Human T-cell leukemia virus type 1 Tax requires direct access to DNA for recruitment of CREB binding protein to the viral promoter

Abstract

Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5'-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter.

FIG. 1

Tax transactivation in vitro is dependent upon CREB and the viral CRE. (A) Schematic representation of the linearized promoter templates used in the in vitro transcription reactions. The HTLV-1 promoter carries the full transcriptional control region of the virus, including the three 21-bp repeats (viral CREs). The heterologous promoters each contained three tandem copies of either the viral CRE (from the promoter-proximal 21-bp repeat) or the cellular CRE (from the human chorionic gonadotropin gene), cloned immediately upstream of the herpesvirus thymidine kinase (TK) minimal promoter (at position −32). (B) In vitro transcription assay showing that Tax activation of transcription is dependent upon the viral CREs. Transcription reaction mixtures contained the HTLV-1 template fragment (50 ng) (lanes 2 to 4), the viral CRE promoter template fragment (100 ng) (lanes 5 to 7), or the cellular CRE promoter template fragment (25 ng) (lanes 8 to 10) in the presence of 32 μg of nuclear extract from the HTLV-1-negative human T-lymphocyte cell line CEM. A 50-ng amount of purified, recombinant CREB and/or 125 ng of purified recombinant Tax was added to the indicated reaction mixtures. A labeled DNA fragment was added to each reaction prior to RNA isolation to measure the recovery of the labeled RNA transcript. The positions of the recovery standard, the RNA transcripts, and the size of the radiolabeled DNA markers are indicated.

FIG. 2

Tax protein alters the MPE:Fe footprint pattern of CREB on a viral CRE but not on a cellular CRE. (A) Tax expands and enhances the MPE:Fe footprint of CREB on the noncoding strand of the viral CRE. The binding-reaction mixtures contained 10 fmol of the viral CRE probe, singly end labeled on the noncoding strand, and 25 ng of purified, recombinant CREB (lanes 2 to 5 and 9 to 11). The reaction mixtures also contained 50 ng (lane 3), 100 ng (lane 4), or 200 ng (lanes 5, 7, 8, 10, and 11) of purified recombinant Tax and 200 ng of purified GST-KIX (lanes 8 and 11), as indicated. Maxam-Gilbert sequencing reactions for guanine and guanine/adenine were run adjacent to the MPE:Fe cleavage products to assign specific nucleotides (40). (B) The KIX domain of CBP is required for Tax expansion of the CREB bZIP footprint. Binding-reaction mixtures contained 10 fmol of the viral CRE probe, singly end labeled on the coding strand, and 5 ng of purified CREB bZIP domain (amino acids 254 to 327) (lanes 2 to 4 and 7 to 9). The reaction mixtures also contained 100 ng (lanes 3 and 9) or 200 ng (lane 4) of Tax and 1 μg of GST-KIX (lanes 8 and 9), as indicated. Maxam-Gilbert sequencing markers were run adjacent to the cleavage products to assign specific nucleotides. (C) Tax does not alter the footprint of CREB or CREB bZIP on the cellular CRE. The binding-reaction mixtures contained 10 fmol of the cellular CRE, singly end labeled on the coding strand, and 10 ng of CREB (lanes 2 to 6 and 14) or 2 ng of CREB bZIP (lanes 8 to 12 and 15). The reaction mixtures also contained 200 ng (lanes 3 and 9) or 400 ng (lanes 4 to 6, 10 to 12, 16 and 17) of Tax and 1 μg (lanes 5 and 11) or 2 μg (lanes 6, 12, 14 to 16, and 18) of GST-KIX as indicated. Maxam-Gilbert sequencing reactions were run adjacent to the MPE:Fe cleavage products to assign specific nucleotides. (D) Schematic representation of the relative protection produced by CREB or by CREB and Tax on the viral CRE. To account for Tax enhancement of CREB binding, experiments with high concentrations of CREB were directly compared to experiments with lower concentrations of CREB in the presence of Tax that gave similar degrees of protection in the core CREB binding site. The relative protection from MPE:Fe cleavage in the presence of CREB (open bars) or CREB and Tax (solid bars) was quantitated and averaged from three separate experiments. Bar heights are proportional to the degree of protection from the cleavage agent, with DNA-alone values equal to 1.0 and higher values corresponding to increased protection from MPE:Fe cleavage.

