Histone chaperone CAF‐1 promotes HIV‐1 latency by leading the formation of phase‐separated suppressive nuclear bodies
2021; Springer Nature; Volume: 40; Issue: 10 Linguagem: Inglês
10.15252/embj.2020106632
ISSN1460-2075
AutoresXiancai Ma, Tao Chen, Zhilin Peng, Ziwen Wang, Jun Liu, Tao Yang, Liyang Wu, Guangyan Liu, Mo Zhou, Muye Tong, Yuanjun Guan, Xu Zhang, Yingtong Lin, Xiaoping Tang, Linghua Li, Zhonghui Tang, Ting Pan, Hui Zhang,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle19 March 2021free access Source DataTransparent process Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies Xiancai Ma Xiancai Ma orcid.org/0000-0002-4934-4221 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Tao Chen Tao Chen Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Zhilin Peng Zhilin Peng Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Ziwen Wang Ziwen Wang orcid.org/0000-0002-1678-7624 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Jun Liu Jun Liu Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Tao Yang Tao Yang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Liyang Wu Liyang Wu Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Guangyan Liu Guangyan Liu College of Basic Medical Sciences, Shenyang Medical College, Shenyang, Liaoning, China Search for more papers by this author Mo Zhou Mo Zhou Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Muye Tong Muye Tong Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yuanjun Guan Yuanjun Guan Core Laboratory Platform for Medical Science, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Xu Zhang Xu Zhang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yingtong Lin Yingtong Lin Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Xiaoping Tang Xiaoping Tang Department of Infectious Diseases, Guangzhou 8th People’s Hospital, Guangzhou, Guangdong, China Search for more papers by this author Linghua Li Linghua Li Department of Infectious Diseases, Guangzhou 8th People’s Hospital, Guangzhou, Guangdong, China Search for more papers by this author Zhonghui Tang Zhonghui Tang Department of Bioinformatics, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Ting Pan Ting Pan Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Center for Infection and Immunity Study, School of Medicine, Sun Yat-sen University, Shenzhen, Guangdong, China Search for more papers by this author Hui Zhang Corresponding Author Hui Zhang [email protected] orcid.org/0000-0003-3620-610X Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Xiancai Ma Xiancai Ma orcid.org/0000-0002-4934-4221 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Tao Chen Tao Chen Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Zhilin Peng Zhilin Peng Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Ziwen Wang Ziwen Wang orcid.org/0000-0002-1678-7624 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Jun Liu Jun Liu Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Tao Yang Tao Yang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Liyang Wu Liyang Wu Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Guangyan Liu Guangyan Liu College of Basic Medical Sciences, Shenyang Medical College, Shenyang, Liaoning, China Search for more papers by this author Mo Zhou Mo Zhou Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Muye Tong Muye Tong Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yuanjun Guan Yuanjun Guan Core Laboratory Platform for Medical Science, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Xu Zhang Xu Zhang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yingtong Lin Yingtong Lin Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Xiaoping Tang Xiaoping Tang Department of Infectious Diseases, Guangzhou 8th People’s Hospital, Guangzhou, Guangdong, China Search for more papers by this author Linghua Li Linghua Li Department of Infectious Diseases, Guangzhou 8th People’s Hospital, Guangzhou, Guangdong, China Search for more papers by this author Zhonghui Tang Zhonghui Tang Department of Bioinformatics, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Ting Pan Ting Pan Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Center for Infection and Immunity Study, School of Medicine, Sun Yat-sen University, Shenzhen, Guangdong, China Search for more papers by this author Hui Zhang Corresponding Author Hui Zhang [email protected] orcid.org/0000-0003-3620-610X Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Author Information Xiancai Ma1,2, Tao Chen1,2, Zhilin Peng1,2, Ziwen Wang1,2, Jun Liu1,2, Tao Yang1,2, Liyang Wu1,2, Guangyan Liu3, Mo Zhou1,2, Muye Tong1,2, Yuanjun Guan4, Xu