Depletion Of TIP60 Research Articles

Abstract We022: Depletion of Tip60 After Myocardial Infarction Induces Histone H2A.Z Deacetylation Followed by Cardiomyocyte Dedifferentiation/Cell-Cycle Activation

Myocardial infarction (MI) leads to cardiomyocyte (CM) loss, resulting in cardiac dysfunction and heart failure. Remuscularization of injured myocardium requires proliferation of surviving CMs. However, approaches aimed at inducing CM regeneration following MI have been inadequate. To this end, we are targeting the acetyltransferase Tip60 (Tat-interactive protein 60 kD), a pleiotropic tumor suppressor encoded by the Kat5 gene. Using a murine genetic model, we previously reported that disruption of Kat5 promotes CM cell-cycle re-entry, and protects against the damaging effects of MI. It is becoming increasingly recognized that pre-existing CMs must undergo dedifferentiation accompanied by reversion to glycolytic metabolism in order to resume proliferation. The site-specific acetylation of the histone variant H2A.Z at lysine K4/K7 (H2A.Zac K4/K7 ) has been shown to maintain the differentiated state in hematopoietic stem cells (HSCs), neurons, and skeletal myocytes. Here, we report that Tip60 depletion post-MI results in the near-complete absence of H2A.Zac K4/K7 in CMs. This is associated with the enrichment of differentially expressed genes (DEGs) in epithelial to mesenchymal transitioning (EMT), cytoskeletal disassembly, extracellular matrix (ECM) softening, and metabolic reprogramming. To assess therapeutic relevance, we have begun to evaluate the potential cardioprotective effects of the anti-parasitic drug pentamidine, a known Tip60 inhibitor. Systemic administration of pentamidine on days 3-16 post-MI preserved cardiac function, accompanied by CM cell-cycle activation. These data suggest that pentamidine treatment enhances remuscularization, thereby promoting the retention of post-MI function, supporting the translational promise of targeting Tip60 as an innovative therapy for ischemic heart disease.

Tip60/Kat5 may be a novel candidate histone acetyltransferase for the regulation of liver iron localization via acetylation.

Hepcidin (HAMP), an iron regulatory hormone synthesized by liver hepatocytes, works together with ferritin (FTH) and ferroportin (FPN) in regulating the storage, transport, and utilization of iron in the cell. Epigenetic mechanisms, especially acetylation, also play an important role in the regulation of iron metabolism. However, a target protein has not been mentioned yet. With this preliminary study, we investigated the effect of histone acetyltransferase TIP60 on the expression of HAMP, FTH, and FPN. In addition, how the depletion of Tip60, which regulates the circadian system, affects the daily expression of Hamp was examined at six Zeitgeber time (ZT) points. For this purpose, liver-specific Tip60 knockout mice (mutant) were produced with tamoxifen-inducible Cre/lox recombination and an iron overload model in mice was generated. While HAMP and FTH expressions decreased, FPN expression increased in the mutant group. Interestingly, there was no change in the iron content. A significant increase was observed in the expressions of HAMP, FTH, and FPN and total liver iron content in the liver tissue of the iron overload group. Since intracellular iron concentration is involved in regulating the circadian clock, temporal expression of Hamp was investigated in control and mutant groups at six ZT points. In the control group, Hamp accumulated in a circadian manner with maximal and minimal levels reaching around ZT16 and ZT8, respectively. In the mutant group, there was a significant reduction in Hamp expression in the light phase ZT0 and ZT4 and in the dark phase ZT16. These data are the first findings demonstrating a possible relationship between Tip60 and iron metabolism.

Conditional depletion of the acetyltransferase Tip60 protects against the damaging effects of myocardial infarction

Injury from myocardial infarction (MI) and consequent post-MI remodeling is accompanied by massive loss of cardiomyocytes (CM), a cell type critical for contractile function that is for all practical purposes non-regenerable due to its profound state of proliferative senescence. Identification of factors that limit CM survival and/or constrain CM renewal provides potential therapeutic targets. Tip60, a pan-acetyltransferase encoded by the Kat5 gene, has been reported to activate apoptosis as well as multiple anti-proliferative pathways in non-cardiac cells; however, its role in CMs, wherein it is abundantly expressed, remains unknown. Here, using mice containing floxed Kat5 alleles and a tamoxifen-activated Myh6-MerCreMer recombinase transgene, we report that conditional depletion of Tip60 in CMs three days after MI induced by permanent coronary artery ligation greatly improves functional recovery for up to 28 days. This is accompanied by diminished scarring, activation of cell-cycle transit markers in CMs within the infarct border and remote zones, reduced expression of cell-cycle inhibitors pAtm and p27, and reduced apoptosis in the remote regions. These findings implicate Tip60 as a novel, multifactorial target for limiting the damaging effects of ischemic heart disease.

Evidence that the acetyltransferase Tip60 induces the DNA damage response and cell-cycle arrest in neonatal cardiomyocytes

Tip60, a pan-acetyltransferase encoded by the Kat5 gene, is enriched in the myocardium; however, its function in the heart is unknown. In cancer cells, Tip60 acetylates Atm (Ataxia-telangiectasia mutated), enabling its auto-phosphorylation (pAtm), which activates the DNA damage response (DDR). It was recently reported that activation of pAtm at the time of birth induces the DDR in cardiomyocytes (CMs), resulting in proliferative senescence. We therefore hypothesized that Tip60 initiates this process, and that depletion of Tip60 accordingly diminishes the DDR while extending the duration of CM cell-cycle activation. To test this hypothesis, an experimental model was used wherein a Myh6-driven Cre-recombinase transgene was activated on postnatal day 0 (P0) to recombine floxed Kat5 alleles and induce Tip60 depletion in neonatal CMs, without causing pathogenesis. Depletion of Tip60 resulted in reduced numbers of pAtm-positive CMs during the neonatal period, which correlated with reduced numbers of pH2A.X-positive CMs and decreased expression of genes encoding markers of the DDR as well as inflammation. This was accompanied by decreased expression of the cell-cycle inhibitors Meis1 and p27, activation of the cell-cycle in CMs, reduced CM size, and increased numbers of mononuclear/diploid CMs. Increased expression of fetal markers suggested that Tip60 depletion promotes a fetal-like proliferative state. Finally, infarction of Tip60-depleted hearts at P7 revealed improved cardiac function at P39 accompanied by reduced fibrosis, increased CM cell-cycle activation, and reduced apoptosis in the remote zone. These findings indicate that, among its pleiotropic functions, Tip60 induces the DDR in CMs, contributing to proliferative senescence.

Abstract 4670: TIP60 regulates alternative splicing of Integrin subunit alpha 6

Abstract Differences in RNA processing, specifically RNA splicing, contribute to enormous diversity that can be generated from the eukaryotic genome. Given the increasing evidence to show that splicing can occur co-transcriptionally, chromatin structure and histone modifications have been reported to affect the choice of a splice site. For instance, HDAC inhibitor treatment has been shown to affect alternative splicing. TIP60 is a haploinsufficient tumor suppressor gene that is frequently downregulated in cancers. TIP60 is a lysine acetyltransferase that acetylates histone and non-histone proteins. Since TIP60 is a writer of histone and non-histone modifications, we investigated TIP60's role in modulating alternative splicing. To understand TIP60's role in alternative splicing, RNA sequencing was performed in MCF10A cells that were (a) transiently depleted of TIP60 (b) stably overexpressing siRNA-resistant wild-type TIP60 (MCF10A*WT) and (c) stably overexpressing siRNA-resistant catalytically dead TIP60 (MCF10A*KD). Differential splicing analysis was performed using MISO program. We found 130 genes to be differentially spliced when TIP60 was depleted and 59 genes were differentially spliced in MCF10A*KD compared to MCF10A*WT. For both comparisons, the majority of the differential splicing events were skipped exon events. Upon overlapping the differential skipped exon events, we found significant exon 25 skipping of ITGA6, resulting in an isoform that has a shorter cytoplasmic domain. TIP60 depletion results in an increase in the ITGA6 isoform that lacks exon 25, which has been implicated to promote tumor initiation in breast cancer. To identify the splicing factor that regulates this event, we performed a siRNA screen of over 70 splicing factors and identified ESRP2 to regulate splicing of ITGA6 exon 25. The consequence and biological significance of TIP60-ESRP2 mediated alternative splicing will be discussed at the conference. Citation Format: Shreshtha Sailesh Bhatia, H. Phillip Koeffler, Sudhakar Jha. TIP60 regulates alternative splicing of Integrin subunit alpha 6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4670.

Abstract 400: Tip60 Depletion in Neonatal Cardiomyocytes Reduces the DNA Damage Response and Increases Cell-cycle Activation

Tip60 ( T at i nteractive p rotein, 60 kD) is a tumor suppressor encoded by the Kat5 gene that functions as a pan-acetylase. Among its targets, Atm ( a taxia- t elangiectasia m utated) undergoes auto-phosphorylation (pATM) when acetylated by Tip60 to induce the DNA damage response (DDR). Because pAtm-induced activation of the DDR is known to cause proliferative senescence in neonatal cardiomyocytes (CMs), we hypothesize that Tip60 is one of the upstream components in this pathway. Using mice containing floxed Kat5 alleles and a tamoxifen-inducible Cre-recombinase transgene driven by Myh6 (Kat5 flox/flox;Myh6-merCremer ), we assessed the effect of depleting Tip60 from neonatal CMs via a single injection of tamoxifen on postnatal day 0 (P0). Immunofluorescent microscopy revealed reduced pAtm-positive CMs at three postnatal stages (P7, P12, P39), accompanied by activation of the cell-cycle as indicated by significantly increased percentages of 5’-bromodeoxyuridine (BrdU)-, phosphohistone H3 (pH3)- and Ki67-positive CMs. In addition to cell-cycle activation, Tip60 depletion promoted nuclear division followed by cytokinesis, as indicated by increased percentage of mononuclear diploid CMs at P12. Accordingly, qPCR revealed that Tip60 depletion increased expression of mRNAs encoding the fetal markers Nppa, Nppb, Myh7 , and Acta1 at P39, which in the absence of CM hypertrophy (assessed by WGA staining) suggested maintenance of a proliferation-competent state. These effects occurred concomitant with depressed levels of mRNAs encoding cell-cycle inhibitors ( Meis1, p27 ) and a trend toward increased levels of G 2 -phase cell-cycle activators ( cyclins A2, B1 ; Cdk1 ) in Tip60-depleted hearts. These findings suggest that depletion of Tip60 at birth inhibits the DDR and delays replicative senescence in neonatal CMs.

Abstract 399: Tip60 Depletion in Adult Cardiomyocytes Promotes Proliferation and Preserves Cardiac Function After Myocardial Infarction

Cardiac disease and injury are accompanied by significant cardiomyocyte (CM) loss. CMs are essentially non-regenerable, preventing re-muscularization and resulting in permanent dysfunction during the healing process. The identification of factors from multiple layers of inhibitors that induce and maintain CM’s pronounced state of proliferative senescence provides potential therapeutic targets. Tip60 ( T at- i nteractive p rotein, 60 kD), a pan-acetylase tumor suppressor encoded by the Kat5 gene, activates multiple anti-proliferative pathways in other cell types. Tip60 is known to acetylate Atm and p53, which respectively activate the DNA damage response and apoptosis. And, Tip60 regulates intracellular levels of p21 and Tert polymerase in a fashion designed to maintain proliferative senescence. Although these functions promote CM proliferative senescence and Tip60 is strongly expressed in the myocardium, whether Tip60 regulates these activities in CMs remains unknown. This study tests the hypothesis that Tip60 inhibits CM cell-cycle activation, promotes apoptosis in the myocardium, and prevents regeneration after cardiac injury. In adult mice containing LoxP -flanked Kat5 alleles and a tamoxifen-inducible Cre-recombinase transgene driven by Myh6 (Kat5 flox/flox;Myh6-merCremer ) , Tip60 depletion three days after myocardial infarction (MI) preserved cardiac function as assessed by echocardiography at 10, 21, and 28 days post-MI. Histologic evaluation revealed that depletion of Tip60 in CMs significantly diminished scarring and increased CM cell-cycle activation, as indicated by increased numbers of Ki67-, BrdU- and pH3-positive CMs at 28 days post-MI. This was accompanied by the presence of CMs in the infarct border zone expressing smooth muscle α-actin, indicative of CM de-differentiation, and by reduced apoptosis in the remote zone as assessed by TUNEL and cleaved caspase-3 staining, suggesting reduced cardiac remodeling. These findings suggest that genetic depletion of Tip60 protects CMs from ischemia-induced cell death while promoting CM proliferation, implicating Tip60 as a novel candidate for cardiac therapeutics.

Abstract A39: Understanding poly-ADP-ribose polymerase (PARP) inhibitor resistance in BRCA2-deficient cells through dual CRISPR knockout and activation screens

Abstract Cells deficient in the DNA repair pathway homologous recombination (HR) show particular sensitivity to inhibitors of poly-ADP-ribose polymerases (PARPi). Based on this observation, several PARPi, such as olaparib, have been FDA approved for the treatment of BRCA2-mutated ovarian cancer. Indeed, clinical trials have demonstrated that use of these agents significantly improved progression-free survival (PFS), for example, from 5.5 months to 19 months in one cohort of ovarian cancer patients harboring BRCA1/2 mutations (SOLO2 trial). However, even in this trial, only 65% of patients who were predicted to be genetically susceptible to this treatment attained 12 months PFS. This indicates that sensitivity to PARPi is mediated by more than simply BRCA2 status. Moreover, investigations into mechanisms governing sensitivity and resistance to PARPi continue to further our understanding of basic DNA repair and replication pathways. To this end, we conducted dual CRISPR screens in BRCA2-deficient HeLa cells as an unbiased approach for identifying proteins whose loss or overexpression confers resistance to PARPi. First, over 19,000 genes were individually knocked out in BRCA2-deficient cells. Then, these cells were treated or not with olaparib. Sequencing and computational analysis were used to identify which genes were lost most often in cells surviving olaparib treatment, yielding hundreds of potential hits. Hits were tested in multiple cell lines using cellular viability, apoptosis, and clonogenic survival assays. Several of these hits were successfully validated, including the histone acetyltransferase TIP60 (KAT5) and the ubiquitin ligase HUWE1. Mechanistically, we show that the rescue of PARPi sensitivity caused by TIP60 depletion is dependent on the downstream proteins 53BP1 and REV7, proteins known to inhibit end resection, thus promoting NHEJ; this indicates that the role of TIP60 as a modulator of double-strand break repair pathway choice underlies this resistance. The finding that increased 53BP1 binding contributes to PARPi resistance in BRCA2-deficient cells is particularly interesting given that loss of 53BP1 has been shown to cause resistance to PARPi in BRCA1-deficient cells. This indicates that pathways that govern sensitivity to PARPi in BRCA1-deficient cells may have opposite effects on this phenotype in BRCA2-deficient cells. To further understand the networks governing PARPi resistance in BRCA2-deficient cells, we also completed a CRISPR activation screen using the same experimental design as the knockout screen. By combining the knockout and activation screens for the first time in this context, we are able to better construct overarching networks regulating PARPi sensitivity in BRCA2-deficient cells. We have identified many potential mediators of PARPi resistance in BRCA2-deficient cells by combining CRISPR-knockout and CRISPR-activation screens. Our work identifies TIP60 as a novel potential biomarker for PARPi response in BRCA2-deficient cells. Citation Format: Kristen E. Clements, Anastasia Hale, Nathanial J. Tolman, George-Lucian Moldovan. Understanding poly-ADP-ribose polymerase (PARP) inhibitor resistance in BRCA2-deficient cells through dual CRISPR knockout and activation screens [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A39.

