Evaluating the Cellular Roles of the Lysine Acetyltransferase Tip60 in Cancer: A Multi-Action Molecular Target for Precision Oncology.

Precision (individualized) medicine relies on the molecular profiling of tumors' dysregulated characteristics (genomic, epigenetic, transcriptomic) to identify the reliance on key pathways (including genome stability and epigenetic gene regulation) for viability or growth, and then utilises targeted therapeutics to disrupt these survival-dependent pathways. Non-mutational epigenetic changes alter cells' transcriptional profile and are a key feature found in many tumors. In contrast to genetic mutations, epigenetic changes are reversable, and restoring a normal epigenetic profile can inhibit tumor growth and progression. Lysine acetyltransferases (KATs or HATs) protect genome stability and integrity, and Tip60 is an essential acetyltransferase due to its roles as an epigenetic and transcriptional regulator, and as master regulator of the DNA double-strand break response. Tip60 is commonly downregulated and mislocalized in many cancers, and the roles that mislocalized Tip60 plays in cancer are not well understood. Here we categorize and discuss Tip60-regulated genes, evaluate Tip60-interacting proteins based on cellular localization, and explore the therapeutic potential of Tip60-targeting compounds as epigenetic inhibitors. Understanding the multiple roles Tip60 plays in tumorigenesis will improve our understanding of tumor progression and will inform therapeutic options, including informing potential combinatorial regimes with current chemotherapeutics, leading to improvements in patient outcomes.

SPIN1 facilitates chemoresistance and HR repair by promoting Tip60 binding to H3K9me3.

The tandem Tudor-like domain-containing protein Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. However, the involvement of SPIN1 in DNA damage repair has remained unclear. Our study shows that SPIN1 is recruited to DNA lesions through its N-terminal disordered region that binds to Poly-ADP-ribose (PAR), and facilitates homologous recombination (HR)-mediated DNA damage repair. SPIN1 promotes H3K9me3 accumulation at DNA damage sites and enhances the interaction between H3K9me3 and Tip60, thereby promoting the activation of ATM and HR repair. We also show that SPIN1 increases chemoresistance. These findings reveal a novel role for SPIN1 in the activation of H3K9me3-dependent DNA repair pathways, and suggest that SPIN1 may contribute to cancer chemoresistance by modulating the efficiency of double-strand break (DSB) repair.

Understanding the role of BRD8 in human carcinogenesis.

The bromodomain is a conserved protein-protein interaction module that functions exclusively to recognize acetylated lysine residues on histones and other proteins. It is noteworthy that bromodomain-containing proteins are involved in transcriptional modulation by recruiting various transcription factors and/or protein complexes such as ATP-dependent chromatin remodelers and acetyltransferases. Bromodomain-containing protein 8 (BRD8), a molecule initially recognized as skeletal muscle abundant protein and thyroid hormone receptor coactivating protein of 120 kDa (TrCP120), was shown to be a subunit of the NuA4/TIP60-histone acetyltransferase complex. BRD8 has been reported to be upregulated in a subset of cancers and implicated in the regulation of cell proliferation as well as in the response to cytotoxic agents. However, little is still known about the underlying molecular mechanisms. In recent years, it has become increasingly clear that the bromodomain of BRD8 recognizes acetylated and/or nonacetylated histones H4 and H2AZ, and that BRD8 is associated with cancer development in both a NuA4/TIP60 complex-dependent and -independent manner. In this review, we will provide an overview of the current knowledge on the molecular function of BRD8, focusing on the biological role of the bromodomain of BRD8 in cancer cells.

Tip60-FOXO regulates JNK signaling mediated apoptosis in Drosophila.