Zhang1,2, Yingtong Lin1,2, Xiaoping Tang5, Linghua Li5, Zhonghui Tang6, Ting Pan1,7 and Hui Zhang *,1,2 1Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China 2Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China 3College of Basic Medical Sciences, Shenyang Medical College, Shenyang, Liaoning, China 4Core Laboratory Platform for Medical Science, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China 5Department of Infectious Diseases, Guangzhou 8th People’s Hospital, Guangzhou, Guangdong, China 6Department of Bioinformatics, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China 7Center for Infection and Immunity Study, School of Medicine, Sun Yat-sen University, Shenzhen, Guangdong, China *Corresponding author. Tel: +86 137 1063 5612; E-mail: [email protected] The EMBO Journal (2021)40:e106632https://doi.org/10.15252/embj.2020106632 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract HIV-1 latency is a major obstacle to achieving a functional cure for AIDS. Reactivation of HIV-1-infected cells followed by their elimination via immune surveillance is one proposed strategy for eradicating the viral reservoir. However, current latency-reversing agents (LRAs) show high toxicity and low efficiency, and new targets are needed to develop more promising LRAs. Here, we found that the histone chaperone CAF-1 (chromatin assembly factor 1) is enriched on the HIV-1 long terminal repeat (LTR) and forms nuclear bodies with liquid–liquid phase separation (LLPS) properties. CAF-1 recruits epigenetic modifiers and histone chaperones to the nuclear bodies to establish and maintain HIV-1 latency in different latency models and primary CD4+ T cells. Three disordered regions of the CHAF1A subunit are important for phase-separated CAF-1 nuclear body formation and play a key role in maintaining HIV-1 latency. Disruption of phase-separated CAF-1 bodies could be a potential strategy to reactivate latent HIV-1. Synopsis One major obstacle to achieving a functional cure of AIDS is HIV-1 latency. Multi-omics screening and physicochemical assays reveal that phased-separated CAF-1 nuclear bodies is a core factor contributing to HIV-1 latency. The histone chaperone CAF-1 contributes to HIV-1 latency in different latency models and primary CD4+ T cells. Epigenetic modifiers and histone chaperones are recruited by CAF-1 to nuclear bodies. CAF-1 bodies are phase-separated hetero-condensates. Phase separation properties of CAF-1 play a key in maintaining HIV-1 latency. Introduction HIV-1/AIDS is incurable because of HIV-1 latency (Chun et al, 1997; Finzi et al, 1997; Wong et al, 1997). HIV-1 proviruses are temporarily silenced within infected resting CD4+ T cells and reactivated along with CD4+ T-cell activation. Although combined antiretroviral therapy (cART) suppresses HIV-1 effectively, the treatment has to be lifelong, as HIV-1 viremia quickly rebounds upon the interruption of cART. Thus, complete eradication of HIV-1-infected cells seems to be the best strategy to cure patients. To this end, several functional cure strategies have been proposed to achieve long-term suppression of HIV-1 replication and remission of HIV-1 viremia (Deeks, 2012; Liu et al, 2015; Elsheikh et al, 2019). Nevertheless, no matter what kinds of functional cure strategies will be adopted, comprehensively elucidating the mechanisms of HIV-1 latency is the prerequisite to develop new or improve existed therapeutic interventions. The mechanisms of HIV-1 latency refer to multiple aspects including the specificities of integration sites, epigenetic regulations, transcriptional control, and post-transcriptional regulations (Mbonye & Karn, 2017; Khoury et al, 2018). Most of the proviruses tend to integrate into the intron of actively transcribed genes. Some latently infected cells with integration hotspots undergo clonal expansion (Schröder et al, 2002; Maldarelli et al, 2014; Wagner et al, 2014; Cohn et al, 2015). To enter latent status for HIV-1 proviruses, the expression of both viral Tat and cellular transcription factors including NF-κB, Sp1, AP-1, NFAT1, and TFIIH should be decreased, and transcription suppressors such as LSF, YY1, CTIP2, and TRIM28 could be recruited to HIV-1 promoters (Nabel & Baltimore, 1987; Perkins et al, 1993; Kinoshita et al, 1998; Yang et al, 1999; Ping & Rana, 2001; He & Margolis, 2002; Kim et al, 2006; Marban et al, 2007; Razooky et al, 2015; Ma et al, 2019). Along with transcriptional inhibition, the HIV-1 promoter undergoes multiple suppressive epigenetic modifications. Chromatin “eraser” modifiers, such as histone deacetylases HDAC1 and HDAC2, remove