Structure of the Papillomavirus DNA-Tethering Complex E2:Brd4 and a Peptide that Ablates HPV Chromosomal Association  Eric A. Abbate, Christian Voitenleitner, Michael R. Botchan  Molecular Cell  Volume 24, Issue 6, Pages 877-889 (December 2006) DOI: 10.1016/j.molcel.2006.11.002 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 A Schematic Map of the E2 and Brd4 Proteins and Colocalization of BPV-E2 and Brd4 in BPV-Transformed C127 Cells (A) The PV E2 and human Brd4 open reading frames indicating domain boundaries. AD, activation domain; DBD, DNA-binding domain; BD, bromodomain; ET, extra-terminal domain. (B) The E2 protein is shown in red, and Brd4 is shown in green. In the merged panel (lower right of each set of four), spots that colocalize appear in yellow. Cellular DNA was detected by DAPI staining and is shown in blue. The three different panels where each panel is a set of four depict cells at three distinct stages of mitosis. Molecular Cell 2006 24, 877-889DOI: (10.1016/j.molcel.2006.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Localization of HPV-16 and HPV-31 DNA in Epithelial Cells (A and B) Metaphase spreads of W12 cells (A), or 9E cells (B), stably maintaining the HPV-16 or HPV-31 genome, respectively. Chromosomes are colored in red, and the viral DNA was detected by FISH. In these merged images, HPV DNA that colocalizes with mitotic chromosomes is yellow, while unassociated plasmids are green. Identical spots on each sister chromatid, which is indicative of an integration event of viral DNA into the host cell genome, are indicated by arrows. The number of viral DNA spots that colocalized with chromosomes from metaphase spreads was counted, binned, and graphed in increments of 50. Molecular Cell 2006 24, 877-889DOI: (10.1016/j.molcel.2006.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Structure of the E2:Brd4 Complex (A) Cartoon representation of the E2:Brd4 complex. The N-terminal α-helical domain of E2 is colored green, and the C-terminal β strand domain is depicted in red. The linker segment between these two domains is in yellow. Brd4 is colored blue. The N and C termini of the proteins are labeled, as are helices of E2. (B) Surface rendering of E2 in green, with residues contributing to the E2:Brd4 interaction colored orange. Brd4 is depicted in blue as a cartoon representation, and side chains making contact with E2 are shown. In yellow are side chains from the nonpolar face of the Brd4 helix that bind in a shallow canyon on the surface of E2. (C) E2 (colored green) side chains (colored orange) and Brd4 (colored blue) side chains (colored yellow) that make up the interaction surface. Important residues are identified. Phe1349 and Phe1357 are labeled to give orientation relative to (B). Molecular Cell 2006 24, 877-889DOI: (10.1016/j.molcel.2006.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Residues Involved in E2:Brd4 Complex Formation (A) (Top) Protein sequence alignment of the N-terminal helical domain from high-risk (HPV-16, -31, and -18), low-risk (HPV-11 and -6), and BPV E2. Amino acid boundaries for each variant are indicated, and residues are colored according to the amino acid type. The secondary structure from HPV-16 E2 is depicted above the alignment, and elements are labeled as in Figure 1A. Helices are represented by rectangles, strands by arrows, and coil regions by black lines. Residues with side chains making contact with Brd4 are indicated by black arrows. Residues making contact with the second Brd4 molecule in the tetramer are designated by red arrows. The ruler above the alignment delineates 10 amino acid increments. (Bottom) Sequence alignment of the extreme C termini of Brd4 homologs from various organisms. The secondary structure from the solved E2:Brd4 complex is shown above the alignment and labeled as above. Residues with side chains making contact with E2 are indicated by black arrows. (B) Pull-down experiments with GST-Brd4 and untagged E2 activation domain (1–201). The Brd4 construct spans amino acids 1313–1362 for the WT and point mutations (left). The labels 1360 and 1357 correspond to deletions of the last two and five amino acids of Brd4, respectively. For E2 mutants (right), pull-down (P), and input E2 (I), lanes are labeled. The percent of E2 pulled down relative to WT is indicated. The F1349A and D1352A mutants that alter the hydrophobic character or charge of the protein run anomalously on the gel. (C) Cartoon representation of the E2:Brd4 tetramer. One heterodimer is colored green (E2) and blue (Brd4), and the other is colored red (E2) and yellow (Brd4). Molecular Cell 2006 24, 877-889DOI: (10.1016/j.molcel.2006.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Design and Properties of GFP-tat-Brd4 Peptide (A) The C-terminal 20 amino acids of Brd4, which encompass the E2-binding site of Brd4 (red box) were fused to the protein transduction domain of HIV-1 Tat protein (Tat) and the GFP protein (green box). (B) Western blot of GFP-tat-Brd4 peptide uptake. W12 cells were incubated with 2 μM peptide for 2 hr. Nuclear extracts of trypsinized cells (5, 10, and 15 μl) were analyzed by immunoblotting with anti-GFP. (C) Cellular localization of GFP-tat-Brd4 peptide. W12 cells grown on coverslips were incubated with 2 μM peptide for 2 hr, fixed with paraformaldehyde, and stained with DAPI. GFP (left) or DNA (right) was visualized by fluorescence microscopy. Molecular Cell 2006 24, 877-889DOI: (10.1016/j.molcel.2006.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Viral DNA Localization in Cells Expressing Wild-Type or Mutant GFP-tat-Brd4 (A and B) FISH and quantitation of HPV-16 (A) and -31 (B) GFP-tat-Brd4-WT. The left panels show representative metaphase spreads. The mitotic chromosomes are shown in red, the viral HPV-16 DNA spots are green, and colocalized spots appear yellow. The arrows show integrated DNA. The right panels depict the quantitative results of 20 metaphase spreads. Due to the low number of spots on the chromosomes, the binning here was changed to increments of 2. (C) Dose dependency of GFP-tat-Brd4-WT. W12 cells were incubated with different amounts of Brd4 peptide. For each concentration of Brd4 peptide, the number of viral DNA spots on mitotic chromosomes of 20 metaphase spreads was averaged and plotted against the concentration of GFP-tat-Brd4. Molecular Cell 2006 24, 877-889DOI: (10.1016/j.molcel.2006.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Characterization of GFP-tat-Brd4D Mutants Metaphase spread of W12 cells treated with GFP-tat-Brd4-F1349A or D1352A (A) or GFP-tat-Brd4-L1354A (B). The mitotic chromosomes are shown in red, and viral DNA is colored in green. The merged image shows colocalized spots in yellow. For quantitation, the number of spots on chromosomes was counted and graphed in binned increments of 50 or 10. Integrated DNA is indicated with arrows. Molecular Cell 2006 24, 877-889DOI: (10.1016/j.molcel.2006.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions