1. Volume 38, Issue 5, Pages 700-711 (June 2010)
Destabilization of TIP60 by Human Papillomavirus E6 Results in Attenuation of TIP60- Dependent Transcriptional Regulation and Apoptotic Pathway 
Sudhakar Jha, Scott Vande Pol, Nilam Sanjib Banerjee, Arun Brendan Dutta, Louise T. Chow, Anindya Dutta 
Molecular Cell 
Volume 38, Issue 5, Pages (June 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2010 38, 700-711DOI: (10.1016/j.molcel.2010.05.020)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 TIP60 Is Destabilized by Human Papillomaviral Protein E6
(A) Inhibition of proteasome in HeLa cells by MG132 decreases basal phosphoH2AX levels.
(B) Stabilization of TIP60 and decrease of basal phosphoH2AX after addition of MG132 are specific for HeLa cells. Cells were incubated with MG132 for 4 hr, and lysates were probed with indicated antibodies.
(C) TIP60 is stabilized and phosphoH2AX decreased in E6/E7-depleted cells. HeLa cells were transfected with indicated siRNA and lysates prepared from cells 72 hr after transfection were probed for indicated proteins.
(D) Stabilization of TIP60 after depletion of HPV E6/E7 in HeLa cells is seen even when p53 is also knocked down, so the stabilization of TIP60 is not secondary to the increase in p53. The rest is as in (C).
(E) HPV18 E6 and E7 mRNA levels are decreased in HeLa cells after transfection of siRNA to HPV18 E6/E7. Also, TIP60 mRNA is not increased by E6/E7 knockdown. The level of a given mRNA was measured by Q-RT-PCR relative to that of Actin mRNA: mean and standard deviation of three experiments, each done in triplicate (n = 9) are shown (see also Figure S1).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 2
TIP60 Is Destabilized by HPV18 E6 in a Proteasome-Dependent Manner
(A) 293T cells were transfected with indicated plasmids and cells were harvested 48 hr after transfection. Immunoblot with indicated antibodies are shown. Of C18E6 plasmid, 0.1, 0.25, 0.5 and 1.0 μg were transfected. A plasmid expressing GST was cotransfected to show that the degradation was specific to TIP60.
(B) TIP60 is degraded by HPV16 E6, but not HPV16 E7. The rest is as in (A).
(C) Inhibition of proteasome restores TIP60 in HPV18 E6-transfected cells. 293T cells were transfected with indicated plasmids, and MG132 was added for 4 hr before cells were harvested. Lysates were probed with indicated antibodies.
(D) TIP60 and p53 are decreased in PHKs containing HPV18 genomic DNA. Addition of MG132 restores the levels of TIP60 and p53. Lysates from PHK with or without HPV18 genome were resolved and immunoblotted with indicated antibodies (see also Figure S2).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 3 HPV E6 Destabilizes TIP60 In Vivo and In Vitro
(A) Depletion of HPV E6 increases the half-life of TIP60. HeLa cells were transfected with indicated siRNA. Seventy-two hours posttransfection, cells were harvested (0 hr), or at indicated times after cycloheximide addition. More protein was loaded in the siControl lanes so that the TIP60 signal was comparable in the two 0 hr lanes. (Graph) Levels of TIP60 in each lane were quantitated using Gene Tool software (Syngene). The level of TIP60 in each 0 hr lane is held at 100%.
(B) In vitro-transcribed and -translated HPV18 E6 protein. Rabbit reticulocyte lysate was incubated with either empty vector or plasmid expressing HPV18 E6 in presence of S35 methionine. Fluorograph for the presence of HPV18 E6 proteins is shown.
(C) TIP60 is degraded in vitro by HPV18 E6. Flag-TIP60 complex was purified from cells and used as a substrate in an in vitro degradation assay with increasing quantities of in vitro-translated HPV18 E6. Immunoblot for levels of TIP60 is shown. Asterisk indicates a cross-reacting band that serves as a loading control. MG132 blocks the in vitro degradation of TIP60 by HPV18 E6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 4
TIP60 Is Destabilized by HPV E6 that Is Defective in Binding to p53, E6AP, and PDZ Domain Proteins
(A) Schematic representation of various mutant forms of HPV16 E6 tested to delineate the region of E6 responsible for destabilizing TIP60. ZnF indicates zinc finger domain. “A” indicates alanine substitution. Summary for data presented in Figures 4B–4D. (−) represents failure to degrade, and (+) represents ability to degrade the indicated protein. The parenthetical signs represent expected results based on the published literature.
(B and C) Screening of HPV E6 mutants for TIP60 destabilization. Myc-p53 or HA-TIP60 was transfected along with expression plasmids of GST and various mutant forms of HPV E6 in 293T cells. Cells were harvested after 48 hr of transfection, and lysates were resolved and immunoblotted for indicated proteins.
(D) N-terminal region of HPV E6 is sufficient for destabilization of TIP60. The rest is as in (B).
(E) HPV18 E6∗I destabilizes TIP60, but not p53, in PHKs. Lysates from PHK with or without mutant HPV18 genomic plasmid expressing E6∗I, but not full-length E6, were resolved and immunoblotted with indicated antibodies.
(F) TIP60 interacts with HPV E6 in vitro. GST pull-down experiments were performed using bacterial lysate coexpressing GST or GST-16E6 along with TIP60. Proteins bound to glutatione beads were resolved on SDS-PAGE and blotted for indicated antibodies.