The JNK signaling pathway plays crucial roles in various physiological processes, including cell proliferation, differentiation, migration, apoptosis, and stress response. Dysregulation of this pathway is closely linked to the onset and progression of numerous major diseases, such as developmental defects and tumors. Identifying and characterizing novel components of the JNK signaling pathway to enhance and refine its network hold significant scientific and clinical importance for the prevention and treatment of associated cancers. This study utilized the model organism Drosophila and employed multidisciplinary approaches encompassing genetics, developmental biology, biochemistry, and molecular biology to investigate the interplay between Tip60 and the JNK signaling pathway, and elucidated its regulatory mechanisms. Our findings suggest that loss of Tip60 acetyltransferase activity results in JNK signaling pathway activation and subsequent induction of JNK-dependent apoptosis. Genetic epistasis analysis reveals that Tip60 acts downstream of JNK, paralleling with the transcription factor FOXO. The biochemical results confirm that Tip60 can bind to FOXO and acetylate it. Introduction of human Tip60 into Drosophila effectively mitigates apoptosis induced by JNK signaling activation, underscoring conserved regulatory role of Tip60 in the JNK signaling pathway from Drosophila to humans. This study further enhances our understanding of the regulatory network of the JNK signaling pathway. By revealing the role and mechanism of Tip60 in JNK-dependent apoptosis, it unveils new insights and potential therapeutic avenues for preventing and treating associated cancers.

TIP60 acetylation of Bub1 regulates centromeric H2AT120 phosphorylation for faithful chromosome segregation.

Proper function of the centromeres ensures correct attachment of kinetochores to spindle microtubules and faithful chromosome segregation in mitosis. Defects in the integrity and function of centromeres can result in chromosome missegregation and genomic instability. Bub1 is essential for the mitotic centromere dynamics, yet the underlying molecular mechanisms remain largely unclear. Here, we demonstrate that TIP60 acetylates Bub1 at K424 and K431 on kinetochores in early mitosis. This acetylation increases the kinase activity of Bub1 to phosphorylate centromeric histone H2A at T120 (H2ApT120), which recruits Aurora B and Shugoshin 1 (Sgo1) to regulate centromere integrity, protect centromeric cohesion, and ensure the subsequent faithful chromosome segregation. Expression of the non-acetylated Bub1 mutant reduces its kinase activity, decreases the level of H2ApT120, and disrupts the recruitment of centromere proteins and chromosome congression, leading to genomic instability of daughter cells. When cells exit mitosis, HDAC1-regulated deacetylation of Bub1 decreases H2ApT120 levels and thereby promotes the departure of centromeric CPC and Sgo1, ensuring timely centromeres disassembly. Collectively, our results reveal a molecular mechanism by which the acetylation and deacetylation cycle of Bub1 modulates the phosphorylation of H2A at T120 for recruitment of Aurora B and Sgo1 to the centromeres, ensuring faithful chromosome segregation during mitosis.

Negative regulation of HDAC3 transcription by histone acetyltransferase TIP60 in colon cancer.

BACKGROUND: Colon cancer is the third most common cancer globally. The expression of histone deacetylase 3 (HDAC3) is upregulated, whereas the expression of tat interactive protein, 60 kDa (TIP60) is downregulated in colon cancer. However, the relationship between HDAC3 and TIP60 in colon cancer has not been clearly elucidated. OBJECTIVE: We investigated whether TIP60 could regulate the expression of HDAC3 and suppress colon cancer cell proliferation. METHODS: RNA sequencing data (GSE108834) showed that HDAC3 expression was regulated by TIP60. Subsequently, we generated TIP60-knockdown HCT116 cells and examined the expression of HDAC3 by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined the expression pattern of HDAC3 in various cancers using publicly available datasets. The promoter activity of HDAC3 was validated using a dual-luciferase assay, and transcription factors binding to HDAC3 were identified using GeneCards and Promo databases, followed by validation using chromatin immunoprecipitation-quantitative polymerase chain reaction. Cell proliferation and apoptosis were assessed using colony formation assays and fluorescence-activated cell sorting analysis of HCT116 cell lines. RESULTS: In response to TIP60 knockdown, the expression level and promoter activity of HDAC3 increased. Conversely, when HDAC3 was downregulated by overexpression of TIP60, proliferation of HCT116 cells was inhibited and apoptosis was promoted. CONCLUSION: TIP60 plays a crucial role in the regulation of HDAC3 transcription, thereby influencing cell proliferation and apoptosis in colon cancer. Consequently, TIP60 may function as a tumor suppressor by inhibiting HDAC3 expression in colon cancer cells.

