FBI-1 can stimulate HIV-1 Tat activity and is targeted to a novel subnuclear domain that includes the Tat-P-TEFb-containing nuclear speckles

Abstract

FBI-1 is a cellular POZ-domain-containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor-rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.

Figure 1

(A) Schematic diagram of the insert for the reporter constructs pHIV-1/TAR/Luc and pHIV-1/SLIIB/Luc containing HIV-1 sequences from −157 to +82, a region that includes 1) the HIV-1 promoter with the enhancer (ENH), the three Sp1 binding sites, and the TATA box and 2) R sequences from positions +1 to +82 with IST sites and the TAR. The SLIIB insert (B) is described in Tiley et al. (1992). (B) Schematic diagram showing the sequence of the TAR and SLIIB inserts. Tat binds to TAR along with its cellular cofactors CDK9 and Cycling T. Replacement of TAR with SLIIB allows the targeting of Rev fusion proteins such as Rev-VP16 to the R region.

Figure 2

Expressed FBI-1 FL synergizes with HIV-1 Tat at the HIV-1 LTR. (A, C, and D) HeLa cells were transiently transfected with 2 μg of the reporter plasmids HIV-1/TAR/Luc (A and C) or HIV-1/SLIIB/Luc (D), along with empty expression vector (No FBI-1) or the indicated HA-tagged expression plasmids at 1× and 3× relative amounts. The amounts of expression plasmids (100–900 ng) were calibrated to express equal amounts of proteins as judged by anti-HA immunoblot (our unpublished results). Amounts transfected were as follows: For (A) 100 ng of pcgn Tat, 200 ng (1) and 600 ng (3) pcgn FBI-1 FL, 100 ng (1) and 300 ng (3) pcgn Oct 1, 300 ng (1) and 900 ng pcgn (3) BCL6; for (C) 100 ng Tat, 200 ng (1) and 600 ng (3) pcgn FBI-1 FL and ΔZF and 150 ng (1) and 450 ng (3) pcgn ΔPOZ; for (D) 500 ng FBI-1 FL, 500 ng pcRev, 100 ng pcgn Tat, 500 ng pcTat-Rev, and 800 ng pc Rev-VP16. 500 ng of β-globin-Renilla expression vector was cotransfected as an internal control for transfection. Depicted activation levels are the results of three independent experiments, are normalized to β-globin expression, and are relative to No FBI-1, − Tat levels arbitrarily set to 1. Error bars represent SD calculated by Microsoft Excel. (B) FBI-1 and FBI-1 mutant constructs. FL FBI-1 corresponds to FBI-1 amino acids 1–584; ΔPOZ, amino acids 122–584; ZF, amino acids 377–584; ZF1, FL FBI-1 with the mutations C384A + C387A; ZF2, FL FBI-1 with the mutations C412A + C415A; ZF3, FL FBI-1 with the mutations C440A + C443A; ZF4, FL FBI-1 with the mutations C468A + C471A; ΔZF, amino acids 1–382 and 485–584.

Figure 3

The ZF domain of FBI-1 is necessary and sufficient for Tat association in vivo. (A) Constructs expressing T7FL, T7ΔPOZ, T7ZF, or T7ΔZF and HA-Tat were expressed alone or in combination as indicated above the lanes into HeLa cells. Thirty-six hours posttransfection, extracts from transfected cells were prepared and incubated with protein A-agarose beads cross-linked to anti-HA 12CA5 monoclonal antibodies (lanes 2–9). The immunoprecipitated proteins were fractionated on a 12.5% SDS-polyacrylamide gel, transferred to nitrocellulose and blotted with anti-T7 antibody. Lane 1: one tenth the starting material for the T7FL + HA-Tat immunoprecipitation (IP; lane 6). Lanes 2–9: the IPs. The position of T7FL, T7-ΔPOZ, and T7-ZF are indicated. For lanes 6–9, an anti-HA immunoblot of a separate gel demonstrated that the HA-tagged proteins were expressed and immunoprecipitated (our unpublished results). For lanes 6–9, an anti-T7 immunoblot of a separate gel loaded with one tenth the starting material for the IP demonstrated that the mutant proteins were expressed to a similar level (B). (C) Constructs expressing T7ZF and wild-type HA-Tat, HA-18IS Tat, or HA-C30, 31A Tat were transfected alone or in combination as indicated above the lanes into HeLa cells. The experiment was performed as in (A). Lanes 1–4 show one tenth the starting materials; lanes 5–8 show the IPs.

