70 ng of the mutant trimeric kinase. D, transcription assays were performed as in B and C except either increasing concentrations of rat TFIIH (1X represents 20 ng) or human CAK (phenyl-Superose; 1X represents 20 ng) were added to each reaction. nt, nucleotide.

The Mechanism of CAK-mediated Repression Is Independent of Its Kinase Activity-- To address whether the kinase activity of CDK7 plays a role in CAK-mediated inhibition, we analyzed a kinase-deficient mutant of CDK7, in which lysine 41 was replaced by alanine (Fig. 3A, lane 3). The purified dimeric CAK/K41A, produced in insect cells, was mixed with the p36/MAT1 subunit, produced in insect cells, and analyzed in a reconstituted transcription system. As Fig. 4C indicates, the addition of kinase-deficient CAK resulted in a potent inhibition of transcription (compare lane 1 to lanes 2 and 3). These results indicate that CAK-mediated inhibition is not because of the kinase activity of CAK and may result from CAK physically destabilizing the preinitiation complex.

Excess TFIIH Can Overcome the Inhibitory Effect of CAK-- Because TFIIH displays a stimulatory activity in transcription by stabilizing the preinitiation complex, we analyzed whether increasing concentrations of TFIIH can relieve the CAK-mediated repression. As Fig. 4D reveals, the addition of excess TFIIH can overcome the inhibitory activity of CAK (compare lanes 2-4 to 6-8). These results indicate that TFIIH and CAK are in a competition for the preinitiation complex. Whereas TFIIH stimulates transcription by stabilizing the preinitiation complex formation, CAK exerts an inhibitory effect by disrupting its formation.

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INTRODUCTION
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The novelty of this work lies in the following. First, it demonstrates transcriptional inhibitory activity for trimeric CAK in a fully defined system, comprised of essentially homogeneous basal factors and RNA polymerase II. Second, it shows that CAK inhibits transcription by preventing RNA polymerase II and TFIIF entry into the preinitiation complex. Third, it presents evidence for the requirement of p36/MAT1 in the CAK-mediated inhibitory effect. Finally, it demonstrates that the inhibitory activity is independent of the kinase activity of CDK7.

The trimeric CAK complex was initially identified as the kinase complex responsible for phosphorylation and consequent activation of other cyclin-dependent kinases from mammalian cells (1-3). It was later discovered that CDK7, cyclin H, and MAT1 were also components of the basal transcription factor TFIIH (4-7). Furthermore, it was observed that TFIIH can be dissociated into two subcomplexes, one containing the core TFIIH subunits (XPB, p62, p51, p44, and p34) and the other containing XPD and the three CAK subunits (18, 19). We found that the trimeric CAK purified from HeLa cells contained a transcriptional inhibitory activity in a highly purified reconstituted transcription system. The inhibitory activity associated with the HeLa fractions was demonstrated to be mediated by the trimeric CAK complex, because the recombinant trimeric CAK produced in insect cells inhibited transcription. Interestingly, the p36/MAT1 subunit of the trimeric CAK was required for the transcriptional inhibition. This observation lends further support to the physiological relevance of CAK-mediated inhibition, as the predominant form of CAK in mammalian extracts not associated with TFIIH contains the p36/MAT1 subunit (19, 21). The transcriptional inhibition by the CAK complex did not result from CDK7 kinase activity, because the kinase-deficient mutant of CAK is also a potent inhibitor of transcription. Our studies revealed that CAK inhibited transcription by preventing the formation of the TBPolF complex. Therefore, either preforming the TBPolF complex or the addition of excess TFIIF, RNAPII, or TFIIH was able to stabilize the complex and to overcome the inhibitory effect of CAK.

A number of studies have concluded that the trimeric CAK complex represents the form of CAK involved in cell cycle control (1-3, 21). Here we present evidence indicating that the trimeric CAK complex displays a novel role in transcription distinct from that of its function when associated with TFIIH. Our findings are a further support for CAK as a regulator of transcription in addition to the function of CAK in cell cycle control.

    ACKNOWLEDGEMENTS

We thank Yuying Zhang for expert technical assistance and P. Lieberman for critical comments on the manuscript. We thank the following people for providing reagents used for this study: Y. Xiong for pET-CDK7 and pET-MAT1; R. Roeder for pHT7MAT1; and D. Morgan for baculoviruses carrying CDK7, cyclin H, or p36/MAT1.

    FOOTNOTES

* This work was supported by Grant GM55059 from the National Institutes of Health and grants from Life and Health Insurance Medical Research Fund, New York Community Trust, and Hirschl Trust (to Z.-Q. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger Supported by a start up fund from the Wistar Institute. To whom correspondence should be addressed: Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3896; Fax: 215-898-3868; E-mail: shiekhattar{at}wistar.upenn.edu.

    ABBREVIATIONS

The abbreviations used are: CDK7, cyclin-dependent kinase 7; CAK, cdk-activating kinase; TF, transcription repair factor; RNAPII, RNA polymerase II; holo, holoenzyme; CTD, carboxyl-terminal domain; HTLV, human T-cell leukemia virus; TBP, TATA-binding protein; TB, TBP·TFIIB.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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