Polyploidy Promotes Hypertranscription, Apoptosis Resistance, and Ciliogenesis in Cancer Cells and Mesenchymal Stem Cells of Various Origins: Comparative Transcriptome In Silico Study.

Mesenchymal stem cells (MSC) attract an increasing amount of attention due to their unique therapeutic properties. Yet, MSC can undergo undesirable genetic and epigenetic changes during their propagation in vitro. In this study, we investigated whether polyploidy can compromise MSC oncological safety and therapeutic properties. For this purpose, we compared the impact of polyploidy on the transcriptome of cancer cells and MSC of various origins (bone marrow, placenta, and heart). First, we identified genes that are consistently ploidy-induced or ploidy-repressed through all comparisons. Then, we selected the master regulators using the protein interaction enrichment analysis (PIEA). The obtained ploidy-related gene signatures were verified using the data gained from polyploid and diploid populations of early cardiomyocytes (CARD) originating from iPSC. The multistep bioinformatic analysis applied to the cancer cells, MSC, and CARD indicated that polyploidy plays a pivotal role in driving the cell into hypertranscription. It was evident from the upregulation of gene modules implicated in housekeeping functions, stemness, unicellularity, DNA repair, and chromatin opening by means of histone acetylation operating via DNA damage associated with the NUA4/TIP60 complex. These features were complemented by the activation of the pathways implicated in centrosome maintenance and ciliogenesis and by the impairment of the pathways related to apoptosis, the circadian clock, and immunity. Overall, our findings suggest that, although polyploidy does not induce oncologic transformation of MSC, it might compromise their therapeutic properties because of global epigenetic changes and alterations in fundamental biological processes. The obtained results can contribute to the development and implementation of approaches enhancing the therapeutic properties of MSC by removing polyploid cells from the cell population.

Securin acetylation prevents precocious separase activation and premature sister chromatid separation.

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

An iron rheostat controls hematopoietic stem cell fate.

Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.

Evidence of Histone H2A.Z Deacetylation and Cardiomyocyte Dedifferentiation in Infarcted/Tip60-depleted Hearts.

Myocardial infarction (MI) in the human heart causes death of billions of cardiomyocytes (CMs), resulting in cardiac dysfunction that is incompatible with life or lifestyle. In order to re-muscularize injured myocardium, replacement CMs must be generated via renewed proliferation of surviving CMs. Approaches designed to induce proliferation of CMs after injury have been insufficient. Toward this end, we are targeting the Tip60 acetyltransferase, based on the rationale that its pleiotropic functions conspire to block the CM cell-cycle at several checkpoints. We previously reported that genetic depletion of Tip60 in a mouse model after MI reduces scarring, retains cardiac function, and activates the CM cell-cycle, although it is unclear whether this culminates in the generation of daughter CMs. For pre-existing CMs in the adult heart to resume proliferation, it is becoming widely accepted that they must first dedifferentiate, a process highlighted by loss of maturity, epithelial to mesenchymal transitioning (EMT), and reversion from fatty acid oxidation to glycolytic metabolism, accompanied by softening of the myocardial extracellular matrix. Findings in hematopoietic stem cells, and more recently in neural progenitor cells, have shown that Tip60 induces and maintains the differentiated state via site-specific acetylation of the histone variant H2A.Z. Here, we report that genetic depletion of Tip60 from naïve or infarcted hearts results in the near-complete absence of acetylated H2A.Z in CM nuclei, and that this is accordingly accompanied by altered gene expressions indicative of EMT induction, ECM softening, decreased fatty acid oxidation, and depressed expression of genes that regulate the TCA cycle. These findings, combined with our previous work, support the notion that because Tip60 has multiple targets that combinatorially maintain the differentiated state and inhibit proliferation, its transient therapeutic targeting to ameliorate the effects of cardiac injury should be considered.

Combination of Resveratrol and PARP inhibitor Olaparib efficiently deregulates homologous recombination repair pathway in breast cancer cells through inhibition of TIP60-mediated chromatin relaxation.