(G) Chargeless mutant of 16E6 (1–43) disrupts this interaction. GST or GST-16E6 (1–43) or GST-16E6 (1–43)-A was expressed together with TIP60. The rest is as in (F) (see also Figure S3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 5 TIP60 Represses HPV18 E6 mRNA
(A) Increase in HPV18 E6 mRNA after depletion of TIP60. HeLa cells were transfected with indicated siRNA, and RNA was prepared from cells harvested 72 hr after transfection. Q-RT-PCR was performed and normalized to actin, and relative levels of HPV18 E6 mRNA are shown. Mean ± SD of three experiments, each done in triplicate (n = 9).
(B) Overexpression of TIP60 in HeLa cells. Flag-TIP60 was overexpressed in HeLa cells after infecting with a retrovirus and selecting the cells on puromycin.
(C) HPV18 E6 mRNA is repressed in TIP60-overexpressing cells. Relative change in HPV18 E6 mRNA shown in the cells expressing either vector alone or Flag-TIP60. Mean ± SD of two experiments, each done in triplicate (n = 6) (∗p ≤ 0.05 by Student's t test). The rest is as in (A) and (B).
(D) HPV18 promoter is repressed by TIP60. Control firefly luciferase plasmid (pGL3C) or plasmid expressing firefly luciferase driven by the HPV18 major early promoter (pGL3C-HPV) was transfected into mock or TIP60-overexpressing HeLa cells. The ratio of the firefly luciferase to cotransfected Renilla luciferase is calculated, and normalized values are shown. Mean ± SD of two experiments, each done in triplicate (n = 6) (∗∗p ≤ 0.05 by Student's t test).
(E) TIP60 binds to HPV18 promoter. ChIP assay was performed in HeLa cells to examine binding of TIP60 on HPV18 promoter. Ratio of Q-PCR signal in TIP60 immunoprecipitate relative to preimmune (negative control) immunoprecipitate is plotted: mean ± SD of three experiments, each done in triplicate (n = 9).
(F) TIP60 acetylates histone H4 on HPV18 promoter. Level of acetylated histone H4 determined by ChIP assay and comparison between the control and TIP60 depleted cells is shown. The rest is as in (E).
(G) Depletion of TIP60 decreases trimethylation on lysine 9 of histone H3. The rest is as in (E) (see also Figure S4).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

8. Figure 6 TIP60-YY1-Brd4 Are Involved in Repressing HPV Promoter
(A) Schematics of amplicon used to map the binding site of TIP60 and YY1 on HPV promoter. Details are in Figure S5A.
(B and C) TIP60 and YY1 bind to the proximal HPV promoter. The rest is as in Figure 5E.
(D) Depletion of YY1 decreases TIP60. HeLa cells were transfected with indicated siRNA, and lysates were probed for indicated antibodies.
(E) Codepletion of YY1 and E6 stabilizes TIP60. The rest is as in (D).
(F) YY1 is required for binding of TIP60 to the HPV promoter. ChIP assay was performed from chromatin prepared from control or YY1-E6 codepleted cells (as in Figure 6E). Binding of TIP60 was significantly decreased in the absence of YY1. The rest is as in Figure 5E.
(G) Increase in HPV18 E6 mRNA after depletion of Brd4 in HeLa cells. The rest is as in Figure 5A.
(H) TIP60 augments binding of Brd4 on the HPV18 promoter. Brd4 occupancy on HPV18 promoter was determined using ChIP assay with anti-Brd4 antibody. The rest is as in Figure 5E. Depletion of TIP60 decreases binding of Brd4 to the HPV18 promoter.
(I) TIP60 depletion does not affect Brd4 protein levels. Immunoblots with indicated antibodies are shown in control and TIP60-depleted HeLa cells.
(J) ATF4 and DKC1 are negatively regulated by TIP60. Knockdown of HPV18 E6 by siRNA represses ATF4 expression, and this is dependent on TIP60, as depletion of TIP60 restores ATF4 level. ATF4 mRNA was measured by Q-RT-PCR, and levels relative to actin mRNA are shown: mean ± SD of two experiments, each done in triplicate (n = 6).
(K) TIP60-mediated repression of ATF4 and DKC1 is HPV independent. HCT116 p53−/− cells were depleted of TIP60 using siRNA, and levels relative to actin mRNA are shown. The rest is as in Figure 6J. Mean ± SD of two experiments, each done in triplicate (n = 6) (see also Figure S5).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

9. Figure 7 TIP60 Is Destabilized by Both High- and Low-Risk HPV E6
(A) p53 is degraded only by high-risk (HPV18 and HPV16) HPV E6. 293T cells were transfected with indicated plasmids, and cells were harvested 48 hr after transfection. Lysates were resolved on SDS-PAGE and probed for indicated antibodies.
(B) TIP60 is degraded by both high- (HPV16 and HPV18) and low-risk (HPV11) HPV E6 and by the cutaneous HPV (HPV8) E6. The rest is as in (A).
(C) p53-dependent activation of the apoptotic gene PUMA is attenuated by low-risk HPV. H1299 cells were transfected with indicated plasmids and doxorubicin (5 μM) added 24 hr after transfection. Cells were harvested 18 hr after doxorubicin treatment. Relative mRNA levels of PUMA or p21 after normalizing to actin are shown: mean ± SD of two experiments, each done in triplicate (n = 6).
(D and E) TIP60 is required for p53-dependent expression of PUMA, but not p21, after DNA damage. Depletion of TIP60 using siRNA in H1299 cells abrogates induction of proapoptotic gene, PUMA. The rest is as in (C) (see also Figure S6).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions