Fig. 2. FRET analysis of protein–protein interactions in vivo. (A) Visualization of FRET. The plasmid constructs indicated on top of each column were transfected in HL3T1 cells; transfected cells were visualized by transmitted light in Nomarski configuration (panels in row a), by excitation at 480 nm and collection at 520 nm, showing EGFP fluorescence after direct EGFP excitation (panels in row b), and by excitation at 350 nm and collection at 520 nm, showing EGFP fluorescence after BFP excitation, indicating FRET (panels in row c). (B) Quantification of FRET. Fluorescent emission at 520 nm from individual cells transfected with the indicated constructs was recorded after excitation at 350 or 480 nm, and integrated intensities over the whole cell were evaluated. Plotted values (indicated by dots) represent the ratio between these two measurements: higher values indicate more efficient resonant energy transfer between BFP and EGFP. Ten consecutively analysed cells were considered for each transfection; both their individual fluorescence ratio and their percentile box-plot distribution are shown. Horizontal lines of the percentile box plot distribution of FRET values, from top to bottom, mark the 10th, 25th, 50th, 75th and 90th percentile, respectively.

Fig. 4. In vitro mapping of the cyclin T1–PML association. (A) Schematic representation of human cyclin T1 and deletion mutants used in this work. Positions of the Tat recognition motif (TRM) in cyclin T1 and of Cys261 critical for Tat binding are indicated. Several other potential functional domains in cyclin T1 have been described, including the cyclin box, a coiled-coil region (coil), a histidine-rich domain (his) and a C-terminal PEST sequence (PEST). (B) PML-IV binds the C-terminus of cyclin T1. Each binding reaction contained 2–5 µg of GST fusions immobilized to glutathione–CL4 beads and in vitro translated, ³⁵S-labelled PML-IV in NETN buffer. After binding at 4°C, beads were washed extensively prior to loading onto a 10% SDS–polyacrylamide gel. Bound material is indicated as a percentage of input material. (C) Schematic representation of human PML-IV, PML-III and PML-RARα. R, RING finger domain; RBCC motif (RING + B-boxes + coiled-coil). Alterations of PML-III present in the mutants used for this experiment are also indicated. (D) Cyclin T1 binds within the coiled-coil domain of the RBCC motif of PML. Binding reactions were conducted as described in (B).

Fig. 6. Transcriptional activity of cyclin T1 on Tat-mediated LTR transactivation. (A) Titration of HA-tagged cyclin T1 on the HIV-1 LTR in HL3T1 cells, a HeLa derivative carrying an integrated LTR-CAT cassette. The experiments were performed by calcium phosphate transfection of a fixed limiting amount of pcDNA3-Tat86 (50 ng) and increasing amounts of pCMV-HA-CycT1. (B) Titration of HA-tagged cyclin T1 on the HIV-1 LTR in CHO cells. Cells were co-transfected with 500 ng of pU3R-III (LTR-CAT reporter), pcDNA3-Tat86 (100 ng) and increasing amounts of pCMV-HA-CycT1, as indicated. (C) Titration of EGFP-tagged cyclin T1 on the HIV-1 LTR in PML–/– MEFs. Cells were co-transfected as in (B) using an LTR-luciferase reporter (500 ng). (D) Transcriptional activity of full-length cyclin T1 and of the 1–300 deletion mutant on the HIV LTR promoter. CHO cells were co-transfected as in (B). Expression of the two constructs was monitored by immunoblot (anti-EGFP).

Fig. 1. Co-localization of human cyclin T1 and PML in nuclear bodies. In rows (A) and (B), HL3T1 and U2OS cells (as indicated) were transfected with pcDNA3-EGFP-cyclin T1 (EGFP, green) and pcDNA3-PML-IV (TRITC, red). In row (C), U2OS cells were transfected only with pcDNA3-EGFP-cyclin T1 (EGFP, green) to visualize endogenous PML (TRITC, red). In row (D), co-localization of endogenous cyclin T1 (Alexa fluor 488, green) and endogenous PML (Alexa fluor 594, red) is shown in HL3T1 cells. The inset shows an enlargement of two PML bodies, one of which also contains cyclin T1 (yellow dot). In row (E), U2OS cells were transfected with pcDNA3-HA-cyclin T1 (TRITC, red) and pcDNA3-PML (FITC, green). In row (F), control plasmid fibrillarin-EGFP (green), which localizes into nucleoli, was co-transfected with pcDNA3-PML-IV (red). In row (G), another control plasmid expressing pEGFP-SF2/AFP (green) was co-transfected with pcDNA3-PML-IV (red). In row (H), U2OS cells were transfected with pcDNA3-Tat86-EGFP (green) and pcDNA3-PML-IV (red).

