PCAF-mediated acetylation of ISX recruits BRD4 to promote epithelial-mesenchymal transition

Abstract
Epigenetic regulation is important for cancer progression; however, the underlying mechanisms, particularly those involving protein acetylation, remain to be fully understood. Here, we show that p300/CBP-associated factor (PCAF)-dependent acetylation of the transcription factor intestine-specific homeobox (ISX) regu- lates epithelial–mesenchymal transition (EMT) and promotes cancer metastasis. Mechanistically, PCAF acetylation of ISX at lysine 69 promotes the interaction with acetylated bromodomain- containing protein 4 (BRD4) at lysine 332 in tumor cells, and the translocation of the resulting complex into the nucleus. There, it binds to promoters of EMT genes, where acetylation of histone 3 at lysines 9, 14, and 18 initiates chromatin remodeling and subse- quent transcriptional activation. Ectopic ISX expression enhances EMT marker expression, including TWIST1, Snail1, and VEGF, induces cancer metastasis, but suppresses E-cadherin expression. In lung cancer, ectopic expression of PCAF–ISX–BRD4 axis compo- nents correlates with clinical metastatic features and poor prog- nosis. These results suggest that the PCAF–ISX–BRD4 axis mediates EMT signaling and regulates tumor initiation and metastasis.

Introduction
Epigenetic regulation has been broadly defined as alteration of gene expression through chromatin structure modification without chang- ing the underlying nucleotide sequences [1]. Histone acetylation, a type of epigenetic modification, plays an important role in gene regulation during embryonic development and human disease progression [2]. Many histone acetyltransferases (HATs), including p300/CBP-associated factor (PCAF), p300/CREB binding protein (CBP), TIP60, and hMOF, are involved in histone acetylation, which has been linked to transcriptionally active chromatin and subse- quent gene transcription [2,3]. Studies have also suggested the involvement of HAT dysregulation in cancer progression, especially in cancer metastasis and recurrence [3,4]. PCAF, a member of the GCN5-related N-acetyltransferase family of protein acetyltrans- ferases, has been shown to be involved in the modulation of dif- ferentiation, angiogenesis, cell cycle progression, gluconeogenesis, and carcinogenesis; however, the pathological functions of PCAF in cancer progression remain controversial [5,6]. Through its ability to interact with p300/CBP, PCAF forms a multimeric acetylase complex that remodels chromatin and facilitates downstream gene expression [7]. Moreover, PCAF acetylates not only histones to promote gene transcription but also certain non-histone transcrip- tion factors (TFs), such as p53 and STAT3, to directly promote their transcriptional activity [6–8]. Evidence suggests that PCAF functions as a key regulator of these non-histone proteins, which coordinate many carcinogenic and tumor suppression processes, such as cell cycle progression, DNA damage response, and apoptosis [9]. Although PCAF exerts important effects on the functions of nucleo- somes and TFs, the underlying mechanisms of these effects in the cytosol and nuclei remain largely unknown.

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extraterminal (BET) protein family, plays an important regulatory role in early embryonic development and human disease progression [10]. A recent report demonstrated that BRD4 controlled tumor metastasis via stability and expression of Snail in breast cancer [11]. Blocking BRD4 interactions by small- molecule inhibitors has been shown to effectively inhibit cell prolif- eration in cancers [12,13], and many potential candidates have been devoted to human clinical trials. As an organizer of super-enhancers (SEs) of hyper-acetylated promoter nucleosomes through a bromod- omain-mediated recruitment mechanism, BRD4 has been shown to interact with TFs that facilitate downstream gene expression [14,15], including oncogenes [12]. Moreover, histone acetylation at H3 lysine residues 9 and 27 was found to be important in BRD4- mediated SE-associated nucleosome organization [12,16]. Accord- ingly, the loss of BRD4 abolished enhancer-mediated gene expression. Although genome-wide studies have indicated that BRD4 is widely distributed along the genome, selective gene expression patterns and how recruitment of HAT-containing co-activators to specific promoters modulates TF-associated nucleosome organization remain largely undefined.The present study shows that PCAF acetylation of intestine-specific homeobox (ISX) recruits BRD4 to promoter nucleosome movement at EMT initiator, such as TWIST1 and Snail1, thereby facilitating chro- matin remodeling and transcriptional initiation via histone H3 acety- lation. Enhanced expression of EMT initiators leads to tumor microenvironment remodeling, thereby promoting metastasis in vitro and in vivo. Ectopic expression of PCAF–ISX–BRD4 axis components correlates with clinical metastatic features and poor prognosis, suggest- ing that the PCAF–ISX–BRD4 axis is an important regulator of tumor metastasis and cell plasticity in a tumorigenic microenvironment.

Results
To study the potential effects of ISX on poor prognosis among patients with cancer [17,18], EMT regulator expression as well as EMT characteristics were initially evaluated in cancer cells express- ing ISX. Lung cancer cells (A549 and H1299) showed increased mRNA and protein levels of mesenchymal cell markers, such as TWIST1, Snail, Slug, ZEB1, Bmi1, fibronectin, N-cadherin, vimentin, and VEGF, after ISX induction using doxycycline (Dox.; 1 lg/ml) but decreased expression of epithelial cell marker E-cadherin (Fig 1A and B). Promoter analysis showed that ISX-GFP transactivated luci- ferase activity driven by Snail1 and TWIST1 promoters (—71 to —87, and —75 to —89 bp, respectively, relative to the transcription start site) in A549 cells (Fig 1C and D). Moreover, ISX-GFP showed high binding activity to the above promoter elements of both Snail1 and TWIST1 with ISX cis-binding element (IBE; the sequence “CGCCGC” is a potential ISX-binding cis-element; [17,19]) in lung cancer cells (Figs 1E and EV1A). Conversely, promoters without the IBE showed no or lesser promoter activity and binding than that with GFP expression control (Figs 1C–E and EV1A). The ISX-induced transac- tivation was completely abolished upon deletion of the IBE in lung cancer cells (Figs 1F and EV1B). The endogenous binding activity of ISX on promoters of EMT markers was verified by Chromatin Immu- noprecipitation (ChIP), and the results showed endogenous ISX bound directly to the promoters of genes involved in EMT, and IL6 promoted the promoter-binding activity of ISX (Fig 1G). The migra- tion and invasion characteristics of ISX in lung cancer cells were further accessed using wound-healing and Transwell invasion assays. As predicted from the previous results, cells with forced ISX expression had higher migration and invasion activities compared with mock-transfected A549 lung cancer cells. However, cancer cells with ISX knockdown did not exhibit enhanced cell migration and invasion induced by ISX (Fig 1H and I). These results suggested that ISX expression transcriptionally upregulated EMT regulators and promoted EMT characteristics in lung cancer cells.

To elucidate the transactivation mechanisms of ISX on EMT regula- tors, co-immunoprecipitation coupled with two-dimensional gel electrophoresis (2-DE) and liquid chromatography–mass spectrome- try was conducted to identify ISX-interacting proteins in lysates of the aggressive lung cancer cell line A549. This approach yielded 11 candidate proteins from three independent 2-DE experiments. One of the oligopeptides, NH2-KADTTTPTTIDPIHEPPSLPPEPK-COOH, from differential expression protein was sequenced via liquid chro- matography–mass spectrometry and was identified as BRD4 (gi 71052031; amino acids 291–314 on BRD4) (Fig 2A). To confirm the interaction between ISX and BRD4 in vivo, immunoprecipitates from A549 and H1299 cells obtained using anti-ISX polyclonal antibody were pulled down and examined by immunoblotting. As predicted, BRD4 was detected in ISX immunoprecipitates, whereas ISX was detected in anti-BRD4 immunoprecipitates (Figs 2B and EV1C). Interestingly, PCAF, an acetylation regulator, was detected in both ISX and BRD4 immunoprecipitates. Moreover, analysis of six lung cancer cell lines (A549, H358, H441, H1299, H1435, and H1437) revealed that the mRNA and protein expression patterns of ISX, PCAF, and BRD4 were co-expressed in lung cancer cells relative to those in human diploid lung fibroblasts (WI38; Fig EV1D and E). The interaction between ISX, PCAF, and BRD4 was further evalu- ated in tumors and adjacent healthy lung tissues from patients with lung cancer. A significant amount of BRD4 and PCAF was detected in anti-ISX immunoprecipitates of lung tumor tissues from patients with non-small-cell lung carcinoma (NSCLC), whereas low levels of BRD4, but not PCAF, RNA pol II, and CBP/CREB, were detected in anti-ISX immunoprecipitates of adjacent healthy lung tissues from patients with lung cancer (Fig 2C). Confocal fluorescence imaging and proximity ligation assay (PLA) were then used to examine the interaction between ISX, PCAF, and BRD4 in A549 cells. Results showed that both BRD4 (red) and PCAF (pink) proteins were co- localized with ISX (green) in the cytosol and nuclei (blank arrow) of A549 cells, and PLA analysis showed positive PLA signals (red) for both BRD4 and PCAF with ISX (Figs 2D and EV1F).

