HIV1-TAT interactive protein (TIP60) is a haploinsufficient tumor suppressor. However, the potential mechanisms endowing its tumor suppressor ability remain incompletely understood. It plays a vital role in virus-induced cancers where TIP60 down-regulates the expression of human papillomavirus (HPV) oncoprotein E6 which in turn destabilizes TIP60. This intrigued us to identify the role of TIP60, in the context of a viral infection, where it is targeted by oncoproteins. Through an array of molecular biology techniques such as Chromatin immunoprecipitation, expression analysis and mass spectrometry, we establish the hitherto unknown role of TIP60 in repressing the expression of the catalytic subunit of the human telomerase complex, TERT, a key driver for immortalization. TIP60 acetylates Sp1 at K639, thus inhibiting Sp1 binding to the TERT promoter. We identified that TIP60-mediated growth suppression of HPV-induced cervical cancer is mediated in part due to TERT repression through Sp1 acetylation. In summary, our study has identified a novel substrate for TIP60 catalytic activity and a unique repressive mechanism acting at the TERT promoter in virus-induced malignancies.

A common feature of all malignant cells is their ability of continued proliferation while avoiding replicative senescence. Tumors either activate expression of telomerase reverse transcriptase, TERT (in the case of ~ 85% of tumors) or use a recombination based alternative lengthening of telomeres (remaining 15% of tumors) to maintain their telomere length and thus ensure continued cell replication. Most viruses which cause tumors such as human papillomavirus (HPV) and Hepatitis B virus (HBV) also activate the expression of telomerase and result in malignancy. In this study, we have identified a novel cellular repressor of telomerase, TIP60. TIP60 which is targeted for degradation by HPV oncoprotein E6, acetylates Sp1, a positive regulator of TERT. We show that TIP60-mediated acetylation of Sp1, inhibits Sp1 function at the TERT promoter, thereby reducing TERT expression and thus the growth of HPV-positive cervical cancer cells, HeLa.

Funding: SJ was supported by grants from National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centers of Excellence initiative to the Cancer Science Institute of Singapore (R-713-006-014-271), National Medical Research Council (NMRC CBRG-NIG BNIG11nov001) and Ministry of Education Academic Research Fund (MOE AcRF Tier 1 T1-2012 Oct -04 and T1-2016- Apr-01). SJ, DK and DGT were supported by the RNA Biology Center at the Cancer Science Institute of Singapore, NUS, as part of funding under the Singapore Ministry of Education’s Tier 3 grants (MOE2014-T3-1-006). DGT was supported by a STaR Investigator Award and an RCE Core grant from the NRF, Singapore, and NIH grants CA66996, CA197697, and HL131477. DR and SH were supported by a post-graduate fellowship awarded by Yong Loo Lin School of Medicine, National University of Singapore. KKL, YZ, SSB, and BHC were supported by a postgraduate fellowship awarded by the Cancer Science Institute of Singapore, National University of Singapore. HSK was supported by a post-graduate fellowship awarded by NGS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

E6 collaborates with the transcription factor c-MYC to upregulate TERT in certain cervical cancer cell lines [ 24 ]. It also interacts with a cellular ubiquitin ligase E6AP to destabilize a repressor, NFX1-91, on the TERT promoter in a proteasome dependent manner [ 22 , 25 , 26 ]. The collaborative role of c-MYC and another transcription factor, Specificity Protein 1 (Sp1), to activate telomerase has also been reported [ 27 ]. This regulation occurs directly at the TERT promoter, which contains E boxes to enable binding of c-MYC and GC boxes that provide binding sites for Sp1 [ 28 ]. The dynamic relationship between E6 and TIP60, the reduction in the tumor size upon overexpression of TIP60 as well as reports of TIP60 being present at the TERT promoter [ 29 ], have led us to question if TIP60 can regulate TERT expression and if this regulation is dependent on E6. This study indeed identifies the hitherto unknown mechanism of TERT expression regulation by TIP60. We show that TIP60, in contrary to its role in acetylating histones leading to transcriptional activation, serves as a negative regulator of TERT expression by inhibition of Sp1 function. We show that TIP60 acetylates residue K639 on Sp1, which prevents Sp1 binding to the TERT promoter. Moreover, the biological significance of TERT repression by TIP60 is demonstrated by the ability of ectopically expressed TERT to rescue growth defects seen upon TIP60 overexpression in colony forming assays. Thus, this study identifies how TIP60 levels regulate TERT expression by limiting Sp1 binding to the TERT promoter.