Recently, we reported that a combination of a natural, bioactive compound Resveratrol (RES) and a PARP inhibitor Olaparib (OLA) deregulated the homologous recombination (HR) pathway, and enhanced apoptosis in BRCA1-wild-type, HR-proficient breast cancer cells. Upon DNA damage, chromatin relaxation takes place, which allows the DNA repair proteins to access the DNA lesion. But whether chromatin remodeling has any role in RES + OLA-mediated HR inhibition is not known. By using in vitro and ex vivo model systems of breast cancer, we have investigated whether RES + OLA inhibits chromatin relaxation and thereby blocks the HR pathway. It was found that RES + OLA inhibited PARP1 activity, terminated PARP1-BRCA1 interaction, and deregulated the HR pathway only in the chromatin fraction of MCF-7 cells. DR-GFP reporter plasmid-based HR assay demonstrated marked reduction in HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. RES + OLA efficiently trapped PARP1 at the DNA damage site in the chromatin of MCF-7 cells. Unaltered expressions of HR proteins were found in the chromatin of PARP1-silenced MCF-7 cells, which confirmed that RES + OLA-mediated DNA damage response was PARP1-dependent. Histone Acetyltransferase (HAT) activity and histone H4 acetylation assays showed reduction in HAT activity and H4 acetylation in RES + OLA-treated chromatin fraction of cells. Western blot analysis showed that the HAT enzyme TIP60, P400 and acetylated H4 were downregulated after RES + OLA exposure. In the co-immunoprecipitation assay, it was observed that RES + OLA caused abolition of PARP1-TIP60-BRCA1 interaction, which suggested the PARP1-dependent TIP60-BRCA1 association. Unaltered expressions of PAR, BRCA1, P400, and acetylated H4 in the chromatin of TIP60-silenced MCF-7 cells further confirmed the role of TIP60 in PARP1-mediated HR activation in the chromatin. Similar results were obtained in ex vivo patient-derived primary breast cancer cells. Thus, the present study revealed that RES + OLA treatment inhibited PARP1 activity in the chromatin, and blocked TIP60-mediated chromatin relaxation, which, in turn, affected PARP1-dependent TIP60-BRCA1 association, resulting in deregulation of HR pathway in breast cancer cells.

BAP18 acting as a novel peroxisome proliferator-activated receptor α co-regulator contributes to hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide with a poor prognosis. The therapeutic outcomes of HCC patients are urgently needed to be improved, and predictive biomarkers for the optimal treatment selection remains to be further defined. In the present study, our results showed that BPTF-associated protein of 18 KDa (BAP18) was highly expressed in HCC tissues. In cultured HCC cells, BAP18 regulated a subset of down-stream genes involved in different functions, particularly including peroxisome proliferator-activated receptor (PPAR) pathway and lipid metabolism. Furthermore, BAP18 co-activated PPARα-mediated transactivation and facilitated the recruitment of nucleosome acetyltransferase of H4 (NuA4)/tat interacting protein 60 (TIP60) complex, thereby increasing histone H4 acetylation on stearoyl-CoA desaturase 1 (SCD1) loci. In addition, BAP18 promoted HCC cell proliferation, increased intracellular lipid levels and enhanced cell survival under the metabolic stress conditions, such as glucose limitation or tyrosine kinase inhibitors (TKIs) treatment. Importantly, higher BAP18 expression was positively correlated with the postoperative recurrence and the poor disease-free survival in clinical patients receiving sorafenib treatment. Altogether, we discovered that BAP18 plays an oncogenic role in the survival and proliferation of HCC cells, and BAP18 may serve as a predictive biomarker for adjunct TKIs treatment in patients with HCC, and further facilitate the precise treatment.

The Tudor-knot Domain of KAT5 Regulates Nucleosomal Substrate Acetylation.