An Interplay Between MRTF-A and the Histone Acetyltransferase TIP60 Mediates Hypoxia-Reoxygenation Induced iNOS Transcription in Macrophages.

Cardiac ischemia-reperfusion injury (IRI) represents a major pathophysiological event associated with permanent loss of heart function. Several inter-dependent processes contribute to cardiac IRI that include accumulation of reactive oxygen species (ROS), aberrant inflammatory response, and depletion of energy supply. Inducible nitric oxide synthase (iNOS) is a pro-inflammatory mediator and a major catalyst of ROS generation. In the present study we investigated the epigenetic mechanism whereby iNOS transcription is up-regulated in macrophages in the context of cardiac IRI. We report that germline deletion or systemic inhibition of myocardin-related transcription factor A (MRTF-A) in mice attenuated up-regulation of iNOS following cardiac IRI in the heart. In cultured macrophages, depletion or inhibition of MRTF-A suppressed iNOS induction by hypoxia-reoxygenation (HR). In contrast, MRTF-A over-expression potentiated activation of the iNOS promoter by HR. MRTF-A directly binds to the iNOS promoter in response to HR stimulation. MRTF-A binding to the iNOS promoter was synonymous with active histone modifications including trimethylated H3K4, acetylated H3K9, H3K27, and H4K16. Further analysis revealed that MRTF-A interacted with H4K16 acetyltransferase TIP60 to synergistically activate iNOS transcription. TIP60 depletion or inhibition achieved equivalent effects as MRTF-A depletion/inhibition in terms of iNOS repression. Of interest, TIP60 appeared to form a crosstalk with the H3K4 trimethyltransferase complex to promote iNOS trans-activation. In conclusion, we data suggest that the MRTF-A-TIP60 axis may play a critical role in iNOS transcription in macrophages and as such be considered as a potential target for the intervention of cardiac IRI.

Abstract 152: Tip60 Depletion Promotes Cardiomyocyte Proliferation and Attenuates Ischemic Injury in the Adult Heart

The pathogenesis of myocardial infarction (MI) is largely attributed to the loss of cardiomyocytes (CMs) and their insufficient regeneration. Inducing the proliferation of pre-existing CMs has emerged as a potential therapeutic strategy for cardiac repair. Results in our laboratory indicate that Tip60 (Tat-interactive protein 60 kD), a pan-acetylase protein encoded by the Kat5 gene, inhibits CM proliferation consequent to its induction of the DNA damage response (DDR) at neonatal stages, which has recently been shown to cause CM replicative senescence. To determine whether Tip60 depletion permits re-entry of adult CMs into the cell-cycle and confers protection from MI, we are employing a line of Kat5 flox/flox mice wherein Tip60 is conditionally and specifically depleted in CMs via tamoxifen-induced activation of a Myh6 -driven merCremer transgene. In uninjured hearts, Tip60 depletion results in transient thickening of the left ventricular walls, accompanied by markedly increased expression of G 2 /M-phase cell cycle regulators (cyclins A2 & B1, Cdk1) and de-differentiation markers (Myh7, Osm, OsmR, Runx1), diminished CM size, decreased expression of cell-cycle inhibitors (p27, Meis1), and remarkable increases in Ki67 and pH3-positive CMs as well as non-CMs. In hearts infarcted by permanent ligation, tamoxifen administration increases fractional shortening, ejection fraction, and anterior wall thickening within 7 days, conditions that are sustained for at least 18 additional days, when reduced scarring is indicated by trichrome staining. Taken together, these results indicate that Tip60 depletion in adult heart may preserve cardiac performance after MI by inducing CM regeneration. These findings should help advance our understanding of the molecular mechanisms that keep CMs in replicative senescence, establishing a possible therapeutic target for maintaining and restoring cardiac muscle after MI.