Fig. 3. Co-immunoprecipitation of cyclin T1 with PML. (A) Endo genous PML and cyclin T1. Total cell lysates from 293 cells were used for immunoprecipitation with rabbit antisera against PML, cyclin T1 or with a control antiserum against human VEGF, as indicated. Western blot analysis was performed to reveal immunoprecipitated cyclin T1. (B) Transfected PML and cyclin T1. 293 cells were transfected with c-Myc-tagged cyclin T1 and EGFP-tagged PML-IV expression plasmids, as indicated. The top panel shows immunoprecipitiation with an anti-EGFP antiserum followed by western blotting with an anti- c-Myc antibody to reveal immunoprecipitated c-Myc-cyclin T1. The bottom two panels are western blottings on total cell lysates from the same cells reacted with anti-c-Myc or anti-EGFP antibodies to reveal expression of the transfected plasmids.

Fig. 5. In vivo analysis of the cyclin T1 C-terminal deletion mutants. (A) Subcellular localization of EGFP–cyclin T1 in human HL3T1 cells and PML–/– MEFs. The same mutants depicted in Figure 4A were also fused to EGFP and transfected in HL3T1 cells. Cells were fixed in paraformaldehyde and analysed by confocal microscopy. (B) Visualization of the interaction between cyclin T1 deletion mutants and PML in vivo by FRET. HL3T1 cells were transfected with the cyclin T1 deletion mutants together with BFP–PML-IV. FRET was measured as indicated in Figure 2A. Despite PML-driven re-localization of the mutants into nuclear bodies (panels in row b, EGFP fluorescence), no FRET is observed between the deleted cyclin T1 proteins and PML-IV (panels in row c, FRET). (C) Quantification of FRET for the interaction between cyclin T1 deletion mutants and PML. Data were analysed and plotted as indicated for Figure 2B. (D) Quantification of FRET in different subnuclear compartments. NP, nucleoplasm; NB, nuclear bodies. Data were analysed and plotted as indicated for Figure 2B.

Fig. 7. Recruitment of cyclin T1 and PML to the HIV-1 LTR upon transcriptional activation. (A) Chromosomal regions analysed by quantitative ChIP. These include the HIV LTR and the B13 and B48 DNA segments in the lamin B2 gene domain, a single-copy region of the human genome. For each of these regions, specific primers were selected (small, converging arrows). The diagrams schematically indicate the location of relevant genomic elements (transcription start site and transcription factor-binding sites in the HIV LTR, lamin B2 gene 3′ end and ppv1 gene) with respect to primer localization. The localizations of transcription factor USF-binding sites in both the LTR and B48 are indicated. (B) Schematic representation of the multicompetitor DNA used for DNA quantification by competitive PCR. This DNA fragment contains all primer recognition sites arranged to generate PCR amplification products of sizes different from, but comparable with those obtained from the amplification of genomic DNA. (C–E) Results of quantitative ChIP in HL3T1 cells. Cells were treated by incubation with GST–Tat86 for 5 h and cross-linked with formaldehyde; chromatin was then sonicated and immunoprecipitated with specific antibodies against cyclin T1 (D) and PML (E). A fixed amount of total sonicated DNA (C) and immunoprecipitated DNA (D and E) was mixed with increasing concentrations of the multicompetitor, as indicated on top of each panel, and PCR-amplified with primers in the LTR, B13 and B48 regions. After amplification, products were resolved by PAGE, stained with ethidium bromide and photographed. Specific bands were quantified by densitometric analysis. The number of DNA molecules corresponding to each genomic region (shown in the tables on the right sides of each panel, together with the LTR/B13 and B48/B13 ratios) were calculated according to the principles of competitive PCR by evaluating the slope of the curve fitting the values corresponding to competitor:genomic ratios (Diviacco et al., 1992). In total genomic DNA from HL3T1 cells, the LTR/B13 ratio is ∼5, indicating that more than one copy of the LTR-CAT cassette is integrated in these cells. M, molecular weight markers; molec., number of DNA molecules; comp., competitor PCR product. (F) The graph reports the results obtained in the experiment shown in (C–E). Enrichment of the B48 (control) and LTR regions over B13 was normalized by dividing the experimental values by those obtained in total input DNA. Consistent results have been observed in at least three independent experiments. (G and H) Results of quantitative ChIP in HIV-infected U1 cells stimulated with TPA. U1 cells were treated with 1 × 10^–7 M TPA for 5 h and then processed as outlined above. After ChIP with an anti-PML antibody, quantification of input and immunoprecipitated DNA was performed as described for HL3T1 cells. (I) The graph reports the results obtained in the experiment shown in (G) and (H). Enrichment of the LTR region after ChIP with anti-PML antibody was calculated after normalization for the DNA quantification values in total input DNA.