The lysine acetyltransferase KAT5 is a pivotal enzyme responsible for catalyzing histone H4 acetylation in cells. In addition to its indispensable HAT domain, KAT5 also encompasses a conserved Tudor-knot domain at its N-terminus. However, the function of this domain remains elusive, with conflicting findings regarding its role as a histone reader. In our study, we have employed a CRISPR tiling array approach and unveiled the Tudor-knot motif as an essential domain for cell survival. The Tudor-knot domain does not bind to histone tails and is not required for KAT5's chromatin occupancy. However, its absence leads to a global reduction in histone acetylation, accompanied with genome-wide alterations in gene expression that consequently result in diminished cell viability. Mechanistically, we find that the Tudor-knot domain regulates KAT5's HAT activity on nucleosomes by fine-tuning substrate accessibility. In summary, our study uncovers the Tudor-knot motif as an essential domain for cell survival and reveals its critical role in modulating KAT5's catalytic efficiency on nucleosome and KAT5-dependent transcriptional programs critical for cell viability.

3, 3'-diindolylmethane, a natural aryl hydrocarbon receptor agonist, alleviates ulcerative colitis by enhancing "glycolysis-lactate-STAT3″ and TIP60 signals-mediated Treg differentiation.

BACKGROUND: Aryl hydrocarbon receptor (AhR) plays an important role in the occurrence and development of ulcerative colitis (UC). In this study, the effect and mechanism of 3, 3'-diindolylmethane (DIM), the classical AhR agonist, on UC was investigated from the angle of recovering the balance of Th17/Treg. METHODS: The in vivo colitis model was established in mice by using dextran sulfate sodium, and CD4+ T cells were used to simulate the in vitro differentiation of Treg and Th17 cells. The proportions and related factors of Th17 and Treg cells were measured using flow cytometry, Q-PCR and western blotting. The glycolysis was evaluated by examining the glucose uptake, glucose consumption and lactate production using kits or immunofluorescence. The activation of AhR was detected by western blotting and the XRE-luciferase reporter gene. The co-immunoprecipitation, transfection or other methods were selected to investigate and identify the signaling molecular pathway. RESULTS: DIM significantly attenuated symptoms of colitis mice by rebuilding the balance of Th17/Treg in anoxic colons. In hypoxia, a more potent promotion of Treg differentiation was showed by DIM relative to normoxia, and siFoxp3 prevented DIM-suppressed Th17 differentiation. DIM repressed the excessive glycolysis in hypoxia evidenced by down-regulated glucose uptake, lactate production, Glut1 and HK2 levels. Interestingly, IL-10, the function-related factor of Treg cells, showed the feedback effect of DIM-suppressed glycolysis. Besides, 2-deoxy-D-glucose, HK2 plasmid and IL-10 antibody prevented increase of DIM on the expression of Foxp3 at the transcriptional level and subsequent Treg differentiation through the lactate-STAT3 pathway, and reasons for the direct improvement of DIM on Foxp3 protein was attributed to promoting the formation of HIF-1α/TIP60 complexes as well as subsequent acetylation and protein stability. Finally, AhR dependence and mechanisms for DIM-improved Treg differentiation in vitro and in vivo were well confirmed by using plasmids or inhibitors. CONCLUSIONS: DIM enhances activation of AhR and subsequent "glycolysis-lactate-STAT3″ and TIP60 signals-mediated Treg differentiation.

Molecular mechanisms of TWIST1-regulated transcription in EMT and cancer metastasis.

TWIST1 induces epithelial-to-mesenchymal transition (EMT) to drive cancer metastasis. It is yet unclear what determines TWIST1 functions to activate or repress transcription. We found that the TWIST1 N-terminus antagonizes TWIST1-regulated gene expression, cancer growth and metastasis. TWIST1 interacts with both the NuRD complex and the NuA4/TIP60 complex (TIP60-Com) via its N-terminus. Non-acetylated TWIST1-K73/76 selectively interacts with and recruits NuRD to repress epithelial target gene transcription. Diacetylated TWIST1-acK73/76 binds BRD8, a component of TIP60-Com that also binds histone H4-acK5/8, to recruit TIP60-Com to activate mesenchymal target genes and MYC. Knockdown of BRD8 abolishes TWIST1 and TIP60-Com interaction and TIP60-Com recruitment to TWIST1-activated genes, resulting in decreasing TWIST1-activated target gene expression and cancer metastasis. Both TWIST1/NuRD and TWIST1/TIP60-Com complexes are required for TWIST1 to promote EMT, proliferation, and metastasis at full capacity. Therefore, the diacetylation status of TWIST1-K73/76 dictates whether TWIST1 interacts either with NuRD to repress epithelial genes, or with TIP60-Com to activate mesenchymal genes and MYC. Since BRD8 is essential for TWIST1-acK73/76 and TIP60-Com interaction, targeting BRD8 could be a means to inhibit TWIST1-activated gene expression.