Evidence that Tip60 Induces the DDR & Cardiomyocyte Replicative Senescence in the Neonatal Heart

Tip60 (Tat interactive protein, 60 kD) is a tumor suppressor protein that acetylates histone and non‐histone proteins. Among the latter, ATM (Ataxia Telangiectasia Mutated), a kinase that induces the DNA damage response (DDR), is activated in cancer cells by Tip60‐mediated acetylation. We are investigating the function of Tip60 in the heart, using global and conditional knockout mouse models to disrupt the Kat5 gene encoding Tip60. Because global knockout (i.e. Tip60−/−) causes early embryonic lethality, we used Tip60+/− heterozygotes to show that, when under cardiac stress, modest Tip60 depletion correlates with re‐activation of the cardiomyocyte (CM) cell‐cycle in the adult heart. More recently, using a knockout model wherein Tip60 is constitutively and specifically depleted in CMs, we observed CM dropout and lethality in three‐month‐old mice, wherein hearts exhibited features of mitotic catastrophe. Because the latter phenotype was preceded by increased CM density, it was suggested that Tip60‐depletion may extend the duration of CM proliferation during neonatal stages of heart development. Moreover, considered in the context of recent reports that ATM is activated at birth to induce the DDR, which in turn causes replicative senescence of CMs by mid‐neonatal stages, these findings suggest that Tip60 induces these events. To examine these possibilities we are conditionally depleting Tip60 in CMs of Tip60F/F;Cre neonatal mice by injecting tamoxifen at postnatal day 0 (P0). To date, immunostaining of sections from neonatal hearts with antibodies that recognize 5′‐BrdU, phospho‐histone‐H3, and phospho‐ATM (pATM) has revealed that Tip60 depletion significantly increases CM proliferation as early as P7, by which time CM proliferation is normally diminished, and that this is accompanied by significantly decreased pATM levels, indicative of a diminished DDR. These findings provide further support for the notion that DDR activation at the time of birth induces replicative senescence of neonatal CMs, implicating Tip60 as the inducer of this process.Support or Funding InformationNIH HL131788 (JL & JA), and a grant from the MCW Cardiovascular CenterThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TIP60 Complex Inhibits Hepatitis B Virus Transcription.

Hepatitis B virus (HBV) is a global major health problem, with over one million deaths annually caused by chronic liver damage. Understanding host factors that modulate HBV replication may aid the development of anti-HBV therapies. Our recent genome-wide small interfering RNA screen using recombinant HBV demonstrated that TIP60 inhibited HBV infection. Here, we show that TIP60 complex contributes to anti-HBV defense. The TIP60 complex bound to the HBV promoter and suppressed HBV transcription driven by the precore/core promoter. The silencing of EP400, TRRAP, BAF53a, RUVBL1, and RUVBL2, which form the TIP60 complex, also resulted in increased HBV transcription. These results contribute to our enhanced understanding of the molecular mechanism of HBV transcription associated with the chromatin structure of HBV covalently closed circular DNA (cccDNA). Exploiting these intrinsic cellular defenses might help develop new anti-HBV agents.IMPORTANCE Investigating the molecular mechanism of HBV replication is important to understand the persistent nature of HBV infection and to aid the development of new HBV agents, which are currently limited to HBV polymerase inhibitors. Previously, we developed a new reporter HBV. By screening host factors using this recombinant virus, we identified several gene products that regulate HBV infection, including TIP60. Here, we showed that TIP60, a catalytic subunit of the NuA4 complex, inhibited HBV replication. Depletion of TIP60 increased the level of HBV mRNA. Moreover, TIP60 localized in the HBV cccDNA chromatin complex catalyzed the acetylation of histone H4 to recruit Brd4. These results suggest that TIP60, in concert with other cellular factors, plays an important role in the regulation of the HBV chromatin structure by acting as a critical component of the intrinsic antiviral defense, which sheds new light on the regulation of HBV replication.

Tip60 is associated with resistance to X-ray irradiation in prostate cancer.

