Identification of poly(ADP-ribose) polymerase as a transcriptional coactivator of the human T-cell leukemia virus type 1 Tax protein

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose activity is believed to contribute significantly to cellular transformation. Tax stimulates transcription from the proviral promoter as well as from promoters for a variety of cellular genes. The mechanism through which Tax communicates to the general transcription factors and RNA polymerase II has not been completely determined. We investigated whether Tax could function directly through the general transcription factors and RNA polymerase II or if other intermediary factors or coactivators were required. Our results show that a system consisting of purified recombinant TFIIA, TFIIB, TFIIE, TFIIF, CREB, and Tax, along with highly purified RNA polymerase II, affinity-purified epitope-tagged TFIID, and semipurified TFIIH, supports basal transcription of the HTLV-1 promoter but is not responsive to Tax. Two additional activities were required for Tax to stimulate transcription. We demonstrate that one of these activities is poly(ADP-ribose) polymerase (PARP), a molecule that has been previously identified to be the transcriptional coactivator PC1. PARP functions as a coactivator in our assays at molar concentrations approximately equal to those of the DNA and equal to or less than those of the transcription factors in the assay. We further demonstrate that PARP stimulates Tax-activated transcription in vivo, demonstrating that this biochemical approach has functionally identified a novel target for the retroviral transcriptional activator Tax.

FIG. 1

HeLa cell nuclear extracts contain factors necessary for the stimulation of transcription by Tax in vitro. (A) Four copies of the HTLV-1 TxRE were placed upstream of the HTLV-1 core promoter. This region contains the natural HTLV-1 TATA box which binds TFIID and allows the assembly of the basal transcription factors. (B) Typical transcription reaction with the plasmid from panel A, HeLa nuclear extract (NE), and 100 ng of recombinant Tax where indicated. The arrow indicates the full-length, 380-base G-less transcript that is activated fivefold by the presence of Tax. For this and all figures, transcription assays were analyzed by PhosphorImager software IQMac version 1.2, and images are presented on a linear scale. (C) Quantitation of transcripts from panel B by PhosphorImager analysis. Values were normalized to full activity with Tax. Fold activation (reaction with Tax divided by reaction without Tax) is indicated (5×).

FIG. 2

Purification scheme of coactivators and TFIID. Starting material for the nuclear extract material was between 60 and 120 liters of cultured cells. Arrows indicate subsequent columns used for purification. Columns shown with horizontal bars were developed with step elutions in buffer with the salt concentrations indicated. Columns shown with slanted bars represent linear gradients with initial and final salt concentrations indicated at the left and right. Semipurified TFIID is the DE-52 0.25 M KCl peak. Purified TFIID eluted from the PureGel SCX column at approximately 150 mM KCl. The coactivator present in the DE-52 0.1 M KCl flowthrough was purified by two separate methods. Purification over a PureGel SCX column yielded the mixed coactivator peak. Purification by gel filtration yielded three activities, LTF1, LTF2, and STF.

FIG. 3

Transcription factors used in in vitro assays. (A) SDS-PAGE analysis of recombinant transcription factors stained with Coomassie blue. Positions of molecular weights of markers are indicated on the left in kilodaltons. Tax and CREB are ∼43 kDa. TFIIA has two polypeptides, ∼55-kDa α/β subunit and ∼12- to 14-kDa γ subunit. TFIIB is ∼30 kDa. TFIIE contains two subunits, ∼34 and ∼56 kDa, and TFIIF contains two subunits, ∼30 and ∼74 kDa. (B) SDS-PAGE analysis of HPLC-purified RNAP II visualized by silver staining. Subunits are indicated on the left. Positions of molecular weights of markers are indicated on the right in kilodaltons.

FIG. 4

Coactivator requirement depends on TFIID source. Radiolabeled RNAs from representative transcription reactions were visualized by PhosphorImager analysis. All reactions contained recombinant TFIIA, TFIIB, TFIIF, CREB, Tax (where indicated), and native, HPLC-purified RNAP II. Purification procedures for semipurified (A) and purified (C) TFIID from HeLa cell nuclear extracts are shown in Fig. 2. Highly purified TFIID (eTFIID) (E) was affinity purified from the phosphocellulose high-salt fraction from HeLa cells expressing epitope-tagged TBP. The mixed coactivator fraction was prepared as shown in Fig. 2. Arrows indicate the full-length transcript. Transcripts were quantitated as in Fig. 1 and shown in panels B, D, and F. Asterisks indicate transcripts at or below background levels.