Experience-dependent Tip60 nucleocytoplasmic transport is regulated by its NLS/NES sequences for neuroplasticity gene control.

Nucleocytoplasmic transport (NCT) in neurons is critical for enabling proteins to enter the nucleus and regulate plasticity genes in response to environmental cues. Such experience-dependent (ED) neural plasticity is central for establishing memory formation and cognitive function and can influence the severity of neurodegenerative disorders like Alzheimer's disease (AD). ED neural plasticity is driven by histone acetylation (HA) mediated epigenetic mechanisms that regulate dynamic activity-dependent gene transcription profiles in response to neuronal stimulation. Yet, how histone acetyltransferases (HATs) respond to extracellular cues in the in vivo brain to drive HA-mediated activity-dependent gene control remains unclear. We previously demonstrated that extracellular stimulation of rat hippocampal neurons in vitro triggers Tip60 HAT nuclear import with concomitant synaptic gene induction. Here, we focus on investigating Tip60 HAT subcellular localization and NCT specifically in neuronal activity-dependent gene control by using the learning and memory mushroom body (MB) region of the Drosophila brain as a powerful in vivo cognitive model system. We used immunohistochemistry (IHC) to compare the subcellular localization of Tip60 HAT in the Drosophila brain under normal conditions and in response to stimulation of fly brain neurons in vivo either by genetically inducing potassium channels activation or by exposure to natural positive ED conditions. Furthermore, we found that both inducible and ED condition-mediated neural induction triggered Tip60 nuclear import with concomitant induction of previously identified Tip60 target genes and that Tip60 levels in both the nucleus and cytoplasm were significantly decreased in our well-characterized Drosophila AD model. Mutagenesis of a putative nuclear localization signal (NLS) sequence and nuclear export signal (NES) sequence that we identified in the Drosophila Tip60 protein revealed that both are functionally required for appropriate Tip60 subcellular localization. Our results support a model by which neuronal stimulation triggers Tip60 NCT via its NLS and NES sequences to promote induction of activity-dependent neuroplasticity gene transcription and that this process may be disrupted in AD.

A Boolean network model of the double-strand break repair pathway choice.

Double strand break (DSB) repair is critical to maintaining the integrity of the genome. DSB repair deficiency underlies multiple pathologies, including cancer, chromosome instability syndromes, and, potentially, neurodevelopmental defects. DSB repair is mainly handled by two pathways: highly accurate homologous recombination (HR), which requires a sister chromatid for template-based repair, limited to S/G2 phases of the cell cycle, and canonical non-homologous end joining (c-NHEJ), available throughout the cell cycle in which minimum homology is sufficient for highly efficient yet error-prone repair. Some circumstances, such as cancer, require alternative highly mutagenic DSB repair pathways like microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA), which are triggered to attend to DNA damage. These non-canonical repair alternatives are emerging as prominent drivers of resistance in drug-based tumor therapies. Multiple DSB repair options require tight inter-pathway regulation to prevent unscheduled activities. In addition to this complexity, epigenetic modifications of the histones surrounding the DSB region are emerging as critical regulators of the DSB repair pathway choice. Modeling approaches to understanding DSBs repair pathway choice are advantageous to perform simulations and generate predictions on previously uncharacterized aspects of DSBs response. In this work, we present a Boolean network model of the DSB repair pathway choice that incorporates the knowledge, into a dynamic system, of the inter-pathways regulation involved in DSB repair, i.e., HR, c-NHEJ, SSA, and MMEJ. Our model recapitulates the well-characterized HR activity observed in wild-type cells in response to DSBs. It also recovers clinically relevant behaviors of BRCA1/FANCS mutants, and their corresponding drug resistance mechanisms ascribed to DNA repair gain-of-function pathogenic variants. Since epigenetic modifiers are dynamic and possible druggable targets, we incorporated them into our model to better characterize their involvement in DSB repair. Our model predicted that loss of the TIP60 complex and its corresponding histone acetylation activity leads to activation of SSA in response to DSBs. Our experimental validation showed that TIP60 effectively prevents activation of RAD52, a key SSA executor, and confirms the suitable use of Boolean network modeling for understanding DNA DSB repair.

