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The acetyltransferase TIP60 (also known as Kat5) is a member of the MYST family of histone acetyltransferases and was initially identified as a cellular protein. TIP60 acetylates histone and non-histone proteins and is involved in diverse biological processes, including apoptosis, cell cycle, and DNA damage responses. In this study, a specific inhibitor of TIP60 was used to detect the function of TIP60 in porcine parthenogenetic embryos. The results showed that TIP60 inhibition impaired porcine parthenogenetic embryonic development. The mechanism of TIP60 was also determined. We found that the TIP60 inhibition impaired embryonic development by ROS induced DNA damage, as demonstrated by the number of γH2A in the nuclei. TIP60 inhibition triggered DNA damage through the regulation of p53-p21 pathway and TIP60 played a role in DNA repair. TIP60 inhibition decreased the efficiency of DNA repair by regulating 53BP1-dependent repair after DNA damage. Inhibition of TIP60 also increased the adaptive response, autophagy, by modulating LC3. Therefore, TIP60 plays a role in early porcine parthenogenetic embryonic development by regulating DNA damage and repair., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The acetyltransferase TIP60 is regulated by phosphorylation, and we have previously shown that phosphorylation of TIP60 on S86 by GSK-3 promotes p53-mediated induction of the BCL-2 protein PUMA. TIP60 phosphorylation by GSK-3 requires a priming phosphorylation on S90, and here, we identify CDK9 as a TIP60S90 kinase. We demonstrate that a phosphorylation-deficient mutant, TIP60 S90A , exhibits reduced interaction with chromatin, histone 3 and RNA Pol II, while its association with the TIP60 complex subunit EPC1 is not affected. Consistently, we find a diminished association of TIP60 S90A with the MYC gene. We show that cells expressing TIP60 S90A , but also TIP60 S86A , which retains S90 phosphorylation, exhibit reduced histone 4 acetylation and proliferation. Thus, our data indicate that, during transcription, phosphorylation of TIP60 at two sites has different regulatory effects on TIP60, whereby S90 phosphorylation controls association with the transcription machinery, and S86 phosphorylation is regulating TIP60 HAT activity., (© 2018 The Authors.)

Tumor cells often encounter hypoglycemic microenvironment due to rapid cell expansion. It remains elusive how tumors reprogram the genome to survive the metabolic stress. The tumor suppressor TIP60 functions as the catalytic subunit of the human NuA4 histone acetyltransferase (HAT) multi-subunit complex and is involved in many different cellular processes including DNA damage response, cell growth and apoptosis. Attenuation of TIP60 expression has been detected in various tumor types. The function of TIP60 in tumor development has not been fully understood. Here we found that suppressing TIP60 inhibited p53 K120 acetylation and thus rescued apoptosis induced by glucose deprivation in hepatocellular cancer cells. Excitingly, Lys-104 (K104), a previously identified lysine acetylation site of TIP60 with unknown function, was observed to be indispensable for inducing p53-mediated apoptosis under low glucose condition. Mutation of Lys-104 to Arg (K104R) impeded the binding of TIP60 to human NuA4 complex, suppressed the acetyltransferase activity of TIP60, and inhibited the expression of pro-apoptotic genes including NOXA and PUMA upon glucose starvation. These findings demonstrate the critical regulation of TIP60/p53 pathway in apoptosis upon metabolic stress and provide a novel insight into the down-regulation of TIP60 in tumor cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background/aims: Aberrant expression of Tip60 is associated with progression in many cancers. However, the role of Tip60 in cancer progression remains contradictory. The aim of this study was to investigate the clinical significance, biological functions and underlying mechanisms of Tip60 deregulation in cholangiocarcinoma (CCA) for the first time., Methods: Quantitative real-time PCR (QRT-PCR), western blotting and immunohistochemistry staining (IHC) were carried out to measure Tip60 expression in CCA tissues and cell lines. Kaplan-Meier analysis and the log-rank test were used for survival analysis. In vitro, cell proliferation was evaluated by flow cytometry and CCK-8, colony formation, and EDU assays. Migration/ invasion was evaluated by trans-well assays. Phosphokinase array was used to confirm the dominant signal regulated by Tip60. Tumor growth and metastasis were demonstrated in vivo using a mouse model., Results: Tip60 was notably downregulated in CCA tissues, which was associated with greater tumor size, venous invasion, and TNM stage. Down-regulation of Tip60 was associated with tumor progression and poorer survival in CCA patients. In vitro and in vivo studies demonstrated that Tip60 suppressed growth and metastasis throughout the progression of CCA. We further identified the PI3K/AKT pathway as a dominant signal of Tip60 and suggested that Tip60 regulated CCA cell proliferation and metastasis via PT3K-AKT pathway. Pearson analysis revealed that PTEN was positively correlated with the Tip60 level in CCA tissues., Conclusion: Tip60, as a tumor suppressor in CCA via the PI3K/AKT pathway, might be a promising therapeutic target or prognostic marker for CCA., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

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.

Background: The nuclear epigenetic integrator UHRF1 is known to play a key role with DNMT1 in maintaining the DNA methylation patterns during cell division. Among UHRF1 partners, TIP60 takes part in epigenetic regulations through its acetyltransferase activity. Both proteins are involved in multiple cellular functions such as chromatin remodeling, DNA damage repair and regulation of stability and activity of other proteins. The aim of this work was to investigate the interaction between UHRF1 and TIP60 in order to elucidate the dialogue between these two proteins., Methods: Biochemical (immunoprecipitation and pull-down assays) and microscopic (confocal and fluorescence lifetime imaging microscopy; FLIM) techniques were used to analyze the interaction between TIP60 and UHRF1 in vitro and in vivo. Global methylation levels were assessed by using a specific kit. The results were statistically analyzed using Graphpad prism and Origin., Results: Our study shows that UHRF1, TIP60 and DNMT1 were found in the same epigenetic macro-molecular complex. In vitro pull-down assay showed that deletion of either the zinc finger in MYST domain or deletion of whole MYST domain from TIP60 significantly reduced its interaction with UHRF1. Confocal and FLIM microscopy showed that UHRF1 co-localized with TIP60 in the nucleus and confirmed that both proteins interacted together through the MYST domain of TIP60. Moreover, overexpression of TIP60 reduced the DNA methylation levels in HeLa cells along with downregulation of UHRF1 and DNMT1., Conclusion: Our data demonstrate for the first time that TIP60 through its MYST domain directly interacts with UHRF1 which might be of high interest for the development of novel oncogenic inhibitors targeting this interaction.

Mitochondria are highly dynamic organelles that change size and morphology by fusing together or dividing through fission. In response to cellular cues, signaling cascades may post-translationally modify mitochondria-shaping proteins, which lead to a change in mitochondria morphology. Here we show that nicotinamide (NAM), an inhibitor of sirtuin deacetylases, promotes degradation of mitochondria fusion protein mitofusin 1 (MFN1), suggesting that acetylation status of MFN1 is important for its protein stability. TIP60 but not PCAF acetyltransferase caused a reduction of MFN1 level. Meanwhile, siRNA-mediated knockdown of SIRT1 deacetylase caused a significant reduction of MFN1 whereas over-expression of SIRT1 increased its level in 293T cells. In vitro acetylation experiments showed that TIP60 increased the acetylation of MFN1 that was abolished by co-existence of SIRT1. Notably, MFN1 and SIRT1 levels were accumulated, along with mitochondria elongation under hypoxic conditions. Thus, the data suggest that mitochondria elongation under hypoxic condition is regulated through SIRT1-mediated MFN1 deacetylation and accumulation. The data provide an insight in the maintenance of cellular homeostasis through mitochondria morphological change., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome., (Copyright © 2017 American Society for Microbiology.)

Histone acetyltransferases (HATs) play vital functions in the tumorigenesis of many solid organ malignancies. We previously screened a human HATs cDNA library and identified Tat-interactive protein-60KDa (TIP60) as a candidate critical HATs in the origination of lung cancer. In this study, our data suggested that overexpression of TIP60 inhibited the cell viability of A549 and H1299 cells since day 2. Compared to the control group, the viability of these two lung cancer cells was inhibited by 25% and 19% at day 6 with the overexpression of TIP60. It increased by 36% and 26% when TIP60 was knockdown for 6 days. The invasion ability of these two cells was also restrained when TIP60 was overexpressed. While knockdown of TIP60 had the opposite effect. Inhibition of TIP60 significantly promoted the expression of molecules in AKT signaling pathway especially c-Myc. Furthermore, compared to paired tumor-adjacent tissue, lung cancer tumors had low expression of TIP60. Therefore, we concluded that TIP60 might inhibit the viability and invasion ability of lung cancer cells through down-regulation of AKT signaling pathway., Competing Interests: Competing Interests: None.

Although histone-modifying enzymes are generally assumed to function in a manner dependent on their enzymatic activities, this assumption remains untested for many factors. Here, we show that the Tip60 (Kat5) lysine acetyltransferase (KAT), which is essential for embryonic stem cell (ESC) self-renewal and pre-implantation development, performs these functions independently of its KAT activity. Unlike ESCs depleted of Tip60, KAT-deficient ESCs exhibited minimal alterations in gene expression, chromatin accessibility at Tip60 binding sites, and self-renewal, thus demonstrating a critical KAT-independent role of Tip60 in ESC maintenance. In contrast, KAT-deficient ESCs exhibited impaired differentiation into mesoderm and endoderm, demonstrating a KAT-dependent function in differentiation. Consistent with this phenotype, KAT-deficient mouse embryos exhibited post-implantation developmental defects. These findings establish separable KAT-dependent and KAT-independent functions of Tip60 in ESCs and during differentiation, revealing a complex repertoire of regulatory functions for this essential chromatin remodeling complex., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

UHRF2 is a ubiquitin-protein ligase E3 that regulates cell cycle, genomic stability and epigenetics. We conducted a co-immunoprecipitation assay and found that TIP60 and HDAC1 interact with UHRF2. We previously demonstrated that UHRF2 regulated H3K9ac and H3K14ac differentially in normal and cancer cells. However, the accurate signal transduction mechanisms were not clear. In this study, we found that TIP60 acted downstream of UHRF2 to regulate H3K9ac and H3K14ac expression. TIP60 is stabilized in normal cells by UHRF2 ubiquitination. However, TIP60 is destabilized in cancer cells. Depletion or inhibition of TIP60 disrupts the regulatory relationship between UHRF2, H3K9ac and H3K14ac. In summary, the findings suggest that UHRF2 mediated the post-translational modification of histones and the initiation and progression of cancer.

The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

Histone acetyltransferases (HATs) are epigenetic drivers that catalyze the acetyl transfer from acetyl-CoA to lysines of both histone and non-histone substrates and thereby induce transcription either by chromatin remodeling or direct transcription factor activation. Histone deacetylases (HDACs) conduct the reverse reaction to counter HAT activity. Physiological processes such as cell cycle progression or apoptosis require a thoroughly balanced equilibrium of the interplay between acetylation and deacetylation processes to maintain or, if required, alter the global acetylome status. Aberrant HAT activity has recently been demonstrated to play a crucial role in the progression of various diseases such as prostate, lung, and colon cancers as well as glioblastomas and neurodegenerative diseases. Recent investigations have aimed for the identification of HAT modulators to further decipher the complexity of acetyl transferase related signaling cascades and discover potential leads for drug design approaches. HDACs have been extensively characterized and targeted by small molecules, including four FDA-approved HDAC inhibitors; in contrast, HATs have not been active targets for therapeutic development. This review will summarize the status of HAT associated diseases and the arsenal of currently known and available HAT inhibitors with respect to their discovery, further improvements, and current applications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Tip60, the founding member of MYST histone acetyltransferase family, was originally identified as a cellular acetyltransferase protein that interacts with HIV-1 Tat. Tip60 plays roles in many processes such as cellular signaling transmission, DNA damage repair, cell cycle and checkpoint control and apoptosis by acetylating its histone or non-histone substrates., Results: Dysfunction of Tip60 could promote or suppress diseases including different kinds of cancers., Conclusion: Here, main functions and its known inhibitors were summarized., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The germ line efficiently combats numerous genotoxic insults to ensure the high fidelity propagation of unaltered genomic information across generations. Yet, germ cells in most metazoans also intentionally create double-strand breaks (DSBs) to promote DNA exchange between parental chromosomes, a process known as crossing over. Homologous recombination is employed in the repair of both genotoxic lesions and programmed DSBs, and many of the core DNA repair proteins function in both processes. In addition, DNA repair efficiency and crossover (CO) distribution are both influenced by local and global differences in chromatin structure, yet the interplay between chromatin structure, genome integrity, and meiotic fidelity is still poorly understood. We have used the xnd-1 mutant of Caenorhabditis elegans to explore the relationship between genome integrity and crossover formation. Known for its role in ensuring X chromosome CO formation and germ line development, we show that xnd-1 also regulates genome stability. xnd-1 mutants exhibited a mortal germ line, high embryonic lethality, high incidence of males, and sensitivity to ionizing radiation. We discovered that a hypomorphic allele of mys-1 suppressed these genome instability phenotypes of xnd-1, but did not suppress the CO defects, suggesting it serves as a separation-of-function allele. mys-1 encodes a histone acetyltransferase, whose homolog Tip60 acetylates H2AK5, a histone mark associated with transcriptional activation that is increased in xnd-1 mutant germ lines, raising the possibility that thresholds of H2AK5ac may differentially influence distinct germ line repair events. We also show that xnd-1 regulated him-5 transcriptionally, independently of mys-1, and that ectopic expression of him-5 suppressed the CO defects of xnd-1 Our work provides xnd-1 as a model in which to study the link between chromatin factors, gene expression, and genome stability., (Copyright © 2016 McClendon et al.)

The adenovirus E1A 243R oncoprotein targets TRRAP, a scaffold protein that assembles histone acetyltransferase (HAT) complexes, such as the NuA4/Tip60 complex which mediates transcriptional activity of the proto-oncogene MYC and helps determine the cancer cell phenotype. How E1A transforms cells through TRRAP remains obscure. We performed proteomic analysis with the N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) and showed that E1A 1-80 interacts with TRRAP, p400, and three other members of the NuA4 complex - DMAP1, RUVBL1 and RUVBL2 - not previously shown to associate with E1A 243R. E1A 1-80 interacts with these NuA4 components and MYC through the E1A TRRAP-targeting domain. E1A 243R association with the NuA4 complex was demonstrated by co-immunoprecipitation and analysis with DMAP1, Tip60, and MYC. Significantly, E1A 243R promotes association of MYC/MAX with the NuA4/Tip60 complex, implicating the importance of the MYC/NuA4 pathway in cellular transformation by both MYC and E1A., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nuclear spheres are protein aggregates consisting of FE65, TIP60, BLM, and other yet unknown proteins. Generation of these structures in the cellular nucleus is putatively modulated by the amyloid precursor protein (APP), either by its cleavage or its phosphorylation. Nuclear spheres were preferentially studied in cell culture models and their existence in the human brain had not been known. Existence of nuclear spheres in the human brain was studied using immunohistochemistry. Cell culture experiments were used to study regulative mechanisms of nuclear sphere generation. The comparison of human frontal cortex brain samples from Alzheimer's disease (AD) patients to age-matched controls revealed a dramatically and highly significant enrichment of nuclear spheres in the AD brain. Costaining demonstrated that neurons are distinctly affected by nuclear spheres, but astrocytes never are. Nuclear spheres were predominantly found in neurons that were negative for threonine 668 residue in APP phosphorylation. Cell culture experiments revealed that JNK3-mediated APP phosphorylation reduces the amount of sphere-positive cells. The study suggests that nuclear spheres are a new APP-derived central hallmark of AD, which might be of crucial relevance for the molecular mechanisms in neurodegeneration., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies., (© 2016 The Author(s).)

Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cellular supply of dNTPs via RNR (ribonucleotide reductase) is crucial for DNA replication and repair. It has been shown that DNA-damage-site-specific recruitment of RNR is critical for DNA repair efficiency in quiescent cells. The catalytic function of RNR produces dNDPs. The subsequent step of dNTP formation requires the function of NDP kinase. There are ten isoforms of NDP kinase in human cells. In the present study, we identified NME3 as one specific NDP kinase that interacts directly with Tip60, a histone acetyltransferase, to form a complex with RNR. Our data reveal that NME3 recruitment to DNA damage sites depends on this interaction. Disruption of interaction of NME3 with Tip60 suppressed DNA repair in serum-deprived cells. Thus Tip60 interacts with RNR and NME3 to provide site-specific synthesis of dNTP for facilitating DNA repair in serum-deprived cells which contain low levels of dNTPs., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Nucleosomes, the basic units of chromatin, are decorated with a myriad of posttranslational modifications (PTMs) by the action of chromatin modifiers. These enzymes function almost exclusively as part of stable protein complexes that assist their recruitment to specific genomic loci, specify their substrate, and provide allosteric control. By altering the interactions within nucleosomes or with neighboring nucleosomes and serving as a platform to engage effector proteins, PTMs deposited by histone-modifying complexes influence virtually every nuclear process and are at the heart of the epigenetic mechanisms. Hence, it is critical to identify their components, define their structures, and characterize their biochemical activities. Here we describe protocols for tandem affinity purification (TAP) of native histone acetyltransferase (HAT) and methyltransferase (HMT) complexes from human cells engineered to express bait proteins from a genomic safe harbor or their endogenous chromosomal genes, using zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems. The approaches presented aim to preserve natural transcriptional and posttranscriptional regulation and minimize biochemical artifacts due to ectopic expression. Near homogenous preparations of native complexes are obtained in sufficient amounts to perform biochemical assays and characterize their components., (© 2016 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are 22- to 24-nucleotide, small, non-coding RNAs that bind to the 3'UTR of target genes to control gene expression. Consequently, their dysregulation contributes to many diseases, including diabetes and cancer. miR-22 is up-regulated in numerous metastatic cancers and recent studies have suggested a role for miR-22 in promoting stemness and metastasis. TIP60 is a lysine acetyl-transferase reported to be down-regulated in cancer but the molecular mechanism of this reduction is still unclear. In this study, we identify TIP60 as a target of miR-22. We show a negative correlation in the expression of TIP60 and miR-22 in breast cancer patients, and show that low levels of TIP60 and high levels of miR-22 are associated with poor overall survival. Furthermore, pathway analysis using high miR-22/low TIP60 and low miR-22/high TIP60 breast cancer patient datasets suggests association of TIP60/miR-22 with epithelial-mesenchymal transition (EMT), a key alteration in progression of cancer cells. We show that blocking endogenous miR-22 can restore TIP60 levels, which in turn decreases the migration and invasion capacity of metastatic breast cancer cell line. These results provide mechanistic insight into TIP60 regulation and evidence for the utility of the combination of TIP60 and miR-22 as prognostic indicator of breast cancer progression.

Acetylation is a major modification that is required for gene regulation, genome maintenance and metabolism. A dysfunctional acetylation plays an important role in several diseases, including cancer. A group of enzymes-lysine acetyltransferases are responsible for this modification and act in regulation of transcription as cofactors and by acetylation of histones and other proteins. Tip60, a member of MYST family, is expressed ubiquitously and is the acetyltransferase catalytic subunit of human NuA4 complex. This HAT has a well-characterized involvement in many processes, such as cellular signaling, DNA damage repair, transcriptional and cellular cycle. Aberrant lysine acetyltransferase functions promote or suppress tumorigenesis in different cancers such as colon, breast and prostate tumors. Therefore, Tip60 might be a potential and important therapeutic target in the cancer treatment; new histone acetyl transferase inhibitors were identified and are more selective inhibitors of Tip60.

Disruption of epigenetic gene control mechanisms involving histone acetylation in the brain causes cognitive impairment, a debilitating hallmark of most neurodegenerative disorders. Histone acetylation regulates cognitive gene expression via chromatin packaging control in neurons. Unfortunately, the histone acetyltransferases (HATs) that generate such neural epigenetic signatures and their mechanisms of action remain unclear. Our recent findings provide insight into this question by demonstrating that Tip60 HAT action is critical for morphology and function of the mushroom body (MB), the learning and memory center in the Drosophila brain. We show that Tip60 is robustly produced in MB Kenyon cells and extending axonal lobes and that targeted MB Tip60 HAT loss results in axonal outgrowth disruption. Functional consequences of loss and gain of Tip60 HAT levels in the MB are evidenced by defects in memory. Tip60 ChIP-Seq analysis reveals enrichment for genes that function in cognitive processes and accordingly, key genes representing these pathways are misregulated in the Tip60 HAT mutant fly brain. Remarkably, increasing levels of Tip60 in the MB rescues learning and memory deficits resulting from Alzheimer's disease associated amyloid precursor protein (APP) induced neurodegeneration. Our studies highlight the potential of HAT activators as a therapeutic option for cognitive disorders.