To determine the potential effects of PCAF on EMT characteris- tics induced by ISX, A549 cells with forced ISX expression were treated with four acetyltransferase inhibitors to evaluate the poten- tial regulatory effects of PCAF on ISX–BRD4 complex formation and EMT characteristics induced by ISX. Garcinol (p300 and PCAF inhi- bitor), C646 (p300/CBP inhibitor), and MB–3 (GCN 5 inhibitor), but not TH1834 (TIP60 inhibitor), were found to abrogate the enhanced expression of EMT regulators (TWIST1, Snail1, and Slug) and mark- ers (fibronectin, N-cadherin, and vimentin) induced by forced ISX expression (Fig 2E). Moreover, the expression of ISX (green) and most BRD4 (red) showed a cytosol localization pattern in A549 cells, while garcinol treatment of the cells abolished PLA signals, as determined through confocal immunofluorescence imaging (Figs 2F and EV1G and H). Also, the ISX-induced expression of EMT markers was abrogated in cells with PCAF knockdown (Figs 2G and EV1I). Further, A549 cells with forced ISX expression showed significantly decreased cell migration and Transwell invasion after garcinol treat- ment (Fig 2H and I). The above results thus suggest that acetylation by the p300/CBP/PCAF complex modulates the expression of EMT regulators and EMT characteristics induced by forced ISX expres- sion.

To further determine the effect of PCAF on ISX-induced EMT, poten- tial PCAF acetylation motifs [6] on ISX were identified, and three point mutations of ISX at positions 38 (AC1), 69 (AC3), and 72 (AC2) were made to examine their impact on the ISX–BRD4 complex formation and EMT regulation (Fig 3A and B). Recombinant PCAF protein significantly acetylated wild-type ISX, AC1, and AC2 mutant proteins. However, PCAF showed no acetylation activity on recom- binant ISX mutant protein at position 69 (AC3) in vitro (Fig 3C). Acetylated wild-type recombinant ISX was then digested with trypsin and sequenced using liquid chromatography–mass spectrometry. The peptide of ISX (NH2-SDMDRPEGPGEEGPGEAAASGSGLEKPPK-COOH, amino acids 44–72) was identified with acetylation lysine at position 69 (y(4): 469.31–511.31 m/z; Fig EV2A and B). A549 and H1299 cells were then transfected with ISX mutants and the expres- sion level and localization of ISX mutants, PCAF, and BRD4 were determined by immunoblotting. PCAF, BRD4, and ISX were detected both in the cytosol and in the nuclei in cells transfected with wild- type ISX, AC1, and AC2 mutants. PCAF and BRD4, as well as the ISX AC3 mutant, were mostly detected in the cytosol fraction, whereas none or low levels were detected in the nuclei of cells transfected with the AC3 ISX mutant (Fig 3D). Compared with A549 cells trans- fected with AC1 or AC2 ISX mutants, no or low levels of PCAF and BRD4 proteins were detected in anti-GFP immunoprecipitates of cells transfected with the ISX AC3 mutant in vivo (Fig 3E). Cells trans- fected with AC3 showed greater suppression in the expression of EMT regulators and markers compared with cells transfected with wild-type ISX and the other AC mutants (Fig EV2C). Acetylation of histones H2, H3, and H4 was assessed in A549 cells with wild-type ISX and AC mutants. Forced expression of wild-type ISX, as well as AC1 and AC2, promoted histone H3 acetylation at positions 9, 14, 18, and 27 (Fig 3F), whereas forced AC3 ISX mutant expression showed no histone H3 acetylation at positions 9, 14, and 18. No acetylation was detected on histones H2 and H4 with forced ISX expression (data not shown).