the active marks acetyls from histone lysine residues (Marban et al, 2007). Deacetylated histone H3 Lysine 9 (H3K9) is further methylated by chromatin “writers”, such as G9a, SUV39H1, and GLP (Chéné et al, 2007; Imai et al, 2010; Ding et al, 2013). H3K27 is methylated by EZH2 (Friedman et al, 2011). H4K20 is methylated by SMYD2 (Boehm et al, 2017). Both H3K9me3 and H3K27me3 as well as H4K20me1 contribute to HIV-1 latency (Ruelas & Greene, 2013). These suppressive epigenetic marks are ultimately maintained by chromatin “readers”, which include three heterochromatin proteins (HP1α, HP1β, HP1γ) for H3K9me3, five CBX paralogs (CBX2, CBX4, CBX6, CBX7, CBX8) for H3K27me3, L3MBTL1 for H4K20me1 (Chéné et al, 2007; Boehm et al, 2017; Khan et al, 2018). Apart from histone modifications, DNA methylation, which is modified by DNMT1 and maintained by MBD2, is also found to contribute to HIV-1 latency in several latency cell lines and HIV-1-infected patients (Blazkova et al, 2009; Kauder et al, 2009; Trejbalová et al, 2016). Cellular processes, as well as HIV-1 latency, have long been thought to be regulated unidimensionally by different cellular components. However, with the rapid development of super-resolution imaging and chromosome conformation capture technologies, a plethora of membrane-less condensates have been found to regulate gene expression in the spatiotemporally multi-dimensional manner (Dekker et al, 2013; Banani et al, 2017). Physicochemical studies of these non-membrane-enclosed compartments have revealed that membrane-less condensates have liquid-like properties and are coalesced via phase separation (Hyman et al, 2014). The driving forces of liquid–liquid phase separation (LLPS) are the weak multivalent interactions which are mediated by low-complexity intrinsically disordered regions (IDRs) within corresponding compartment components (Brangwynne et al, 2015). BRD4, MED1, OCT4, and GCN4, all of which harbor IDRs, form LLPS droplets to link super-enhancers (SEs) and gene activation (Boija et al, 2018; Sabari et al, 2018). HP1, SUV39H1, and TRIM28 form LLPS droplets to link heterochromatic H3K9me3 and gene suppression (Larson et al, 2017; Strom et al, 2017; Sanulli et al, 2019; Wang et al, 2019). More other components with LLPS properties include paraspeckle component NEAT1, Polycomb body component CBX2, PML body component PML (Banani et al, 2016; Yamazaki et al, 2018; Plys et al, 2019). The fate of these LLPS condensates is highly influenced by different modifications. The hyperphosphorylation of RNA polymerase II (RNAP II) C-terminal domain switches RNAP II from transcriptional condensates to splicing condensates (Lu et al, 2018; Guo et al, 2019). The acetylation of DDX3X, the methionine oxidation of Pbp1, and the phosphorylation of FUS inhibit the formation of corresponding phase-separated condensates (Monahan et al, 2017; Kato et al, 2019; Saito et al, 2019). The SUMOylation of PML promotes the formation of phase-separated condensates (Banani et al, 2016). Apart from the LLPS of cellular components, measles virus (MeV) and vesicular stomatitis virus (VSV) also form inclusion bodies with LLPS properties (Heinrich et al, 2018; Zhou et al, 2019). Although multitudes of cellular or viral proteins or RNAs have been found to phase separate into membrane-less organelles, the precise function of LLPS on different cellular and viral processes is still enigmatic (Alberti et al, 2019). Epigenetic modifiers and maintainers, some of which also have LLPS properties as we mentioned above, have been studied intensively these years (Larson et al, 2017; Strom et al, 2017; Plys et al, 2019; Sanulli et al, 2019; Wang et al, 2019). However, how and when distinct epigenetic proteins are recruited to target genes and HIV-1 promoter are still less defined. Histone chaperones, which contribute to epigenetic memory and genome stability, can act as landing pads for multiple epigenetic proteins to alter the global epigenetic landscape (De Koning et al, 2007; Groth et al, 2007). DNA clamp PCNA recruits DNMT1 and HDAC1 to methylate DNA CpGs and deacetylate histone lysines, respectively (Chuang et al, 1997; Milutinovic et al, 2002). Chromatin assembly factor 1 (CAF-1), which deposits newly synthesized H3-H4 onto replicating DNA, has been found to recruit SETDB1, SUV39H1, HDAC1/2, KDM1A, MBD1, and HP1α to the long terminal repeats (LTRs) of endogenous retrotransposons or endogenous retroviruses to establish and maintain the suppressive epigenetic modifications in mouse pluripotent stem cells (Murzina et al, 1999; Reese et al, 2003; Loyola et al, 2009; Cheloufi et