TIP60 can act as a transcriptional activator or a repressor depending on the cellular context. However, little is known about the role of the chromodomain in the functional regulation of TIP60. In this study, we found that TIP60 interacted with H3K4me3 in response to TNF-α signaling. TIP60 bound to H3K4me3 at the promoters of the NF-κB target genes IL6 and IL8. Unlike the wild-type protein, a TIP60 chromodomain mutant did not localize to chromatin regions. Because TIP60 binds to histones with specific modifications and transcriptional regulators, we used a histone peptide assay to identify histone codes recognized by TIP60. TIP60 preferentially interacted with methylated or acetylated histone H3 and H4 peptides. Phosphorylation near a lysine residue significantly reduced the affinity of TIP60 for the modified histone peptides. Our findings suggest that TIP60 acts as a functional link between the histone code and transcriptional regulators.

Disruption of epigenetic gene control mechanisms in the brain causes significant cognitive impairment that is a debilitating hallmark of most neurodegenerative disorders, including Alzheimer's disease (AD). Histone acetylation is one of the best characterized of these epigenetic mechanisms that is critical for regulating learning- and memory- associated gene expression profiles, yet the specific histone acetyltransferases (HATs) that mediate these effects have yet to be fully characterized. Here, we investigate an epigenetic role for the HAT Tip60 in learning and memory formation using the Drosophila CNS mushroom body (MB) as a well-characterized cognition model. We show that Tip60 is endogenously expressed in the Kenyon cells, the intrinsic neurons of the MB, and in the MB axonal lobes. Targeted loss of Tip60 HAT activity in the MB causes thinner and shorter axonal lobes while increasing Tip60 HAT levels cause no morphological defects. Functional consequences of both loss and gain of Tip60 HAT levels in the MB are evidenced by defects in immediate-recall memory. Our ChIP-Seq analysis reveals that Tip60 target genes are enriched for functions in cognitive processes, and, accordingly, key genes representing these pathways are misregulated in the Tip60 HAT mutant fly brain. Remarkably, we find that both learning and immediate-recall memory deficits that occur under AD-associated, amyloid precursor protein (APP)-induced neurodegenerative conditions can be effectively rescued by increasing Tip60 HAT levels specifically in the MB. Together, our findings uncover an epigenetic transcriptional regulatory role for Tip60 in cognitive function and highlight the potential of HAT activators as a therapeutic option for neurodegenerative disorders., (Copyright © 2014 by the Genetics Society of America.)

A balanced deoxyribonucleotide (dNTP) supply is essential for DNA repair. Here, we found that ribonucleotide reductase (RNR) subunits RRM1 and RRM2 accumulated very rapidly at damage sites. RRM1 bound physically to Tip60. Chromatin immunoprecipitation analyses of cells with an I-SceI cassette revealed that RRM1 bound to a damage site in a Tip60-dependent manner. Active RRM1 mutants lacking Tip60 binding failed to rescue an impaired DNA repair in RRM1-depleted G1-phase cells. Inhibition of RNR recruitment by an RRM1 C-terminal fragment sensitized cells to DNA damage. We propose that Tip60-dependent recruitment of RNR plays an essential role in dNTP supply for DNA repair. [ABSTRACT FROM AUTHOR]

Histone acetylation is a key regulatory feature for chromatin that is established by opposing enzymatic activities of lysine acetyltransferases (KATs/HATs) and deacetylases (KDACs/HDACs). Esa1, like its human homolog Tip60, is an essential MYST family enzyme that acetylates histones H4 and H2A and other nonhistone substrates. Here we report that the essential requirement for ESA1 in Saccharomyces cerevisiae can be bypassed upon loss of Sds3, a noncatalytic subunit of the Rpd3L deacetylase complex. By studying the esa1∆ sds3∆ strain, we conclude that the essential function of Esa1 is in promoting the cellular balance of acetylation. We demonstrate this by fine-tuning acetylation through modulation of HDACs and the histone tails themselves. Functional interactions between Esa1 and HDACs of class I, class II, and the Sirtuin family define specific roles of these opposing activities in cellular viability, fitness, and response to stress. The fact that both increased and decreased expression of the ESA1 homolog TIP60 has cancer associations in humans underscores just how important the balance of its activity is likely to be for human well-being., (Copyright © 2014 by the Genetics Society of America.)