A549 cells transfected with AC1 or AC2 ISX mutant, as well as wild-type ISX, significantly promoted EMT characteristics [Fig 3G (migration) and h (invasion)]. However, cells transfected with AC3 ISX mutant showed no enhancement in both migration and Tran- swell invasion compared with cells with forced ISX or AC1 (AC2) expression. To evaluate cell migration, invasion, and metastasis in vivo, constitutively RFP-expressing A549 cells transfected with wild-type ISX or AC3 ISX mutant were directly injected into the lungs of nude mice, after which an in vivo imaging system (IVIS) was used to monitor tumor cell progression every week (Fig 3I). Mice injected with A549 cells having forced wild-type ISX expres- sion developed a detectable tumor at the second week in the lung and subsequent proliferation and metastasis were noted on the third week after injection. Most of mice injected with A549 cells with wild-type ISX were not survived with global tumor cell metastasis from the fourth weeks (Fig 3J and K). Conversely, A549 cells transfected with the AC3 ISX mutant showed no or few detectable tumors at the fourth week, whereas no or minor metastases were detected at the fifth week in nude mice (Fig 3J). Nude mice injected with A549 cells expressing ISX, but not those injected with cells expressing vector or AC3 ISX, showed limited survival and died 3– 6 weeks postinjection (Fig 3K). The above result showed that acety- lation of ISX at lysine residue 69 is essential for ISX-BRD4 complex formation, ISX-induced EMT, and tumor metastasis in lung cancer.

Similarly, His6-tagged wild-type and mutated BRD4 proteins were incubated with recombinant PCAF to evaluate the potential acetylation sites in vitro and determine whether BRD4 is a target protein of PCAF. Four potential lysine acetylation sites on BRD4 [289 (AC2), 291(AC1), 329 (AC3), and 332 (AC4)] were developed and expressed to examine the impact of the ISX–BRD4 complex on EMT in lung cancer cells (Fig 4A and B). PCAF protein showed significant acetylation with wild-type BRD4 and AC1–AC3 BRD4 mutants but not with the AC4 BRD4 mutant(Fig 4C). Acetylated wild-type recombinant BRD4 was then digested with trypsin and sequenced by liquid chromatography– mass spectrometry. The peptide of BRD4 (NH2-ESSRPVKPPKK- COOH, amino acids 323–333) was identified with acetylation lysine at position 332 (y(2): 275.21–317.21 m/z; Fig EV3A and B). Wild-type and mutant BRD4 were then expressed in A549 cells with ISX-GFP expression, and ectopic BRD4 proteins were detected in anti-GFP immunoprecipitates. Compared with cells transfected with wild-type or AC1–AC3 mutants, no or fewer BRD4 proteins were detected in the anti-GFP immunoprecipitates of cells transfected with the AC4 BRD4 mutant in vivo (Figs 4D and EV3C). Similarly, the expression of AC4 BRD4 mutant in A549 cells abolished the mRNA enhancement of TWIST1 and Snail1 induced by forced ISX–BRD4 complex expression (Fig 4E and F), consequently abolishing its high DNA-binding affinity for the promoters of TWIST1 and Snail1 (Fig 4G and H). Moreover, A549 cells expressing the AC4 BRD4 mutant showed significantly decreased EMT characteristics (invasion activity) (Fig 4I).