Tip60, an oncogene, accelerates cell growth by regulating androgen receptor translocation into the nucleus in prostate cancer. However, the mechanism of Tip60 in the response of prostate cancer to radiotherapy, and radioresistance, has not been studied. Using human prostate cancer samples and two human prostate cancer cell lines (LNCaP and DU145), Tip60 protein expression and the acetylation of ataxia telangiectasia mutant (ATM) were analysed by western blotting and immunoprecipitation. Tip60 was downregulated with small interfering RNA. Cells were irradiated using X‐rays at 0.25 Gy·min−1. Cell viability was assessed by the MTT assay. The expression of Tip60 protein was increased in radioresistant prostate cancer tissues in comparison with radiosensitive tissues, which was also confirmed in both irradiated DU145 and LNCaP cells. Furthermore, the acetylation of ATM was also upregulated in a time‐dependent manner after irradiation of both DU145 and LNCaP cells. Additionally, depletion of Tip60 decreased the survival of LNCaP and DU145 cells by inducing apoptosis, reduced the acetylation of ATM and decreased the expression of phosphorylated ATM, Chk2 and cdc25A in both DU145 and LNCaP cells after X‐ray irradiation. The results of this study demonstrated that the expression of Tip60 may be related to the radioresistance of prostate cancer and could serve as a promising predictive factor for prostate cancer patients receiving radiotherapy.

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60.

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

Chronic treatment with cisplatin induces chemoresistance through the TIP60-mediated Fanconi anemia and homologous recombination repair pathways

The Fanconi anemia pathway in coordination with homologous recombination is essential to repair interstrand crosslinks (ICLs) caused by cisplatin. TIP60 belongs to the MYST family of acetyltransferases and is involved in DNA repair and regulation of gene transcription. Although the physical interaction between the TIP60 and FANCD2 proteins has been identified that is critical for ICL repair, it is still elusive whether TIP60 regulates the expression of FA and HR genes. In this study, we found that the chemoresistant nasopharyngeal carcinoma cells, derived from chronic treatment of cisplatin, show elevated expression of TIP60. Furthermore, TIP60 binds to the promoters of FANCD2 and BRCA1 by using the chromatin immunoprecipitation experiments and promote the expression of FANCD2 and BRCA1. Importantly, the depletion of TIP60 significantly reduces sister chromatid exchange, a measurement of HR efficiency. The similar results were also shown in the FNACD2-, and BRCA1-deficient cells. Additionally, these TIP60-deficient cells encounter more frequent stalled forks, as well as more DNA double-strand breaks resulting from the collapse of stalled forks. Taken together, our results suggest that TIP60 promotes the expression of FA and HR genes that are important for ICL repair and the chemoresistant phenotype under chronic treatment with cisplatin.

Depletion of Tip60 from In Vivo Cardiomyocytes Increases Myocyte Density, Followed by Cardiac Dysfunction, Myocyte Fallout and Lethality.

Tat-interactive protein 60 (Tip60), encoded by the Kat5 gene, is a member of the MYST family of acetyltransferases. Cancer biology studies have shown that Tip60 induces the DNA damage response, apoptosis, and cell-cycle inhibition. Although Tip60 is expressed in the myocardium, its role in cardiomyocytes (CMs) is unclear. Earlier studies here showed that application of cardiac stress to globally targeted Kat5+/—haploinsufficient mice resulted in inhibition of apoptosis and activation of the CM cell-cycle, despite only modest reduction of Tip60 protein levels. It was therefore of interest to ascertain the effects of specifically and substantially depleting Tip60 from CMs using Kat5LoxP/-;Myh6-Cre mice in the absence of stress. We report initial findings using this model, in which the effects of specifically depleting Tip60 protein from ventricular CMs, beginning at early neonatal stages, were assessed in 2–12 week-old mice. Although 5’-bromodeoxyuridine immunostaining indicated that CM proliferation was not altered at any of these stages, CM density was increased in 2 week-old ventricles, which persisted in 4 week-old hearts when TUNEL staining revealed inhibition of apoptosis. By week 4, levels of connexin-43 were depleted, and its patterning was dysmorphic, concomitant with an increase in cardiac hypertrophy marker expression and interstitial fibrosis. This was followed by systolic dysfunction at 8 weeks, after which extensive apoptosis and CM fallout occurred, followed by lethality as mice approached 12 weeks of age. In summary, chronic depletion of Tip60 from the ventricular myocardium beginning at early stages of neonatal heart development causes CM death after 8 weeks; hence, Tip60 protein has a crucial function in the heart.

