ZNF10 Inhibits HIV-1 LTR Activity Through Interaction with NF-ÎºB And

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FEBS Letters 589 (2015) 2019–2025

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ZNF10 inhibits HIV-1 LTR activity through interaction with NF-jB and Sp1 binding motifs ⇑ ⇑ Hironori Nishitsuji a,c, , Leila Sawada a, Ryuichi Sugiyama a,d, Hiroshi Takaku a,b,

a Department of Life and Environmental Sciences, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan b Research Institute, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan c Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516, Japan d Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

article info abstract

Article history: Kruppel-associated box-containing zinc finger (KRAB-ZNF) genes constitute the single largest gene Received 2 March 2015 family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated Revised 4 June 2015 52 cDNA clones of KRAB-ZFPs from U1 cell lines and screened them to identify which were capable Accepted 11 June 2015 of regulating HIV-1 gene expression. We identified 5 KRAB-ZFPs that suppressed P50% of HIV-1 LTR. Available online 19 June 2015 Of the 5 identified KRAB-ZFPs, the expression of ZNF10 significantly enhanced the transcriptional Edited by Ivan Sadowski repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV-1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1-gamma binding. These results indicate that ZNF10 could be involved in a potent Keywords: KRAB domain intrinsic antiretroviral defense. Zinc finger protein Ó 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. TRIM28 SETDB1 HP1-gamma HIV-1 LTR

1. Introduction Several groups have reported that artificially engineered KRAB domain-containing zinc finger proteins that bind to HIV-1 The cellular tropism of HIV-1 replication has recently been sequences also induce proviral silencing [7–9]. Furthermore, host found to be controlled by the 50 LTR of HIV-1 [1]. The HIV-1 LTR proteins such as OTK18 suppress HIV-1 Tat-induced LTR activation functions as a typical promoter, containing several different bind- through the negative regulatory element (NRE) and ETS binding ing sites specific for cellular and viral regulatory proteins [2,3].Itis site (EBS). All of these LTR binding elements are important due to regulated not only by viral proteins (Tat, Nef, Vpu) but also by their therapeutic potential in the reactivation of HIV-1 in latently ubiquitously expressed host factors such as Sp1 and TFIID. infected cells [10–12]. Additionally, inducible transcription factors such as NF-jB, AP1, Reynolds et al. reported that a genetically engineered KRAB Sp1, NFAT, USF, and COUP play roles in LTR-driven gene expres- domain containing a C2H2-type zinc finger motif suppressed sion. These inducible regulatory factors vary in response to stimuli Tat-mediated HIV-1 LTR activity, thereby making it an attractive such as hyperthermia, oxidative stress and infection [4–6]. candidate for antiretroviral therapy [8]. In addition, endogenous

Moreover, the Kruppel-associated box (KRAB) zinc finger proteins OTK18, which contains 13 C2H2-type zinc finger motifs, inhibits (ZNFs) have the potential to regulate HIV-1 gene expression. HIV-1 replication. OTK18 was identified by differential display of mRNA from HIV-1-infected macrophages and was shown to interact with and suppress the NRE (255/238) and EBS (150/139) in Author contributions: H.N. and H.T.: conceived and supervised the study; H.N. and the HIV-1 LTR [11,13]. More recently, we showed that ZNF350 H.T.: designed experiments; H.N., L.S., and R.S.: performed experiments and (ZBRK1) is able to repress HIV-1 replication through binding the analyzed data: H.T.: analyzed data. H.T. and H.N. wrote the manuscript. 145 to 126 region of the HIV-1 LTR and ZNF1 in conjunction with ⇑ Corresponding authors at: Research Center for Hepatitis and Immunology, TRIM28 and HDAC2, thereby suppressing HIV-1 LTR-driven gene National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516, Japan expression [14]. Interestingly, the methylation driven by SETDB1 (H. Nishitsuji). Research Institute, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan. Fax: +81 47 478 0407 (H. Takaku). did not influence this mechanism, which probably reflects the fact E-mail addresses: [email protected] (H. Nishitsuji), hiroshi.taka- that each ZNF has a unique pattern of inhibition. TRIM28 (tripartite [email protected], [email protected] (H. Takaku).

http://dx.doi.org/10.1016/j.febslet.2015.06.013 0014-5793/Ó 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. 2020 H. Nishitsuji et al. / FEBS Letters 589 (2015) 2019–2025 motif-containing protein 28, also known as KRAB-associated supernatant was incubated with 1 lg of anti-TRIM28 antibody protein 1, KAP1) is a well-characterized transcriptional repressor. and 40 ll of protein G magnetic beads for 2 h at 4 °C. The beads The inhibition mediated by TRIM28 was proven to suppress endoge- were then washed with PBS containing 0.02% TritonX-100, and nous retroviruses by recruiting the H3K9 methyltransferase ESET the immunocomplexes were eluted by boiling in 20 llof5 sam- (also called SETDB1 or KMT1E) and heterochromatin protein 1 ple buffer and analyzed by SDS–PAGE and Western blotting. (HP1) in mouse ES cells [15,16]. TRIM28-mediated gene-speciﬁc transcriptional repression requires a ZNF protein such as ZFP809 2.6. Luciferase assay to directly recognize integrated viral DNA [17]. Furthermore, TRIM28 inhibits HIV-1 integration through a cellular pathway The luciferase assay details are provided in the Supplementary targeting acetylated IN [18]. Materials and Methods section. In this study, we investigated the role of KRAB-zinc ﬁnger proteins in the transcriptional repression of HIV-1. We found that 2.7. Measurement of HIV-1 p24 antigen ZNF10 highly represses HIV LTR-mediated transcription compared with other ZNFs. We also show that ZNF10 in conjunction with The p24 antigen measurement details are provided in the TRIM28, SETDB1 and HP1-gamma suppresses HIV-1 LTR-mediated Supplementary Materials and Methods section. transcription. 2.8. HIV-1 challenge and culture assay 2. Materials and methods The HIV-1 challenge and culture assay details are provided in 2.1. Plasmids the Supplementary Materials and Methods section.

The details of the plasmid constructs used in this study are pro- 3. Results and discussion vided in the Supplementary Materials and Methods section. 3.1. Screening results 2.2. Cells We obtained the coding DNA sequences (CDSs) of 134 human 293T cells were maintained in Dulbecco’s modified Eagle’s med- ZNFs and queried the NCBI protein database. We then isolated ium supplemented with 10% FBS, 100 units/ml penicillin, and 134 cDNAs encoding Kruppel-related zinc finger genes from a cell 100 lg/ml streptomycin. MT4 and U1 cells were grown in RPMI line latently infected with HIV-1 (U1). To evaluate the level of 1640 supplemented with L-glutamine (Sigma Aldrich, St. Louis, expression of the 134 ZNFs in U1 cells, we performed RT-PCR anal- MO, USA), 10% fetal bovine serum (FBS) (Biosera, Nuaille, France) ysis. We observed no detectable mRNAs encoding 82 of the 134 and 1% antibiotics (Sigma Aldrich). The cultures were maintained ZNFs in U1 cells (data not shown). Therefore, we focused on the at 37 °C/5% CO2. On day 4, the U1 cells were collected and cen- other 52 ZNFs as candidate transcriptional repressors of the trifuged at 1000 rpm for 3 min. The supernatant was discarded, HIV-1 LTR. and RNA was extracted from the pellets using RNeasy Plus Mini Next, we determined whether these 52 ZNFs could inhibit kits (Qiagen, Venlo, Netherlans). The RNA concentrations were HIV-1 LTR promoter activity. 293T cells were co-transfected with measured with a spectrophotometer (Thermo Scientific, the KRAB-ZNF expression vectors and with the HIV-1 LTR-driven Wilmington, MA, USA), and RNA quality (absence of RNA degrada- luciferase reporter plasmid. As shown in Fig. 1, ZNF10, ZNF566, tion) was assessed by gel electrophoresis. The levels of HIV-1 p24 ZNF333, ZNF561, and ZNF324 significantly suppressed P50% antigen were determined using chemiluminescence enzyme HIV-1 LTR activity compared with the empty vector control. By immunoassays (CLEIAs) (Lumipulsef, FUJIREBIO, Tokyo, Japan). contrast, ZNF416, ZNF115, and ZNF41 potentially acted as positive regulators of HIV-1, promoting LTR-driven transcription (Fig. 1). 2.3. Western blotting Mysliwice et al. reported that ZFP496 functions as a transcriptional activator when it is tethered to DNA or when it is directly bound to The details of the western blotting analysis are provided in the the DNA-binding motif [19]. However, in this study we did not Supplementary Materials and Methods section. analyze the up-regulation of LTR-driven transcription. To further investigate the effect of KRAB-ZNF (ZNF10, ZNF566, ZNF333, 2.4. Small interfering RNA (siRNA) ZNF561, and ZNF324)-mediated repression of the LTR, we used siRNAs specific for the five identified KRAB-ZNFs. The introduction KRAB-ZNF (ZNF10, ZNF566, ZNF333, ZNF561, and ZNF324) of each specific siRNA induced efficient knockdown of expression mRNA was analyzed, and specific Stealth RNAi™ predesigned of the corresponding protein (Fig. 2A). Knockdown of ZNF10 siRNAs were ordered (Life Technologies, Gland Island, NY, USA). expression significantly enhanced the transcriptional activity of siTRIM28 and siSETDB1 were purchased from Santa Cruz the LTR compared with the knockdown of other KRAB-ZNFs Biotechnology (Santa Cruz, CA, USA), and siHP1-gamma was pur- (Fig. 2B). The KRAB zinc finger protein 10 (ZNF10) contains two chased from Sigma–Aldrich. KRAB domains (A and B boxes) and nine zinc finger regions [20]. To evaluate the effect of ZNF10 in T cells, MT-4 cells were trans- 2.5. Immunoprecipitation duced with the control or ZNF10-specific short hairpin RNA (shRNA) vectors and then infected with HIV-1. Virus replication 293T cells were transfected with 0.5 lg of pHA-ZNF10 using was then monitored by measuring the production of p24 in the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 48 h supernatant every two days post-infection. The depletion of post-transfection, the transfected cells were harvested and sus- ZNF10 in MT-4 cells resulted in twofold more HIV-1 replication pended in 0.5 ml lysis buffer (20 mM Tris–HCl pH 7.5, 250 mM at four and six days post-infection (Fig. 2C). These results suggest NaCl, 1 mM EDTA, 5% glycerol, 1% TritonX-100). The cell lysates that ZNF10 inhibits HIV-1 gene expression through transcriptional were centrifuged at 15000g for 20 min at 4 °C, after which the repression of the LTR. H. Nishitsuji et al. / FEBS Letters 589 (2015) 2019–2025 2021

Fig. 1. The inhibitory effects of HIV-1 LTR-driven transcription by 52 ZNFs. 293T cells (4 105 cells) were transfected with 1 ng of pLTR-luc, 1 ng of pCMV-Renilla and 200 ng of pHA-ZNFs or p-HA (empty vector) using Lipofectamine 2000 (Invitrogen). At 48 h post-transfection, the levels of luciferase gene expression were determined by measuring the luciferase activity. Fireﬂy luciferase activity was normalized to Renilla luciferase activity. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.

Fig. 2. (A) The knockdown of ZNF expression enhances the transcriptional activity of the HIV-1 LTR. 293T cells were treated with 70 nM of the indicated siRNA or control siRNA for 24 h prior to transfection with 5 ng of pLTR-Luc and 1 ng of pCMV-Renilla-Luc. At 48 h post-transfection, the protein lysates were subjected to SDS–PAGE and probed for the indicated protein. (B) The luciferase activity of the protein lysates in Fig. 2A was analyzed. Fireﬂy luciferase activity was normalized to Renilla luciferase activity. (C) The effect of ZNF10 in T cells. MT-4 cells were infected with a lentiviral vector expressing shControl or shZNF10. At 5 days post-infection, the cells were infected with 2 ng of NL4–3 p24. Virus replication was monitored every 2 days after infection by measuring the amount of p24 viral antigen in the culture supernatant. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.