To analyze the interaction mode of the ISX–BRD4 complex, co- immunoprecipitation was used to identify the interaction domain between ISX and BRD4 in A549 cells. GFP-tagged wild-type and dele- tion mutants of ISX [18] were transiently transfected into A549 cells with HA-tagged BRD4 expression (Fig 5A), after which immunopre- cipitates by anti-GFP or anti-HA antibody were blotted (Fig 5B). HA- tagged BRD4 was detected in cells with wild-type and mutant ISX protein expression but not those with ISX DHD expression (upper panel in Fig 5B). Wild-type and mutant ISX, but not ISX DHD, also showed high binding affinity to HA-tagged BRD4 (lower panel in Fig 5B). Further, interactions between domains on BRD4 and ISX were monitored. Through three-dimensional structure modeling(Fig 5C and D), surface electrostatic forces propensity, and solvation energy (Fig 5E and F), bromodomain 1 (BD1) and 2 (BD2) of BRD4 showed potential binding to the homeobox domain of acetylated ISX. Tyr97 (Y) and Asn140 (N) of the BD1 domain and Tyr390 (Y) and Asn433 (N) of the BD2 domain in BRD4 were critical residues needed for BRD4 to recognize and bind the acetylated lysine peptide on ISX. Wild-type and mutant BRD4 were then transfected into lung cancer cells with ISX-GFP (Fig 6A, left). Wild-type and mutant proteins of BRD4, including DBD1(DB1), 97Y?A, and 140N?A, were detected in immunoprecipitates obtained with anti-GFP. DBD2(DB2), 390Y?A, and 433N?A mutants of BRD4 appeared to have no ISX-binding abil- ity in A549 cells (Fig 6A, right). Moreover, A549 cells expressing BD1(DB1), DBD2(DB2), 390 Y?A, and 433N?A mutants together with ISX-GFP showed significantly decreased DNA-binding activity of Snail1 and TWIST1 promoters (Fig 6B and C). The Hprt1 promoter region (-190-+40 bp; red) was used as a negative control. Subse- quently, the expression of DBD2(DB2), 390Y?A, and 433N?A ISX mutants in A549 cells abolished the enhanced cell migration and invasion activity induced by the ISX–BRD4 complex (Fig 6D and E). The results suggest that interactions between acetylated Lys69 of ISX and Tyr390 and Asn433 in the BD2 domain of BRD4 play a critical role in ISX–BRD4 complex formation.

To explore the clinical impact of PCAF–ISX–BRD4 signals in lung cancer, 157 paired NSCLC tumor samples (tumors along with neigh- boring healthy lung tissues) were obtained and analyzed. Compared with the adjacent normal lung tissues, both ISX (brown) and PCAF (brown) showed a tumor-specific expression pattern in lung tumor masses and were detected in both the cytoplasm and the nuclei of tumor cells (Fig 7A). Confocal fluorescence imaging was then used to examine the interaction between ISX, BRD4, and PCAF in immunostained samples of tumor masses and adjacent healthy lung tissues from patients with NSCLC. The expression of both BRD4 (red; Fig 7B) and PCAF (red; Fig 7C) was co-localized with ISX expression (green) in lung cancer cells (yellow arrow; Fig 7B and C). Moreover, mRNA expression of ISX strongly correlated with those of BRD4 and PCAF in patients with NSCLC (Pearson’s correla- tion coefficient, r = 0.8749 and 0.8156, respectively, P < 0.0001; Fig 7D and E). BRD4 mRNA expression also strongly correlated with PCAF mRNA expression in the same patients (r = 0.8148, P < 0.0001; Fig EV3D). Correlation of the expression for ISX, acety- lated ISX, and invasion marker (vimentin) [20,21] was monitored in NSCLC tumor mass, and the results showed that acetylated ISX and total ISX expression correlated with the expression of vimentin and the level of invasion (Figs 7F and EV3E).

To describe clinical characteristics and evaluate the prognostic value of ISX and BRD4 in NSCLC, 157 patients with follow-up data were analyzed for baseline characteristics. Patients with NSCLC were categorized into “low” and “high” expression groups for ISX and BRD4 using survival receiver operating characteristic curve analysis (Table 1). Signifi- cant differences in tumor sizes, metastases, and cancer stages were observed between patients with high and low levels of ISX or BRD4 expression (Table 1). Analysis of the survival curves indicated that patients with NSCLC having relatively lower ISX expression had a significantly longer survival time than that in patients with NSCLC having relatively higher expression after pulmonary resection (Fig 7G; P = 0.0085). Similarly, patients with NSCLC having rela- tively lower BRD4 or PCAF expression had a significantly longer survival time than that in patients with NSCLC having relatively higher expression after pulmonary resection (Figs 7H and EV3F). These results suggest that the PCAF–ISX–BRD4 axis is involved in NSCLC progression and patient survival.