Figure 4

FBI-1, HIV-1 Tat, and TAK/P-TEFb localization. (A) Epifluorescent image of HeLa cells transfected with HA-Tat expressing plasmid. FBI-1 (panel 1, green) and HA-Tat (panel 2, red) colocalize (panels 3 and 4, yellow) at numerous foci within the nonnucleolar nucleoplasm. Endogenous FBI-1 was detected with a rabbit polyclonal (413) primary antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody (FITC-GAR). HA-Tat was detected with a mouse monoclonal (12CA5) primary antibody and a Texas Red-conjugated Goat anti-mouse secondary antibody (TR-GAM). (B) Epifluorescent image of HeLa cells transfected with 2 μg of HA-Tat expressing plasmid (panel 1) or mock transfected (2 μg of expression vector) HeLa cells (panels 2 and 3). HA-Tat (panel 1, column 1, green), endogenous cyclin T1 (panel 2, column 1, green) and Cdk9 (panel 3, column 1, green) and nuclear speckles (panels 1–3, column 2, red) colocalize (panels 1–3, column 3 and 4, yellow) at numerous irregularly shaped foci within the nonnucleolar nucleoplasm. HA-Tat was detected with 12CA5 primary antibody and an FITC-conjugated goat anti-mouse secondary (FITC-GAM). Cyclin T1 was detected with a rabbit polyclonal (anticyclin T1) antibody and FITC-GAR secondary. Cdk9 was detected with a rabbit polyclonal (anti-Cdk9) antibody and FITC-GAR secondary. Nuclear speckles were detected with a human polyclonal (ANA-S; panel 1) primary antibody or a mouse monoclonal (SC35) primary antibody and with Texas Red–conjugated goat anti-human antibody (TR-GAH) and TR-GAM, respectively. (C) Epifluorescent image of HeLa cells. Endogenous FBI-1 (green) displays a heterogeneous nuclear localization pattern characterized by numerous signal foci that surround and overlap with the nuclear speckle domain (red). Note that often there is a slight decrease in signal within a FBI-1 foci that matches the shape of the corresponding speckle (panel 2, arrowheads). FBI-1 was detected with 413 primary and FITC-GAR secondary antibodies, and nuclear speckles were detected with SC35 primary and TR-GAM secondary antibodies.

Figure 5

Less-soluble FBI-1 displays peripheral-speckle localization. Epifluorescent image of prefixation CSK extracted HeLa cells. Less-soluble endogenous FBI-1 (green) shows strong signal surrounding and connecting the nuclear speckles (red) but shows a much weaker signal at the center of many speckles (large arrowheads) and from regions of the nucleus that have no speckles (narrow arrowheads). FBI-1 was detected with 413 primary antibody or with another rabbit polyclonal antibody specific for FBI-1 (415, our unpublished results) and FITC-GAR secondary antibody. Nuclear speckles were detected with SC35 primary and TR-GAM secondary antibodies. (B) Confocal image of prefixation CSK extracted Hela cells transfected with 2 μg of T7-FBI-1 (FL). T7-FBI-1 (green) signal is enhanced at the periphery of the SC35 nuclear speckles (red). T7-FBI-1 was detected with a mouse monoclonal primary antibody (T7 epitope antibody, Novagen) and FITC-GAM secondary antibody. Nuclear speckles were detected as in A.

Figure 6

The DNA-binding zinc fingers 1 and 2 are required for less-soluble FBI-1's peripheral-speckle pattern of localization. Epifluorescent image of prefixation extracted HeLa cells transfected with 2 μg of the T-FBI-1 mutant-expressing plasmids ΔPOZ (A, panel 1), ΔZF (A, panel 2), ZF1 (B, column 1), ZF2 (B, column 2), ZF3 (B, column 3), and ZF4 (B, column 4). (A) Less-soluble T-FBI-1 ΔPOZ (panel 1, green) shows a peripheral-speckle pattern of localization (Merge), whereas T-FBI-1 ΔZF (panel 2, green) shows an aberrant homogeneous pattern of localization with an increased signal from the nucleoli and no increase in signal near the nuclear speckles (box 2, red and box 3). The T-tagged FBI-1 mutants were detected as T-FBI-1 in Figure 4. The nuclear speckles were detected as in Figure 5. Bright images result from 8× exposure times. (B) Less-soluble T-FBI-1 ZF3 and ZF4 (columns 3 and 4, respectively, green) also show a peripheral-speckle pattern of localization; however, T-FBI-1 ZF1 and ZF2 show aberrant homogeneous patterns of localization (columns 1 and 2, respectively, green). The T-tagged point mutants were detected as T-FBI-1 in Figure 6. Bright images result from 10× exposure times. (C) Summary of results from A and B.

Figure 7

Less soluble FBI-1's peripheral-speckle pattern is dependent on cellular DNA but not on cellular RNA. Epifluorescent image of prefixation extracted HeLa cells treated for 30 min at 37°C with either RNase A (A and B, panel 2), DNase I (A and B, panel 3), or buffer (A and B, panel 1). (A) FBI-1's (green) peripheral-speckle pattern is little disturbed by treatment with buffer (panel 1) or RNase A (panel 2); however, less-soluble FBI-1 signal is drastically reduced upon DNase I treatment (panel 3, column 1), whereas the nuclear speckles (red) are undisturbed. Cdk9, and nuclear speckles were detected as in Figure 5. FBI-1 was detected as in Figure 4. (B) Less soluble Cdk9's (green) localization pattern is relatively undisturbed by all three treatments (column 1) and still colocalizes (yellow) with nuclear speckles (red). DNase I treatment results in a strong reduction of DNA content in the cell as judged by DAPI staining (A and B, blue)

Figure 8

Total FBI-1 is redistributed to enlarged and rounded nuclear speckles, but the less-soluble FBI-1 population and peripheral-speckle pattern is drastically reduced upon inhibition of transcription. Epifluorescent images of nonextracted (panel 1) and prefixation extracted (panel 2) HeLa cells either untreated (column 1) or treated for 5 h with 50 μg/ml of α-amanitin (columns 2–4). Total FBI-1 (panel 1, green) is redistributed into enlarged nuclear speckles (red and yellow). Note that unlike in non–α-amanitin–treated cells (Figure 4C), the FBI-1 signal foci match the shape, size, and relative intensity of the nuclear speckles even in irregularly shaped foci (arrowhead). However, less-soluble FBI-1 signal is almost completely lost (panel 2, column 2). A six times longer exposure (panel 2, column 4, Bright) shows that the little FBI-1 that is left after extraction does not show a peripheral-speckle pattern of localization. Nuclear speckles were detected as in Figure 5. FBI-1 was detected as in Figure 4.