Tip60, the 60 kDa HIV-1 Tat-interactive protein, is a key member of the MYST family of histone acetyltransferases (HATs) and plays critical roles in apoptosis and DNA repair. Potent and selective inhibitors of Tip60 are valuable tools for studying the functions of this potential drug target. In this work, we designed, synthesized and evaluated a new set of substrate-based inhibitors containing multiple binding modalities. In addition to the coenzyme A (CoA) moiety and the histone H3 peptide backbone, mono- and tri-methyl marks were incorporated at Lys 4 and/or Lys 9 sites in the H3 peptide substrate. The biochemical assay results showed that the presence of methyl group(s) on the substrate resulted in more potent inhibitors of Tip60, relative to the parent H3-CoA bisubstrate inhibitor. Importantly, by comparing the inhibitory properties of the ligands against full-length Tip60 and the HAT domain, we determined that the K4me1 and K9me3 marks contributed to the potency augmentation by interacting with the catalytic region of the enzyme., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In embryonic stem cells (ESCs), the Tip60 histone acetyltransferase activates genes required for proliferation and silences genes that promote differentiation. Here we show that the class II histone deacetylase Hdac6 co-purifies with Tip60-p400 complex from ESCs. Hdac6 is necessary for regulation of most Tip60-p400 target genes, particularly those repressed by the complex. Unlike differentiated cells, where Hdac6 is mainly cytoplasmic, Hdac6 is largely nuclear in ESCs, neural stem cells (NSCs), and some cancer cell lines, and interacts with Tip60-p400 in each. Hdac6 localizes to promoters bound by Tip60-p400 in ESCs, binding downstream of transcription start sites. Surprisingly, Hdac6 does not appear to deacetylate histones, but rather is required for Tip60-p400 binding to many of its target genes. Finally, we find that, like canonical subunits of Tip60-p400, Hdac6 is necessary for robust ESC differentiation. These data suggest that Hdac6 plays a major role in the modulation of Tip60-p400 function in stem cells. DOI: http://dx.doi.org/10.7554/eLife.01557.001.

Meiosis is the process by which diploid germ cells produce haploid gametes. A key event is the formation of the synaptonemal complex. In the pachytene stage, the unpaired regions of X and Y chromosomes form a specialized structure, the XY body, within which gene expression is mostly silenced. In the present study, we showed that SYCP3-like X-linked 2 (SLX2, 1700013H16Rik), a novel member of XLR (X-linked Lymphocyte-Regulated) family, was specifically expressed in meiotic germ cells. In the spermatocyte SLX2 was distributed in the nucleus of germ cells at the preleptotene, leptotene and zygotene stages and is then restricted to the XY body at the pachytene stage. This localization change is coincident with that of phosphorylated histone H2AX (γH2AX), a well-known component of the sex body. Through yeast two-hybrid screening and coimmunoprecipitation assays, we demonstrated that SLX2 interacts with synaptonemal complex central element protein 2 (SYCE2), an important component of synaptonemal complex, and histone acetyltransferase TIP60, which has been implicated in remodeling phospho-H2AX-containing nucleosomes at sites of DNA damage. These results suggest that SLX2 might be involved in DNA recombination, synaptonemal complex formation as well as sex body maintenance during meiosis., (© 2013 Elsevier B.V. All rights reserved.)

The transfer of acetyl groups from acetyl coenzyme A to the ε amino group of internal lysine residues is catalyzed by Tip60, which is in the MYST family of nuclear histone acetyltransferases (HATs). The tyrosine phosphorylation of Tip60 seems to be a unique modification. We present evidence that Tip60 is modified on tyrosine 327 by Abl kinase. We show that this causes functional changes in HAT activity and the subcellular localization of TIP60, which forms a complex with Abl kinase. The Tip60 mutation Y327F abolished tyrosine phosphorylation, reduced the inhibition of Tip60 HAT activity, and caused G0-G1 arrest and association with FE65. Thus, our findings for the first time suggested a novel regulation mechanism of Tip60. Regulation was through phosphorylation of tyrosine 327 by Abl tyrosine kinase and depended on environmental conditions, suggesting that the tyrosine residue of Tip60 is important for the activation process.