Discussion
The present study provides evidence that PCAF acetylation of the oncogenic transcription factor ISX recruits the acetylated BET member BRD4 to translocate the complex into the nucleus, promot- ing the formation of organized nucleosomes on the promoters of downstream target genes, whereas PCAF acetylation of histone H3 at lysine residues 9, 14, and 18 initiates chromatin remodeling and subsequent transcription (see Fig EV4 for a model). The PCAF–ISX– BRD4 axis upregulates EMT-associated gene expression and promotes tumor metastasis in vitro and in vivo. These results suggest that PCAF–ISX–BRD4 signaling plays a pivotal role in tumor metasta- sis and can be a potential therapeutic target for ISX-induced tumors.Lung cancer is the leading cause of cancer deaths worldwide, including the United States, and metastasis is the major factor caus- ing these deaths [22,23]. Metastasis is a complex multistep morpho- genic process, and epithelial–mesenchymal transition (EMT) is believed to be the initial step of this process [24]. Despite several signaling molecules (e.g., PI3K, Snail, HIF1a, and SIP1) [25–28] and transcription factors (TWIST1/2, Snail1/2, ZEB1/2, and FOXC2) [29–32] having been identified as major regulators of EMT, a detailed regulatory mechanism for oncogene-induced EMT has yet to be established. The present study shows that the oncogenic tran- scription factor ISX is a key regulator of EMT and tumor metastasis by modulating the expression of EMT regulators, such as TWIST1 and Snail1. ISX, a pair-family homeobox TF, is a pro-inflammatory cytokine (IL-6)-induced homeobox gene that is highly expressed in hepatoma cells from patients with hepatocellular carcinoma (HCC). By directly regulating downstream cell cycle regulators (cyclin D1 and E2F1) [18,19] and immune checkpoint regulators (IDOs, PD-L1, and B7-2) [17], ISX has been shown to promote cell tumorigenic activities and is highly correlated with patient poor prognosis, high- lighting its importance in regulating HCC progression [18].

The find- ings of the present study further interpret the mechanism and correlation between ISX and poor prognosis in lung cancers, provid- ing a new therapeutic target in cancer therapy.PCAF, a member of the GCN5-related N-acetyltransferase family of protein acetyltransferases, has been shown to be involved in the modulation of tumorigenesis and metastasis; however, the patho- genic function of PCAF appears to be controversial [5,6] and is believed to be cofactor-dependent in human disease [7,33]. Although the oncogenic function of PCAF had originally been identi- fied to be induced by adenoviral E1A infection [5], it was also found to promote lung cancer progression by priming EZH2 acetylation [6]. Through acetylation of EZH2, PCAF enhances the protein stabil- ity in order to suppress target gene expression and promote lung cancer cell migration and invasion [6]. Conversely, PCAF has also been proposed to suppress cancer metastasis by restraining the activity of TP53 [34,35] and TFs Gli1 [36] and PTEN [37] through acetylation, thereby regulaying protein stability and inhibiting the EMT of cancer cells. Nonetheless, detailed regulatory mechanisms remain largely unknown. Our findings showed that PCAF acetyla- tion of ISX is the key step to translocate the PCAF–ISX–BRD4 complex into nuclei and subsequent nucleosome formation on promoters of target genes. It provides vehicle-based evidence to interpret how PCAF is transported into nuclei to acetylate target histone proteins and chromatin remodeling.

The importance of BRD4 in tumorigenesis has been demon- strated by its ability to bind to acetylated histones and regulate tran- scription of oncogenic genes involved in several types of cancers [12]. Many small-molecule inhibitors, such as JQ1, were developed to disrupt protein–protein interactions between BRD4 and acetyl lysine, which effectively block cell proliferation and cytokine production in acute inflammation in cancers [38]. As an epigenetic reader, BRD4 recognizes acetylated H3 and H4 lysine residues via its bromodomains and activates downstream gene expression [39,40]. BRD4 is widely distributed along the whole genome, and many TFs and chromatin remodeling proteins have been shown to interact with BRD4 and define its selectivity for target genes in tumorigenesis, such as TP53, c-JUN, C/EBPa and b, c-MYC, and TWIST1 [12,14–16]. Generally, TFs are initially shown to recruit acetyltransferases, such as p300/CBP, that subsequently promote the acetylation of other non-histone proteins in the enhancer nucleo- somes of the target genes. Therefore, recruitment of BRD4 to active enhancers is likely mediated by its interaction with acetylated histones, as well as direct binding to enhancer-associated TFs [10,40]. Here, we provide evidence showing that ISX functions as an enhancer-binding factor in translocating the BRD4–PCAF complex to associate with histone H3-acetylated K27 on targeted nucleosomes, whereas PCAF acetylates histone H3 at 9, 14, and 18 lysine to initiate consequent chromatin remodeling. As shown in structure modeling, the carboxyl group of N-acetyl Lys69 of ISX could provide direct hydrogen bonding with the amide nitrogen of Asn433 in the BD2 domain of BRD4 and a water-mediated hydrogen bond interaction with the hydroxyl group of Tyr390 in the BD2 domain of BRD4 to facilitate ISX–BRD4 complex formation. More- over, single point mutations, either Tyr390 or Asn433, when substi- tuted with alanine, abolished ISX–BRD4 complex formation and led to subsequent gene repression of EMT regulators. Modulating the target gene expression of EMT regulators driven by PCAF–TF (ISX)– BRD4 clearly demonstrates a mechanistically synergistic regulatory effect on cancer metastasis.Collectively, we have shown that the PCAF acetylation of the ISX–BRD4 complex unpacks chromatin and activates the expression of EMT regulators through acetylation of histone H3, subsequently promoting EMT and metastasis. Our findings highlight the important regulatory role of acetylation signaling in the PCAF–ISX–BRD4 axis to promote EMT initiation and regulation during tumor metastasis, highlighting its potential as a therapeutic target for prevention of EMT and metastasis.