al, 2015; Hatanaka et al, 2015; Yang et al, 2015). Some histone chaperones also participate in HIV-1 latency. The chromatin reassembly factor (CRF) FACT contributes to HIV-1 latency by decoying Tat and subsequently interfering with the association of Tat with P-TEFb complex (Huang et al, 2015). Another CRF named Spt6 forms a complex with LEDGF/p75 and Iws1 and aggregates on the latent HIV-1 LTR to maintain suppressive chromatin marks (Gérard et al, 2015). Due to the clustered epigenetic protein-bound nature of histone chaperones, we hypothesize that a pivotal histone chaperone may exist to orchestrate most of the suppressive epigenetic proteins together to synergistically mediate HIV-1 latency. In this study, we revealed that histone chaperone CAF-1 is enriched on HIV-1 LTR and maintains HIV-1 latency in several in vitro latency models and in resting CD4+ T cells from individuals on cART. The depletion of CHAF1A subunit of CAF-1 results in the loss of multiple suppressive epigenetic marks including H3K9me3, H4K20me3, and methyl-CpGs, as well as the accumulation of several active epigenetic marks including H3K4me3, H3K36me2, and acetyl-lysines. Further proteomics, biophysical, and biochemical assays characterized that multitudes of suppressive epigenetic proteins and histone chaperones are recruited by CAF-1 and form nuclear condensates with LLPS properties. We also identified a few of key amino acids within CHAF1A IDRs which mediate the LLPS of CAF-1 body. Most importantly, the mutation of key amino acids not only dissolves CAF-1 body but also eliminates CAF-1 body-mediated HIV-1 latency. We speculate that CAF-1 body could be a core factor which organizes the suppressive elements and maintains HIV-1 latency. Results CAF-1 promotes HIV-1 latency To find potential targets which might contribute to HIV-1 latency, we compared gene expression in unstimulated and TNFα-stimulated HIV-1 latency cell line J-Lat 10.6 which harbors an integrated full-length HIV-1 pseudotyped provirus (Jordan et al, 2003). The expression of GFP, which is inserted into the HIV-1 nef open reading frame, is significantly upregulated upon TNFα stimulation. Utilizing RNA-Seq and mass spectrometry (MS) analyses, we found that several transcriptional factors, including JUN, were significantly upregulated in the presence of TNFα signaling (Figs 1A and B, and EV1A). The expression of SEC16A, the third intron of which harbors HIV-1 pseudotyped provirus integration site, was unchanged. Conversely, several suppressive epigenetic proteins were significantly downregulated. Especially, CHAF1A, CHAF1B, and RBBP4, which are subunits of CAF-1 complex, were simultaneously downregulated (Fig 1A). Given that CAF-1 shows suppressive effect on gene expression, we speculated that CAF-1 might contribute to HIV-1 latency (Murzina et al, 1999; Reese et al, 2003; Loyola et al, 2009; Cheloufi et al, 2015; Hatanaka et al, 2015; Yang et al, 2015). As CHAF1A is the major subunit of CAF-1, hereafter we conducted further experiments on CHAF1A. We knocked out CHAF1A in J-Lat 10.6 and found that the depletion of CHAF1A upregulated HIV-1 expression in the heterogeneous knockout cell line and the homogeneous knockout cell line (Figs 1C and E, and EV1B and C, Appendix Fig S1A and B). HIV-1 reactivation was enhanced much higher when supplemented with histone deacetylase (HDAC) inhibitor SAHA, bromodomain and extra-terminal (BET) domain inhibitor JQ-1, and other analogous LRAs (Fig EV1D). These results were well-repeated in other monoclonal latency model cell lines including J-Lat 6.3, 8.4, 9.2, 15.4, and several heterogeneous latency model cell lines (Fig EV1-EV5, Appendix Fig S1C–E). Figure 1. CAF-1 promotes HIV-1 latency A. RNA-Seq of naïve and TNFα-stimulated J-Lat 10.6 cells. Differentially expressed genes were filtered with log2FC of 1 and P value FDR cutoff of 0.05. Upregulated and downregulated genes were labeled as red and blue dots, respectively. Representative genes were labeled aside corresponding dots. B. RNA-Seq result as in (A). Significantly changed genes were sorted out and plotted as heatmap. SEC16A indicated unchanged gene. C. The GFP-positive percentages of monoclonal sgCHAF1A and sgNT J-Lat 10.6 cell lines were shown in the top right corner of each flow cytometry figure. TNFα, SAHA, and JQ-1 were used as supplements. D. ChIP assay with antibody against CHAF1A was performed in J-Lat 10.6 cells. All the ChIP-qPCR DNA signals were normalized to siNC IgG of G5′. G5′ represented cellular DNA and viral 5′LTR junction; A: Nucleosome 0 assembly site; B: Nucleosome free region; C: Nucleosome 1 