FIG. 5

Activity of LTF1, LTF2, and STF. (A) In vitro transcription reactions were carried out and analyzed as before except that recombinant TFIIE was added to all reactions. Tax (100 ng) and fractions containing LTF1, LTF2, or STF were added as indicated. The arrow indicates the full-length transcript. (B) Transcripts were quantitated as in Fig. 4. (C) In vitro transcription reactions were carried out as in panel A except that CREB was omitted from the first two lanes as indicated. (D) Transcripts from panel C were quantitated as in Fig. 4.

FIG. 6

Purification of LTF2. (A) The various stages of purification of LTF2 were analyzed by SDS-PAGE and visualized by silver staining. The following fractions that contained the peak of LTF2 activity are in the lanes as indicated: 0.3 to 0.5 M KCl step of the phosphocellulose column; 0.1 M KCl flowthrough of the DE-52 column; fractions corresponding to molecular weights of approximately 80 to 130 kDa of the Superdex 200 column; 0.7 to 0.8 M ammonium sulfate fraction of a linear gradient on the phenyl-Superose column; 0.25 to 0.3 M KPO₄ of a linear gradient on the hydroxyapatite column; and the 150 to 180 mM KCl fractions of a linear gradient on an analytical 4.6-mm by 10-cm PureGel SCX column. Molecular masses are indicated (in kilodaltons) on the left. (B) Highly purified PARP from the PureGel SCX column peak shown in panel A were substituted for LTF2 in transcription reactions using conditions similar to those in Fig. 5. STF, PARP, and LTF1 were added as indicated. The arrow indicates the full-length transcript. (C) Transcripts were quantitated as in Fig. 4.

FIG. 7

Identification of the 110-kDa protein in LTF2. (A) LTF2 fractions were obtained as shown in Fig. 2 and further purified over phenyl, Superose, and hydroxyapatite. (B) LTF2 fractions from the hydroxyapatite column were analyzed by SDS-PAGE followed by silver staining. M, markers (molecular masses are indicated at the left); on, the onput to the column. Individual fraction numbers are indicated. The 110-kDa protein that copurifies with LTF2 is indicated by the arrow. Fractions 72 to 74 were pooled and sequenced, and the results are shown in panel C. The 1,014 amino acids of PARP are represented by boxes with the amino acid number indicated below. Using the SwissProt.r34 database, 46 of the 47 amino acids in our 110-kDa protein sequence corresponded to sequences within the protein PARP (entry P09874). The actual sequences of the peptides are displayed in the single-letter code and are positioned over the region to which they mapped onto PARP. The lysine indicated by a smaller K is the one mismatch (arginine in PARP). The position of mass spectrometry peaks that matched the predicted tryptic fragments of PARP are shown by bold black lines. Functional domains within PARP are labeled. Putative Zn finger domains are in the amino-terminal region. The automodification domain (Automod.) is the region that is extensively autoribosylated. The catalytic domain that binds substrate is in the carboxy-terminal region.

FIG. 8

PARP(1-450) can substitute for LTF2 in transcription. (A) Transcription reactions were carried out as before, in the absence of LTF2 or with 40 ng of recombinant PARP(1-450) where indicated. The arrow indicates the full-length transcript. (B) Transcripts were quantitated as in Fig. 4.

FIG. 9

PARP enhances Tax-activated transcription in vivo. A mouse embryonic cell line deficient for PARP was transfected with 10 μg of a luciferase reporter plasmid driven by the HTLV-1 promoter (positions −306 to −1) alone or with 15 μg of an HTLV-1 Tax expression vector; 10 μg of PARP expression vector (pcD-12) was added as indicated. Each reaction received additional pUC DNA to a total of 35 μg of DNA. Luciferase activity was normalized to protein concentration and then divided by the activity in the absence of Tax and PARP; the numerical value is indicated above each bar. Experiment 1 is the average of duplicates, while experiment 2 is the average of triplicates. The positive error bar indicates the standard deviation for each average.