Numerous studies indicate the importance of acetylation in p53-mediated stress responses upon DNA damage. We and others previously showed that TIP60 (Tat-interacting protein of 60 kDa)-mediated acetylation of p53 at K120 is crucial for p53-dependent apoptotic responses. Nevertheless, it remains unclear how TIP60-mediated effects on p53 are dynamically regulated in vivo. Here, we report that UHRF1 (ubiquitin-like with PHD and RING finger domains 1) interacts with TIP60 both in vitro and in vivo and induces degradation-independent ubiquitination of TIP60. Moreover, UHRF1 expression markedly suppresses the ability of TIP60 to acetylate p53. In contrast, RNAi-mediated knockdown of UHRF1 increases the endogenous levels of p53 acetylation at K120 and p53-mediated apoptosis is significantly enhanced in UHRF1-depleted cells. To elucidate the mechanisms of this regulation, we found that the interaction between TIP60 and p53 is severely inhibited in the presence of UHRF1, suggesting that UHRF1 modulates TIP60-mediated functions in both K120 acetylation-dependent and -independent manners. Consistent with this notion, UHRF1 knockdown promotes activation of p21 and PUMA but not MDM2. These findings demonstrate that UHRF1 is a critical negative regulator of TIP60 and suggest that UHRF1-mediated effects on p53 may contribute, at least in part, to its role in tumorigenesis.

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.

The abundant expression of IFNγ in Th-inducing POK (ThPOK)-deficient CD4(+) T cells requires the activation of Eomesodermin (Eomes); however, the underlying mechanism of this phenomenon remains unclear. Here we report that ThPOK binds directly to the promoter region of the Eomes gene to repress its expression in CD4(+) T cells. We identified the histone acetyltransferase TIP60 as a co-repressor of ThPOK-target genes, where ectopically expressed TIP60 increased ThPOK protein stability by promoting its acetylation at its Lys(360) residue to then augment the transcriptional repression of Eomes. Moreover, knockdown of endogenous TIP60 abolished the stabilization of ThPOK in CD4(+) T cells, which led to the transcriptional activation of Eomes and increased production of IFNγ. Our results reveal a novel pathway by which TIP60 and ThPOK synergistically suppresses Eomes function and IFNγ production, which could contribute to the regulation of inflammation.

Sleep disturbances are common in neurodegenerative diseases such as Alzheimer disease (AD). Unfortunately, how AD is mechanistically linked with interference of the body's natural sleep rhythms remains unclear. Our recent findings provide insight into this question by demonstrating that sleep disruption associated with AD is driven by epigenetic changes mediated by the histone acetyltransferase (HAT) Tip60. In this study, we show that Tip60 functionally interacts with the AD associated amyloid precursor protein (APP) to regulate axonal growth of Drosophila small ventrolateral neuronal (sLNv) pacemaker cells, and their production of neuropeptide pigment dispersing factor (PDF) that stabilizes appropriate sleep-wake patterns in the fly. Loss of Tip60 HAT activity under APP neurodegenerative conditions causes decreased PDF production, retraction of the sLNv synaptic arbor required for PDF release and disruption of sleep-wake cycles in these flies. Remarkably, excess Tip60 in conjunction with APP fully rescues these sleep-wake disturbances by inducing overelaboration of the sLNv synaptic terminals and increasing PDF levels, supporting a neuroprotective role for Tip60 in these processes. Our studies highlight the importance of epigenetic based mechanisms underlying sleep disturbances in neurodegenerative diseases like AD.

Herein, we report that the concanavalin A binding of Tip60 (a target of the human immunodeficiency virus type 1-encoded transactivator Tat interacting protein 60 KD; a histone acetyltransferase; HAT) is enhanced as the result of endoplasmic reticulum (ER) stress. The cell expression of Tip60 combined with site-directed mutagenesis analysis was used to identify the glutamine 324 residue as the lecithin binding (Concanavalin A; Con A) site. The Tip60 N324A mutant strain, which seems to be the Con A binding-deficient, was attenuated the protein-protein interactions with FE65 and its protein stability, but its ability of G0-G1 cell cycle arrest was not interrupted. Interestingly, both HAT activity and the nuclear localization of Tip60 N324A mutant were enhanced than those of Tip60 WT. Thus, our results indicate that the Con A binding deficient of Tip60 seems to be one of the most pivotal posttranslational modifications (such as N-glycosylation) for its functional regulation signal, which is generated in response to ER stress.