3.2. Identification of potential ZNF10 response elements in the HIV-1 phosphorylation of the RNA polymerase IICTD by recruiting HIV-1 LTR Tat to the TAR) and PCAF (which is known to possess histone acetyl transferase activity) [21]. To further determine the role of the To determine the LTR sequence responsible for the suppressive cis-elements of the LTR in ZNF10 repression, we introduced muta- function of ZNF10, we generated deletion mutants of the LTR tions into the NF-jB or Sp1 binding sites in the LTR (Fig. 3A). There (Fig. 3A) [14]. The expression of ZNF10 repressed transcription was no significant suppressive effect of ZNF10 upon mutations of from the 335 to +282 (Fig. 3C), 245 to +282 (Fig. 3D), and the NF-jB- or Sp1-binding sites of the LTR (Fig. 3F and G). To deter- 106 to +282 LTR segments (Fig. 3E), as well as from the mine the influence of ZNF10 and of the NF-jB- and Sp1-binding sites full-length LTR (Fig. 3B). These deletion mutants of the negative on HIV-1 LTR repression, 293T cells were transfected with regulatory element (NRE) of HIV-1 LTR did not influence the sup- pcDNA-HA or pcDNA-HA-ZNF10, pLTR-luc, pCMV-Renilla-luc, and pressive effect of ZNF10. In addition, HIV-1 Tat was not required pNF-jB-luc or pSp1-luc using Lipofectamine 2000 (Fig. 3I and J). for the repressive activity of ZNF10 (Fig. 3H). Furthermore, we examined the over-expression of pNF-jB or pSp1 A previous study has shown that transcriptional silencing of the in HIV-1 LTR-Luc and ZNF10-expressing plasmid-transfected cells HIV-1 LTR by HP1-gamma requires Sp1, P-TEFb (which leads to the (Fig. 3K and L). The luciferase reporter assays showed that the 2022 H. Nishitsuji et al. / FEBS Letters 589 (2015) 2019–2025

Fig. 3. The region of ZNF10 that is responsible for the transcriptional repression of the HIV-1 LTR. (A) Schematic representation of the HIV-1 LTR-driven expression of ﬁreﬂy luciferase. The numbers shown are relative to the transcriptional start site nucleotide, +1. (B–G) 293T cells transfected with 1 ng of pCMV-Renilla-Luc and 200 ng of pHA or pHA-ZNF10, along with 5 ng of pLTR-Luc (B) or its corresponding mutants (C–G). The luciferase assay was performed as described in Fig. 1. (H) 293T cells were transfected with 1 ng of pCMV-Renilla-Luc, 5 ng of pLTR-Luc and 100 ng of pCMV-Tat, along with 200 ng of pHA or pHA-ZNF10. The luciferase assay was performed as described in Fig. 1. (I, J) 293T cells were transfected with 200 ng of pHA or pHA-ZNF10, 1 ng of pCMV-Renilla-luc and 10 ng of pNF-jB-luc (Agilent Technologies) or 10 ng of pSp1-luc (Qiagen) using Lipofectamine 2000. The luciferase assay was performed as described in Fig. 1. (K, L) 293T cells were transfected with 200 ng of pHA or pHA-ZNF10, 1 ng of pLTR-luc, 1 ng of pCMV-Renilla-luc and 200 ng of pcDNA-65(NF-jB) or 200 ng of pCIneo-Sp-1 using Lipofectamine 2000. The luciferase assay was performed as described in Fig. 1. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.