TIP60 inhibits metastasis by ablating DNMT1-SNAIL2-driven epithelial-mesenchymal transition program.

HIV-Tat-interacting protein of 60 kDa (TIP60) is a lysine acetyltransferase and known to be downregulated in multiple cancers. Among various signalling pathways, TIP60 is implicated in regulating epithelial-mesenchymal transition (EMT). Here, we show that TIP60 expression abrogates cell migration and metastatic potential of breast cancer cells using in vitro and in vivo models. Mechanistically, we show that this is through its ability to destabilize DNMT1 and inhibit SNAIL2 function (SNAIL2-mediated EMT/cell migration). Depletion of TIP60 stabilizes DNMT1 and increases SNAIL2 levels, resulting in EMT. Recruitment of DNMT1 to the SNAIL2 targets in the absence of TIP60 increases DNA methylation on their promoter region and further represses the expression of epithelial markers. In pathophysiological scenario, we find TIP60 to be significantly downregulated in breast cancer patients with poor overall survival and disease-free survival prognoses. These data suggest that levels of TIP60 can be a prognostic marker of breast cancer progression and stabilization of TIP60 could be a promising strategy to treat cancers.

Control of genetic stability by a new heterochromatin compaction pathway involving the Tip60 histone acetyltransferase.

Pericentric heterochromatin is a highly compacted structure required for accurate chromosome segregation in mitosis. In mammals, it relies on methylation of histone H3K9 by Suv39H enzymes, which provides a docking site for HP1 proteins, therefore mediating heterochromatin compaction. Here we show that, when this normal compaction pathway is defective, the histone acetyltransferase Tip60 is recruited to pericentric heterochromatin, where it mediates acetylation of histone H4K12. Furthermore, in such a context, depletion of Tip60 leads to derepression of satellite transcription, decompaction of pericentric heterochromatin, and defects in chromosome segregation in mitosis. Finally, we show that depletion of BRD2, a double bromodomain-containing protein that binds H4K12ac, phenocopies the Tip60 depletion with respect to heterochromatin decompaction and defects in chromosome segregation. Taking the results together, we identify a new compaction pathway of mammalian pericentric heterochromatin relying on Tip60 that might be dependent on BRD2 recruitment by H4K12 acetylation. We propose that the underexpression of Tip60 observed in many human tumors can promote genetic instability via defective pericentric heterochromatin.

Stabilization of p21 (Cip1/WAF1) following Tip60-dependent acetylation is required for p21-mediated DNA damage response

The molecular mechanisms controlling post-translational modifications of p21 have been pursued assiduously in recent years. Here, utilizing mass-spectrometry analysis and site-specific acetyl-p21 antibody, two lysine residues of p21, located at amino-acid sites 161 and 163, were identified as Tip60-mediated acetylation targets for the first time. Detection of adriamycin-induced p21 acetylation, which disappeared after Tip60 depletion with concomitant destabilization of p21 and disruption of G1 arrest, suggested that Tip60-mediated p21 acetylation is necessary for DNA damage-induced cell-cycle regulation. The ability of 2KQ, a mimetic of acetylated p21, to induce cell-cycle arrest and senescence was significantly enhanced in p21 null MEFs compared with those of cells expressing wild-type p21. Together, these observations demonstrate that Tip60-mediated p21 acetylation is a novel and essential regulatory process required for p21-dependent DNA damage-induced cell-cycle arrest.