Male 8 weeks BALB/c nu/nu mice (N = 30; randomly divided into three groups) were obtained from the National Laboratory of Animal Breeding and Research Center (Taipei, Taiwan) and housed according to the protocols established by the Animal Center of the Kaohsiung Medical University (Kaohsiung, Taiwan). Human lung cancer cell lines (A549, H358, H441, H1299, H1435, and H1437), and human diploid lung fibroblast W138 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained according to their protocols. The study was conducted with approval (IACUC-104181) from the ethics committee of Kaohsi- ung Medical University.Full-length ISX cDNA was amplified from a human testis cDNA library (GIBCO/BRL, Cheshire, UK) using PCR. ISX cDNA and mutant ISX were each subcloned into the pEGFP/C1 vector (Clontech) to express a GFP-tagged ISX. Full-length BRD4 and mutants were inserted into the FLAG (or mCherry)-tagged BRD4. The PLKO.1.puro or.neo vector was used as a backbone for shRNAi constructs targeting ISX (sequence, 50-CAAACTTGCATCCCTGT GCTA-30). The PLKO.1.puro or.neo vector was used as a backbone for shRNAi constructs targeting PCAF(sequence 4, 50-GTTGGCTATA TCAAGGATTAT-30; sequence 6, 50-TGGCATGTCCATTAGCTATTT- 30; sequence 7, 50-TTAATGGGATGTGAGCTAAAT-30). WI38, A549,H358, H441, H1299, H1435, and H1437 cell lines were subcultured and maintained according to ATCC protocols. Transfection was performed using a lipofectamine transfection kit (GIBCO/BRL). Cell lines from both ATCC and BRC have been thoroughly tested and authenticated; morphology, karyotyping, and PCR-based approaches were used to confirm the identity of the original cell lines.

Western blotting staining and immunohistochemical (fluorescence) staining were performed as described previously [17,19]. The primary antibodies used in this study were Snail (1:1,000 dilution; MABE167; Merck, Darmstadt, Germany), E-cadherin (1:1,000 dilu- tion; #3195S; Cell Signaling Technology), PCAF (1:1,000 dilution; #3378S; Cell Signaling Technology), vimentin (1:2,000 dilution; GTX100619; GeneTex), b-actin (1:10,000 dilution; #4967L; Cell Signaling Technology), HA (1:1,000 dilution; #3724S; Cell Signaling Technology), GFP (1:500 dilution; SC-9996; Santa Cruz Biotechnol- ogy), ISX (1:200 dilution; sc-398934; Santa Cruz Biotechnology), TWIST1 (1:200 dilution; ab49254; Abcam), BRD4 (1:1,000 dilution; #13440S; Cell Signaling Technology), acetylated lysine (1:500 dilu- tion; #9441S Cell Signaling Technology), fibronectin (1:500 dilution; GTX112794; GeneTex), mCherry (1:500 dilution; GTX128508; GeneTex), Slug (1:1,000 dilution; GTX128796; GeneTex), VEGF (1:200 dilution; sc-7269; Santa Cruz Biotechnology), and N-cadherin (1:1,000 dilution; GTX127345; GeneTex). FITC-conjugated anti- rabbit IgG, rhodamine-conjugated anti-mouse IgG, and alkaline phosphatase-conjugated anti-rabbit IgG antibody (1:500 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) CCS-1477 were also used. All experiments were repeated at least three times.