NF-jB and Sp1 regions were required for the repression of the LTR by To address the binding of ZNF10 to the LTR, we performed an ZNF10 (Fig. 3I–L). These results suggest that the NF-jB and Sp1 sites electrophoretic mobility shift assay (EMSA). Incubation of the within the LTR contain essential elements for the transcriptional LTR probe containing NF-jB and Sp1 binding sites with nuclear repression of the LTR by ZNF10. extract revealed a shifted band (Fig. S1). The LTR-ZNF10 complex H. Nishitsuji et al. / FEBS Letters 589 (2015) 2019–2025 2023

Fig. 4. TRIM28, HP1-gamma and SETDB1 are required for the repressive activity of ZNF10. (A–D) 293T cells were treated with 70 nM of the indicated siRNA for 24 h prior to transfection with 5 ng of pLTR-Luc, 1 ng of pCMV-Renilla-Luc and 300 ng of pHA or pHA-ZNF10. At 48 h post-transfection, the protein lysates were subjected to SDS–PAGE and probed for the indicated protein. The luciferase activity of the protein lysates was also analyzed. Firefly luciferase activity was normalized to Renilla luciferase activity. (E) 293T cells were transfected with 500 ng of pHA-ZNF10. At 48 h post-transfection, the cell lysates were immunoprecipitated using an anti-HA antibody, followed by Western blotting with an anti-TRIM28 antibody. The results are representative of three independent experiments, and the error bars show the standard deviations from the means. (F) ZNF10 repression is reversed by HDAC inhibitors. 293T-LTR-Luc cells were transfected with 400 ng of pHA or pHA-ZNF10. At 24 h after transfection, the cells were treated with TSA for 24 h. The levels of luciferase gene expression were determined by measuring the luciferase activity. (G) U1 cells were treated with 5 ng/ml of PMA (Sigma Aldrich). After 48 h and 72 h, virus replication was monitored by measuring the amount of p24 viral antigen in the culture supernatant. The results are representative of three independent experiments, and the error bars show the standard deviation from the mean values. The protein lysates were subjected to SDS–PAGE and probed for the indicated protein. 2024 H. Nishitsuji et al. / FEBS Letters 589 (2015) 2019–2025 was observed to be supershifted by an anti-ZNF10 antibody. To fur- Acknowledgments ther confirm this observation, a supershift was also performed using nuclear extracts from 293T cells expressing HA-ZNF10 and We thank Dr. M. Abe, and Mr. H. Sato for their technical assis- an anti-HA antibody. These results indicate that ZNF10 binds to tance. This work was supported in part by a Grant-in-Aid for the NF-jB and Sp1 binding sites of LTR. Science Research (C) from the Japan Society for the Promotion of Science (JSPS), Japan; by a Grant-in-Aid for AIDS research from the Ministry of Health, Labor, and Welfare, Japan; and by a grant 3.3. Transcriptional repression of the HIV-1 LTR by ZNF10 requires from the Strategic Research Foundation Grant-aided Project for TRIM28, HP1-gamma and SETDB1 Private Universities from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT). Although the TRIM28/ZNF complex has been observed in the presence of different ZNFs [22–24], the role of other molecules in LTR-mediated transcriptional inhibition has been intensively stud- Appendix A. Supplementary data ied. TRIM28 mediates gene silencing by recruiting the NuRD histone deacetylase (HDAC) complex, the SETDB1 histone Supplementary data associated with this article can be found, in methyltransferase and the heterochromatin-associated protein the online version, at http://dx.doi.org/10.1016/j.febslet.2015.06. HP1-gamma to target promoters [25–29]. In a previous study, we 013. demonstrated that ZBRK1 in conjunction with TRIM28 and HDAC2 suppresses HIV-1 LTR-driven gene expression [14].To References investigate the mechanism involved in ZNF10-mediated repression [1] Reed-Inderbitzin, E. and Maury, W. (2003) Cellular specificity of HIV-1 of the LTR, we transfected siRNAs specific for TRIM28, HP1-gamma replication can be controlled by LTR sequences. Virology 314, 680–695. or SETDB1 into empty plasmid-transfected or ZNF10-expressing [2] Pereira, L.A., Bentley, K., Peeters, A., Churchill, M.J. and Deacon, N.J. (2000) A plasmid-transfected cells. 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