Telomeres consist of repetitive DNA at the ends of linear chromosomes and their degradation limits the replicative potential of cells [ 12 , 13 ]. This is a consequence of progressive telomere shortening, occurring with every round of cell division due to the combined contributions of the end replication problem and active telomere processing [ 14 , 15 ]. Like normal somatic cells, cancer cells would ultimately face telomere-induced replicative senescence but they (re-)activate one of two telomere maintenance pathways to bypass this fate: about 85% of all tumors reactivate telomerase, whereas the remaining 15% employ a recombination based maintenance mechanism, named Alternative Lengthening of Telomeres (ALT) [ 16 , 17 ]. Telomerase is a reverse transcriptase that can add telomeric repeats de novo, thus counteracting telomere shortening. Its minimal core consists of a catalytic subunit, TERT, and an RNA template for reverse transcription, TERC [ 18 ]. TERT expression is regulated at the epigenetic, transcription and protein level by a multitude of cellular factors [ 19 ]. Additionally, many oncoviral proteins such as HPV E6 and HBV HBx are known to activate the expression of TERT, thus circumventing senescence and serving their purpose of cellular proliferation [ 20 – 23 ].

HIV-1 TAT Interactive protein (TIP60) is a lysine acetyltransferase and a member of the MYST (Moz, Ybf2/Sas3, Sas2 and TIP60) family of evolutionarily related histone acetyltransferases [ 1 ]. TIP60 can acetylate histone and non-histone proteins and is involved in a multitude of cellular functions like altering the chromatin structure, transcription and DNA damage repair response [ 2 – 4 ]. TIP60’s role as a bona fide tumor suppressor has also been documented [ 5 ]. In return, it is targeted for degradation by many viral oncoproteins like the human papillomavirus (HPV) oncoprotein E6 and adenoviral oncoproteins EIB55K and E4orf6 [ 6 – 8 ]. Infection by the oncogenic HPV is the leading cause of cervical cancer, the second most common cancer in women worldwide. High-risk HPV (HPV 16, HPV 18) is implicated in invasive carcinoma while low-risk HPV (HPV6, HPV8, HPV11) is responsible for genital warts and non-malignant lesions [ 9 ]. The mechanism of transformation of normal keratinocytes by HPV is dependent on two oncoproteins, E6 and E7, which interact with and destabilize the tumor suppressors p53 and pRb (Retinoblastoma protein) [ 10 , 11 ]. E6 also destabilizes TIP60 by cooperating with an E3 cellular ubiquitin ligase, EDD1 [ 8 ], and TIP60 in turn represses the E6 promoter, thus maintaining a delicate balance of cellular and viral gene expression [ 7 ]. Interestingly, both high and low-risk HPV are capable of destabilizing TIP60 whereas only the high-risk E6 is capable of destabilizing p53. This suggests that TIP60 degradation is an essential and common mechanism in promoting viral gene expression [ 7 , 11 ].

Results

Acetylation of Sp1 on lysine 639 by TIP60 regulates Sp1’s DNA binding ability As TIP60 is an acetyltransferase that can acetylate histone and non-histone proteins and was found to interact with Sp1, we next investigated whether such a post-translational modification was putatively regulating Sp1 in this context. To test this, we performed pull-down experiments in 293T cells due to their high transient transfection efficiency and robust expression of exogenous protein. 293T cells were transiently transfected with EGFP-Sp1 either alone or with wild-type (MSCV-TIP60WT) or catalytically inactive form of TIP60 (MSCV-TIP60KD). Sp1 was pulled down using GFP TRAP A beads and its acetylation status was determined using a pan-acetyl antibody. We observed that overexpression of wild-type TIP60 increased the acetylation status of Sp1 whereas acetylation levels were unaltered upon overexpression of the catalytically inactive TIP60 (Fig 4B), thereby confirming that Sp1 is indeed a substrate for TIP60’s acetyltransferase activity. In order to identify the lysine residue(s) on Sp1 that are specifically acetylated by TIP60, we performed the GFP pull-down experiments in SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) labeled cells and analyzed the Sp1 acetylation status between cells overexpressing either TIP60WT or TIP60KD quantitatively by mass spectrometry (S6B Fig). First we ensured that incorporation rate of the labeled amino acids in our input samples was satisfactory (S6C Fig). Here, specific acetylation sites displayed a differential SILAC ratio, whereas non-TIP60 dependent acetylation sites should have SILAC ratios of about 1:1. We identified 5 acetylated lysines on Sp1 (Figs 4C and S6B) of which we also identified a previously known residue K703, a substrate of CBP/p300 acetyltransferase activity [33]. The other four acetylation sites on Sp1 have not been previously reported in other studies. The acetylation spectra for these individual peptides was observed, confirming their existence in our system of study (S7A, S7B and S7C Fig). While most acetylation sites have SILAC ratios similar to the total Sp1 protein, the K639 acetylation site has SILAC ratios indicating specific acetylation by TIP60 (Fig 4C). Interestingly, the K639 residue is part of the first of three zinc finger domains which are involved in Sp1 DNA binding (S6C Fig) [34]. This led us to speculate that acetylation of Sp1 residue K639 by TIP60 might inhibit the occupancy of Sp1 on the TERT promoter by interfering with Sp1’s DNA binding ability. In order to test this hypothesis, using site-directed mutagenesis, we generated two mutations of Sp1, one which was non-acetylable (K639R) and another that mimicked constitutive acetylation (K639Q). We then performed in vitro reconstitution DNA pull-downs with wild-type Sp1 as well as the two mutants. We transfected wild-type Sp1 and the two mutants (Sp1 K639R and Sp1 K639Q) in HeLa cells and performed pull-down experiments using probes with either wild-type Sp1 binding sequence or the mutated Sp1 binding site sequence. We observed that wild-type Sp1 binds to the wild-type DNA probe but not to the probe containing mutated Sp1 binding site, demonstrating the specificity of the experiment. Strikingly, the mutant that mimics constitutive acetylation, Sp1 K639Q, could not bind to either the wild-type or mutant DNA. In contrast, Sp1 K639R, non-acetylable mimetic was found to bind non-specifically to both the wild-type and mutant DNA sequences (Fig 4D). To test if this binding behaviors were recapitulated in vivo, we transiently transfected these GFP tagged Sp1 constructs into HeLa cells (Fig 4E) and performed ChIP with GFP antibody to selectively pulldown only the exogenous Sp1 proteins. Consistent with the DNA pulldown results in Fig 4D, wild-type Sp1 protein was found to be enriched at the TERT promoter compared to the distal region (Fig 4F). Similar results were also obtained with Sp1 K639R mutant whereas the SP1 K639Q mutant was less enriched at the promoter region compared to both Sp1 WT and Sp1 K639R. This suggests that TIP60-mediated acetylation on Sp1 inhibits its DNA binding ability to the Sp1 binding sites found in the TERT promoter. This data shows that Sp1 is an acetylation target of TIP60 and that indeed acetylation at K639 inhibits its DNA binding function, thus providing a likely mechanism for TIP60-dependent transcriptional control of TERT.

TIP60-dependent growth inhibition is due to repression of TERT expression HeLa-TIP60 cells showed a significant reduction in colony formation ability in vitro and tumor formation in vivo. We questioned if this tumor suppressor role of TIP60 was dependent on repression of the TERT promoter and telomerase activity. Therefore in order to address the biological significance of TIP60-mediated TERT repression, we transiently transfected HeLa-LPCX and HeLa-TIP60 cells with either pBABE (vector) or pBABE-TERT. In this plasmid, TERT expression is not under the control of its endogenous promoter and could thus serve as a useful system to study if TIP60-mediated growth repression, is in part, facilitated by TERT downregulation and if it can be rescued by expression of exogenous TERT. 24 h post transfection, RNA was isolated from the same set of cells which were used in the colony formation assay (CFA) to study TERT expression and we observed a significant overexpression of TERT in both HeLa-LPCX and HeLa-TIP60 (S8A Fig). There was also a decrease in TERT expression in HeLa-TIP60-pBABE as compared to HeLa-LPCX-pBABE, consistent with our earlier findings (S8B Fig). 2×103 cells were seeded for CFA. Colonies were fixed and stained with crystal violet after 11 days and their sizes were quantified. We identified a significant reduction in the average size and number of colonies in HeLa-TIP60-pBABE as compared to HeLa-LPCX-pBABE (vector), consistent with our earlier findings [8]. Overexpression of TERT in the HeLa-LPCX background did not affect the colony formation ability. Interestingly, overexpression of TERT in HeLa-TIP60 cells showed a significant increase in the average size and number of colonies as compared to HeLa-TIP60-pBABE, thus rescuing the growth defect caused by TIP60 overexpression (Fig 5A, 5B and 5C). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. TIP60-mediated growth defects can be rescued by TERT overexpression and are dependent on Sp1 acetylation. (A) Representative image from Colony Formation Assay (CFA) where HeLa-LPCX and HeLa-TIP60 cells transiently transfected with pBABE (vector) or pBABE-TERT were seeded at a low density (2×103 cells). The cells were allowed to grow for 11 days before fixing and staining with crystal violet. (B, C) Bar graph shows the quantification performed using Image J software for average size and number of colonies respectively. The overexpression of TIP60 reduces average colony size, which is rescued upon overexpression of TERT in HeLa-TIP60. (D) Sp1 plasmids in LHCX vector backbone were transfected into HeLa cells. Twenty four hours post transfection, 2500 cells were seeded for colony formation assay and representative images are shown. 11 days after, they were fixed and stained with crystal violet and (E) number of colonies was quantified using Image J software. Error bars reflect the standard error of mean (SEM) and significance is represented as *, P<0.05, **, P<0.01, ***, P<0.001. https://doi.org/10.1371/journal.ppat.1006681.g005