FIG. 2

Tax protein alters the MPE:Fe footprint pattern of CREB on a viral CRE but not on a cellular CRE. (A) Tax expands and enhances the MPE:Fe footprint of CREB on the noncoding strand of the viral CRE. The binding-reaction mixtures contained 10 fmol of the viral CRE probe, singly end labeled on the noncoding strand, and 25 ng of purified, recombinant CREB (lanes 2 to 5 and 9 to 11). The reaction mixtures also contained 50 ng (lane 3), 100 ng (lane 4), or 200 ng (lanes 5, 7, 8, 10, and 11) of purified recombinant Tax and 200 ng of purified GST-KIX (lanes 8 and 11), as indicated. Maxam-Gilbert sequencing reactions for guanine and guanine/adenine were run adjacent to the MPE:Fe cleavage products to assign specific nucleotides (40). (B) The KIX domain of CBP is required for Tax expansion of the CREB bZIP footprint. Binding-reaction mixtures contained 10 fmol of the viral CRE probe, singly end labeled on the coding strand, and 5 ng of purified CREB bZIP domain (amino acids 254 to 327) (lanes 2 to 4 and 7 to 9). The reaction mixtures also contained 100 ng (lanes 3 and 9) or 200 ng (lane 4) of Tax and 1 μg of GST-KIX (lanes 8 and 9), as indicated. Maxam-Gilbert sequencing markers were run adjacent to the cleavage products to assign specific nucleotides. (C) Tax does not alter the footprint of CREB or CREB bZIP on the cellular CRE. The binding-reaction mixtures contained 10 fmol of the cellular CRE, singly end labeled on the coding strand, and 10 ng of CREB (lanes 2 to 6 and 14) or 2 ng of CREB bZIP (lanes 8 to 12 and 15). The reaction mixtures also contained 200 ng (lanes 3 and 9) or 400 ng (lanes 4 to 6, 10 to 12, 16 and 17) of Tax and 1 μg (lanes 5 and 11) or 2 μg (lanes 6, 12, 14 to 16, and 18) of GST-KIX as indicated. Maxam-Gilbert sequencing reactions were run adjacent to the MPE:Fe cleavage products to assign specific nucleotides. (D) Schematic representation of the relative protection produced by CREB or by CREB and Tax on the viral CRE. To account for Tax enhancement of CREB binding, experiments with high concentrations of CREB were directly compared to experiments with lower concentrations of CREB in the presence of Tax that gave similar degrees of protection in the core CREB binding site. The relative protection from MPE:Fe cleavage in the presence of CREB (open bars) or CREB and Tax (solid bars) was quantitated and averaged from three separate experiments. Bar heights are proportional to the degree of protection from the cleavage agent, with DNA-alone values equal to 1.0 and higher values corresponding to increased protection from MPE:Fe cleavage.

FIG. 2

Tax protein alters the MPE:Fe footprint pattern of CREB on a viral CRE but not on a cellular CRE. (A) Tax expands and enhances the MPE:Fe footprint of CREB on the noncoding strand of the viral CRE. The binding-reaction mixtures contained 10 fmol of the viral CRE probe, singly end labeled on the noncoding strand, and 25 ng of purified, recombinant CREB (lanes 2 to 5 and 9 to 11). The reaction mixtures also contained 50 ng (lane 3), 100 ng (lane 4), or 200 ng (lanes 5, 7, 8, 10, and 11) of purified recombinant Tax and 200 ng of purified GST-KIX (lanes 8 and 11), as indicated. Maxam-Gilbert sequencing reactions for guanine and guanine/adenine were run adjacent to the MPE:Fe cleavage products to assign specific nucleotides (40). (B) The KIX domain of CBP is required for Tax expansion of the CREB bZIP footprint. Binding-reaction mixtures contained 10 fmol of the viral CRE probe, singly end labeled on the coding strand, and 5 ng of purified CREB bZIP domain (amino acids 254 to 327) (lanes 2 to 4 and 7 to 9). The reaction mixtures also contained 100 ng (lanes 3 and 9) or 200 ng (lane 4) of Tax and 1 μg of GST-KIX (lanes 8 and 9), as indicated. Maxam-Gilbert sequencing markers were run adjacent to the cleavage products to assign specific nucleotides. (C) Tax does not alter the footprint of CREB or CREB bZIP on the cellular CRE. The binding-reaction mixtures contained 10 fmol of the cellular CRE, singly end labeled on the coding strand, and 10 ng of CREB (lanes 2 to 6 and 14) or 2 ng of CREB bZIP (lanes 8 to 12 and 15). The reaction mixtures also contained 200 ng (lanes 3 and 9) or 400 ng (lanes 4 to 6, 10 to 12, 16 and 17) of Tax and 1 μg (lanes 5 and 11) or 2 μg (lanes 6, 12, 14 to 16, and 18) of GST-KIX as indicated. Maxam-Gilbert sequencing reactions were run adjacent to the MPE:Fe cleavage products to assign specific nucleotides. (D) Schematic representation of the relative protection produced by CREB or by CREB and Tax on the viral CRE. To account for Tax enhancement of CREB binding, experiments with high concentrations of CREB were directly compared to experiments with lower concentrations of CREB in the presence of Tax that gave similar degrees of protection in the core CREB binding site. The relative protection from MPE:Fe cleavage in the presence of CREB (open bars) or CREB and Tax (solid bars) was quantitated and averaged from three separate experiments. Bar heights are proportional to the degree of protection from the cleavage agent, with DNA-alone values equal to 1.0 and higher values corresponding to increased protection from MPE:Fe cleavage.

FIG. 2

Tax protein alters the MPE:Fe footprint pattern of CREB on a viral CRE but not on a cellular CRE. (A) Tax expands and enhances the MPE:Fe footprint of CREB on the noncoding strand of the viral CRE. The binding-reaction mixtures contained 10 fmol of the viral CRE probe, singly end labeled on the noncoding strand, and 25 ng of purified, recombinant CREB (lanes 2 to 5 and 9 to 11). The reaction mixtures also contained 50 ng (lane 3), 100 ng (lane 4), or 200 ng (lanes 5, 7, 8, 10, and 11) of purified recombinant Tax and 200 ng of purified GST-KIX (lanes 8 and 11), as indicated. Maxam-Gilbert sequencing reactions for guanine and guanine/adenine were run adjacent to the MPE:Fe cleavage products to assign specific nucleotides (40). (B) The KIX domain of CBP is required for Tax expansion of the CREB bZIP footprint. Binding-reaction mixtures contained 10 fmol of the viral CRE probe, singly end labeled on the coding strand, and 5 ng of purified CREB bZIP domain (amino acids 254 to 327) (lanes 2 to 4 and 7 to 9). The reaction mixtures also contained 100 ng (lanes 3 and 9) or 200 ng (lane 4) of Tax and 1 μg of GST-KIX (lanes 8 and 9), as indicated. Maxam-Gilbert sequencing markers were run adjacent to the cleavage products to assign specific nucleotides. (C) Tax does not alter the footprint of CREB or CREB bZIP on the cellular CRE. The binding-reaction mixtures contained 10 fmol of the cellular CRE, singly end labeled on the coding strand, and 10 ng of CREB (lanes 2 to 6 and 14) or 2 ng of CREB bZIP (lanes 8 to 12 and 15). The reaction mixtures also contained 200 ng (lanes 3 and 9) or 400 ng (lanes 4 to 6, 10 to 12, 16 and 17) of Tax and 1 μg (lanes 5 and 11) or 2 μg (lanes 6, 12, 14 to 16, and 18) of GST-KIX as indicated. Maxam-Gilbert sequencing reactions were run adjacent to the MPE:Fe cleavage products to assign specific nucleotides. (D) Schematic representation of the relative protection produced by CREB or by CREB and Tax on the viral CRE. To account for Tax enhancement of CREB binding, experiments with high concentrations of CREB were directly compared to experiments with lower concentrations of CREB in the presence of Tax that gave similar degrees of protection in the core CREB binding site. The relative protection from MPE:Fe cleavage in the presence of CREB (open bars) or CREB and Tax (solid bars) was quantitated and averaged from three separate experiments. Bar heights are proportional to the degree of protection from the cleavage agent, with DNA-alone values equal to 1.0 and higher values corresponding to increased protection from MPE:Fe cleavage.

FIG. 3

Kinetics of CREB bZIP dissociation in the presence of Tax and KIX. The 73-aa bZIP domain of CREB was incubated with the viral CRE probe in the absence or presence of Tax or of Tax plus KIX. Binding-reaction mixtures were then challenged with a 1,000-fold molar excess of unlabeled cellular CRE binding site, and the kinetics of dissociation were analyzed by EMSA and quantitated by ImageQuant. The concentration of protein-DNA complexes remaining bound, relative to the concentration bound at time zero (no added competitor) (B/B₀), was plotted as a function of the time after challenge. The dissociation of CREB bZIP from the viral CRE probe (solid squares) in the presence of Tax (open squares) or in the presence of Tax plus KIX (solid triangles) is shown.

FIG. 4

Chromomycin A₃ binds to the GC-rich flanking sequences and inhibits the association of Tax and KIX with CREB and the viral CRE. (A) MPE:Fe footprinting shows that chromomycin A₃ binds preferentially to the viral CRE flanking sequences. Increasing concentrations of chromomycin A₃ (lanes 2 to 10) were incubated with 10 fmol of the viral CRE probe singly end labeled on the coding strand. Nucleotide positions were assigned based on previous footprinting experiments with Maxam-Gilbert sequencing markers. (B) Chromomycin A₃ inhibits association of Tax with CREB and the viral CRE. Increasing concentrations of chromomycin A₃ were incubated with the viral CRE probe for 20 min before the addition of 5 ng of CREB (lanes 3 to 15) and 50 ng of Tax (lanes 10 to 15), as indicated. The binding-reaction products were analyzed by EMSA. The positions of the free probe and the protein-DNA and drug-DNA complexes are indicated. (C) Chromomycin A₃ inhibits the association of Tax and KIX with the viral CRE. Increasing concentrations of chromomycin A₃ were incubated with the DNA for 20 min before the addition of 5 ng of CREB (lanes 1 to 8), 50 ng of Tax (lanes 2 to 8), and/or 70 ng of GST-KIX (lanes 3 to 8), as indicated. The binding-reaction products were analyzed by EMSA. The positions of the free probe and the protein-DNA and drug-DNA complexes are indicated.

FIG. 4

Chromomycin A₃ binds to the GC-rich flanking sequences and inhibits the association of Tax and KIX with CREB and the viral CRE. (A) MPE:Fe footprinting shows that chromomycin A₃ binds preferentially to the viral CRE flanking sequences. Increasing concentrations of chromomycin A₃ (lanes 2 to 10) were incubated with 10 fmol of the viral CRE probe singly end labeled on the coding strand. Nucleotide positions were assigned based on previous footprinting experiments with Maxam-Gilbert sequencing markers. (B) Chromomycin A₃ inhibits association of Tax with CREB and the viral CRE. Increasing concentrations of chromomycin A₃ were incubated with the viral CRE probe for 20 min before the addition of 5 ng of CREB (lanes 3 to 15) and 50 ng of Tax (lanes 10 to 15), as indicated. The binding-reaction products were analyzed by EMSA. The positions of the free probe and the protein-DNA and drug-DNA complexes are indicated. (C) Chromomycin A₃ inhibits the association of Tax and KIX with the viral CRE. Increasing concentrations of chromomycin A₃ were incubated with the DNA for 20 min before the addition of 5 ng of CREB (lanes 1 to 8), 50 ng of Tax (lanes 2 to 8), and/or 70 ng of GST-KIX (lanes 3 to 8), as indicated. The binding-reaction products were analyzed by EMSA. The positions of the free probe and the protein-DNA and drug-DNA complexes are indicated.

FIG. 4

Chromomycin A₃ binds to the GC-rich flanking sequences and inhibits the association of Tax and KIX with CREB and the viral CRE. (A) MPE:Fe footprinting shows that chromomycin A₃ binds preferentially to the viral CRE flanking sequences. Increasing concentrations of chromomycin A₃ (lanes 2 to 10) were incubated with 10 fmol of the viral CRE probe singly end labeled on the coding strand. Nucleotide positions were assigned based on previous footprinting experiments with Maxam-Gilbert sequencing markers. (B) Chromomycin A₃ inhibits association of Tax with CREB and the viral CRE. Increasing concentrations of chromomycin A₃ were incubated with the viral CRE probe for 20 min before the addition of 5 ng of CREB (lanes 3 to 15) and 50 ng of Tax (lanes 10 to 15), as indicated. The binding-reaction products were analyzed by EMSA. The positions of the free probe and the protein-DNA and drug-DNA complexes are indicated. (C) Chromomycin A₃ inhibits the association of Tax and KIX with the viral CRE. Increasing concentrations of chromomycin A₃ were incubated with the DNA for 20 min before the addition of 5 ng of CREB (lanes 1 to 8), 50 ng of Tax (lanes 2 to 8), and/or 70 ng of GST-KIX (lanes 3 to 8), as indicated. The binding-reaction products were analyzed by EMSA. The positions of the free probe and the protein-DNA and drug-DNA complexes are indicated.

FIG. 5

Tax cross-links to the 5′-flanking region of the viral CRE. (A) Schematic representation of the individual phosphate positions derivatized within the top strand of the viral CRE. The numbers below the sequence refer to the phosphate positions. The position of each cross-linking arm is indicated by arrows above the sequence. For example, p1 represents the oligomer which contains a single azidophenacyl cross-linking moiety attached to the phosphorothioate positioned at the first phosphate in the DNA backbone. (B) SDS-PAGE analysis of protein-DNA cross-linking. Cross-linking reaction mixtures contained 10 ng of CREB and/or 250 ng of Tax in the presence of 25 to 40 fmol of viral CRE. Each binding-reaction mixture contained the photoreactive cross-linking arm in the position indicated below the lane numbers. The positions of the proteins cross-linked to the derivatized viral CRE are indicated, and molecular weight markers (MWM) are shown on the right. (C) Identification of the cross-linked polypeptides with truncated CREB. Cross-linking reaction mixtures contained 10 ng of CREB or 2 ng of CREB Δ57–132 and 250 ng of Tax in the presence of 30 fmol of the viral CRE. The binding-reaction mixtures contained the photoreactive cross-linking arm at position 3. The positions of the proteins cross-linked to the derivatized viral CRE are indicated on the right. (D) Summary of the cross-linking studies. A schematic representation of all the phosphate positions individually examined in this study is shown at the top of the figure. The presence (+) or absence (−) of a CREB or Tax cross-link is indicated below each phosphate position. Boldface type indicates specific positions where Tax enhanced the CREB cross-link. The asterisk indicates that all Tax cross-links were observed only in the presence of CREB. (E) Chromomycin A₃ inhibits cross-linking of Tax to the viral CRE. Reaction mixtures contained 30 fmol of p3 probe (preincubated with the indicated amount of chromomycin A₃) and 10 ng of CREB (lanes 1 to 3) and/or 250 ng of Tax (lanes 4 to 6). The complexes were analyzed by SDS-PAGE.

FIG. 5

Tax cross-links to the 5′-flanking region of the viral CRE. (A) Schematic representation of the individual phosphate positions derivatized within the top strand of the viral CRE. The numbers below the sequence refer to the phosphate positions. The position of each cross-linking arm is indicated by arrows above the sequence. For example, p1 represents the oligomer which contains a single azidophenacyl cross-linking moiety attached to the phosphorothioate positioned at the first phosphate in the DNA backbone. (B) SDS-PAGE analysis of protein-DNA cross-linking. Cross-linking reaction mixtures contained 10 ng of CREB and/or 250 ng of Tax in the presence of 25 to 40 fmol of viral CRE. Each binding-reaction mixture contained the photoreactive cross-linking arm in the position indicated below the lane numbers. The positions of the proteins cross-linked to the derivatized viral CRE are indicated, and molecular weight markers (MWM) are shown on the right. (C) Identification of the cross-linked polypeptides with truncated CREB. Cross-linking reaction mixtures contained 10 ng of CREB or 2 ng of CREB Δ57–132 and 250 ng of Tax in the presence of 30 fmol of the viral CRE. The binding-reaction mixtures contained the photoreactive cross-linking arm at position 3. The positions of the proteins cross-linked to the derivatized viral CRE are indicated on the right. (D) Summary of the cross-linking studies. A schematic representation of all the phosphate positions individually examined in this study is shown at the top of the figure. The presence (+) or absence (−) of a CREB or Tax cross-link is indicated below each phosphate position. Boldface type indicates specific positions where Tax enhanced the CREB cross-link. The asterisk indicates that all Tax cross-links were observed only in the presence of CREB. (E) Chromomycin A₃ inhibits cross-linking of Tax to the viral CRE. Reaction mixtures contained 30 fmol of p3 probe (preincubated with the indicated amount of chromomycin A₃) and 10 ng of CREB (lanes 1 to 3) and/or 250 ng of Tax (lanes 4 to 6). The complexes were analyzed by SDS-PAGE.