assembly site; V5: Viral 5′LTR and gag leader sequence junction; E represented envelope; V3: Viral poly purine tract and 3′LTR junction; G3′ represented viral 3′LTR and cellular DNA junction. E. The statistical graph of the result in (C). F–I. ChIP assays with antibodies against H3K9me3, H4K20me3, H3K4me3, and H3K9Acetyl were performed as in (D). Only “C” position signals were showed and normalized to Input. J, K. J-Lat 8.4 cells were treated with shCHAF1A lentiviruses and 5-aza-dC. The GFP-positive cells and HIV-1 LTR methyl-CpGs percentages of different groups were plotted in (J) and (K), respectively. L. ATAC-Seq was performed in WT and CHAF1A-KO J-Lat 10.6 cells. The relative tag densities of the pseudotyped HIV-1 5′LTR integration site in each group were calculated. The highest tag density was set as 100. Figure showed 2 kb range centered the 5′LTR integration site. Dashed line represented HIV-1 pseudotyped virus 5′LTR integration site. M. ChIP assays with antibodies against CDK9 and Pho-Pol II were performed in siNC and siCHAF1A J-Lat 10.6 cells. ChIP signals in each group were normalized to input. Data information: Data represented mean ± SEM in triplicate. P-values were calculated by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. CAF-1 promotes HIV-1 latency A. Mass spectrometry (MS) result of naïve and TNFα-stimulated J-Lat 10.6 cells. Significantly changed genes were plotted as heatmap. Representative genes were shown in table aside the heatmap. The heatmap scale represented fold change of gene expression (Min: −20 fold; Max: 20 fold). B, C. The GFP-positive percentages of heterogeneous sgCHAF1A and sgNT J-Lat 10.6 cell lines were shown as flow cytometry figure and statistical histogram. TNFα, SAHA and JQ-1 were used as supplements. D. Fifteen LRAs targeting eight signaling pathways were used in heterogeneous and homogeneous CHAF1A-KO J-Lat 10.6 cell lines. E, F. Two pseudotyped HIV-1 latency cell lines (J-Lat-NIB and J-Lat-NPB) were treated with sgCHAF1A lentiviruses, SAHA and JQ-1. The GFP-positive percentages of each group were measured. The backbones of each pseudotyped HIV-1 were also shown. G–J. J-Lat 6.3, 8.4, 9.2 and 15.4 were treated as in Fig 1C. The reactivation efficiencies of each group were indicated by the percentages of GFP-positive cells. Data information: Data represented mean ± SEM in triplicate. P-values were calculated by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. CAF-1 alters the epigenetic status of HIV-1 promoter and enriches SUMOylation system A. ChIP assay with antibody against CHAF1A was performed in TZM-bl cells as in Fig 1D. ChIP-qPCR DNA signals were normalized to Input. G5: cellular DNA and viral 5’LTR junction; L: luciferase region; G3: viral 3′LTR and cellular DNA junction. The other regions were as in Fig 1D. B–E. ChIP assays with antibodies against H3K9me, H3K9me2, H3K27me3 and H3K36me2 were performed as in Fig 1F. F. The schematic of CpGs on HIV-1 LTR of J-Lat 8.4. Eleven CpGs were shown in circles. G, H. The flow cytometry figures and Methyl-CpGs graphs of J-Lat 8.4 which were treated with shCHAF1A and 5-aza-dC, corresponding to statistical histograms in Fig 1J and K. I. RNA-Seq result of wild-type and CHAF1A-KO J-Lat 10.6. Differentially expressed genes were filtered with log2FC of 1 and PvalueFDR cutoff of 0.05. Upregulated and downregulated genes were labeled as red and blue dots, respectively. Representative genes were labeled aside corresponding dots. J. Flag-tagged TRIM28, SUMO1, SUMO2 and SUMO4 were co-overexpressed with HA-tagged CHAF1A. HA-tagged CHAF1A was IP with anti-HA beads. Both total and IP samples were IB with anti-HA, anti-Flag and anti-Actin antibodies. K. HA-tagged CHAF1A and HA-tagged GFP were co-overexpressed with Flag-tagged CDK9. HA-tagged proteins were immunoprecipitated (IP) with anti-HA beads. Both total and IP samples were immunoblotted (IB) with anti-HA, anti-Flag and anti-GAPDH antibodies. L. HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, UBC9, and TRIM28. siRNAs targeting TRIM28 and CHAF1A treated CDK9-overexpressed HeLa cells, respectively. CDK9 was IP with anti-HA beads. Both total and IP samples were IB with anti-HA, anti-Flag and anti-GAPDH antibodies. Data information: Data represented mean ± SEM in triplicate. P-values were calculated by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. CAF-1 forms nuclear bodies A. GFP-tagged CHAF1A was imaged with AF594-tagged antibody against CHAF1A. B. RFP-tagged CHAF1A was imaged with AF488-tagged antibody against CHAF1A. C, D. The en
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