FIG. 5

Tax cross-links to the 5′-flanking region of the viral CRE. (A) Schematic representation of the individual phosphate positions derivatized within the top strand of the viral CRE. The numbers below the sequence refer to the phosphate positions. The position of each cross-linking arm is indicated by arrows above the sequence. For example, p1 represents the oligomer which contains a single azidophenacyl cross-linking moiety attached to the phosphorothioate positioned at the first phosphate in the DNA backbone. (B) SDS-PAGE analysis of protein-DNA cross-linking. Cross-linking reaction mixtures contained 10 ng of CREB and/or 250 ng of Tax in the presence of 25 to 40 fmol of viral CRE. Each binding-reaction mixture contained the photoreactive cross-linking arm in the position indicated below the lane numbers. The positions of the proteins cross-linked to the derivatized viral CRE are indicated, and molecular weight markers (MWM) are shown on the right. (C) Identification of the cross-linked polypeptides with truncated CREB. Cross-linking reaction mixtures contained 10 ng of CREB or 2 ng of CREB Δ57–132 and 250 ng of Tax in the presence of 30 fmol of the viral CRE. The binding-reaction mixtures contained the photoreactive cross-linking arm at position 3. The positions of the proteins cross-linked to the derivatized viral CRE are indicated on the right. (D) Summary of the cross-linking studies. A schematic representation of all the phosphate positions individually examined in this study is shown at the top of the figure. The presence (+) or absence (−) of a CREB or Tax cross-link is indicated below each phosphate position. Boldface type indicates specific positions where Tax enhanced the CREB cross-link. The asterisk indicates that all Tax cross-links were observed only in the presence of CREB. (E) Chromomycin A₃ inhibits cross-linking of Tax to the viral CRE. Reaction mixtures contained 30 fmol of p3 probe (preincubated with the indicated amount of chromomycin A₃) and 10 ng of CREB (lanes 1 to 3) and/or 250 ng of Tax (lanes 4 to 6). The complexes were analyzed by SDS-PAGE.

FIG. 5

Tax cross-links to the 5′-flanking region of the viral CRE. (A) Schematic representation of the individual phosphate positions derivatized within the top strand of the viral CRE. The numbers below the sequence refer to the phosphate positions. The position of each cross-linking arm is indicated by arrows above the sequence. For example, p1 represents the oligomer which contains a single azidophenacyl cross-linking moiety attached to the phosphorothioate positioned at the first phosphate in the DNA backbone. (B) SDS-PAGE analysis of protein-DNA cross-linking. Cross-linking reaction mixtures contained 10 ng of CREB and/or 250 ng of Tax in the presence of 25 to 40 fmol of viral CRE. Each binding-reaction mixture contained the photoreactive cross-linking arm in the position indicated below the lane numbers. The positions of the proteins cross-linked to the derivatized viral CRE are indicated, and molecular weight markers (MWM) are shown on the right. (C) Identification of the cross-linked polypeptides with truncated CREB. Cross-linking reaction mixtures contained 10 ng of CREB or 2 ng of CREB Δ57–132 and 250 ng of Tax in the presence of 30 fmol of the viral CRE. The binding-reaction mixtures contained the photoreactive cross-linking arm at position 3. The positions of the proteins cross-linked to the derivatized viral CRE are indicated on the right. (D) Summary of the cross-linking studies. A schematic representation of all the phosphate positions individually examined in this study is shown at the top of the figure. The presence (+) or absence (−) of a CREB or Tax cross-link is indicated below each phosphate position. Boldface type indicates specific positions where Tax enhanced the CREB cross-link. The asterisk indicates that all Tax cross-links were observed only in the presence of CREB. (E) Chromomycin A₃ inhibits cross-linking of Tax to the viral CRE. Reaction mixtures contained 30 fmol of p3 probe (preincubated with the indicated amount of chromomycin A₃) and 10 ng of CREB (lanes 1 to 3) and/or 250 ng of Tax (lanes 4 to 6). The complexes were analyzed by SDS-PAGE.