Knockdown of DOM/Tip60 Complex Subunits Impairs Male Meiosis of Drosophila melanogaster.

ATP-dependent chromatin remodeling complexes are involved in nucleosome sliding and eviction and/or the incorporation of histone variants into chromatin to facilitate several cellular and biological processes, including DNA transcription, replication and repair. The DOM/TIP60 chromatin remodeling complex of Drosophila melanogaster contains 18 subunits, including the DOMINO (DOM), an ATPase that catalyzes the exchange of the canonical H2A with its variant (H2A.V), and TIP60, a lysine-acetyltransferase that acetylates H4, H2A and H2A.V histones. In recent decades, experimental evidence has shown that ATP-dependent chromatin remodeling factors, in addition to their role in chromatin organization, have a functional relevance in cell division. In particular, emerging studies suggested the direct roles of ATP-dependent chromatin remodeling complex subunits in controlling mitosis and cytokinesis in both humans and D. melanogaster. However, little is known about their possible involvement during meiosis. The results of this work show that the knockdown of 12 of DOM/TIP60 complex subunits generates cell division defects that, in turn, cause total/partial sterility in Drosophila males, providing new insights into the functions of chromatin remodelers in cell division control during gametogenesis.

The effect of silencing the Tip60 gene on the response to radiotherapy in breast cancer cells.

Since patients with triple-negative breast cancer do not respond to hormone therapy, the main treatment method is the combination of chemotherapy and radiotherapy. Because the DNA of the tumor cell is the target in both some chemotherapeutics and radiotherapy, problems may occur in individuals with a high DNA repair pathway. It is suggested that high expression of the Tip60 gene, which has an important role in repairing DNA damage, will increase the repair of DNA double-strand breaks in tumor cells, especially during radiotherapy treatment, thus reducing the response to treatment and adversely affecting treatment. In this study, for the first time, the role of the silenced and active Tip60 gene in response to radiotherapy in MDA-MB-231 and MCF-7 cells was investigated. For this purpose, the Tip60 gene was silenced by applying siRNA to the cell lines and UV was applied. In the study, cytotoxicity and DNA breaks were measured by MTT and COMET methods, and mRNA and protein expression values were measured by PCR and Raman spectrophotometer in silenced, unsilenced, UV-treated, and non-UV-treated cell lines. According to the results of the study, increased DNA damage was observed in MCF-7 cell lines in which the Tip60 gene was silenced, and radiotherapy was applied, compared to the cell lines with the Tip60 gene active. It was observed that DNA damage in MDA-MB-231 cell lines was less than in cell lines with the active Tip60 gene.

TIP60 is required for tumorigenesis in non-small cell lung cancer.

Histone modifications play crucial roles in transcriptional activation, and aberrant epigenetic changes are associated with oncogenesis. Lysine (K) acetyltransferases 5 (TIP60, also known as KAT5) is reportedly implicated in cancer development and maintenance, although its function in lung cancer remains controversial. Here we demonstrate that TIP60 knockdown in non-small cell lung cancer cell lines decreased tumor cell growth, migration, and invasion. Furthermore, analysis of a mouse lung cancer model with lung-specific conditional Tip60 knockout revealed suppressed tumor formation relative to controls, but no apparent effects on normal lung homeostasis. RNA-seq and ChIP-seq analyses of inducible TIP60 knockdown H1975 cells relative to controls revealed transglutaminase enzyme (TGM5) as downstream of TIP60. Investigation of a connectivity map database identified several candidate compounds that decrease TIP60 mRNA, one that suppressed tumor growth in cell culture and in vivo. In addition, TH1834, a TIP60 acetyltransferase inhibitor, showed comparable antitumor effects in cell culture and in vivo. Taken together, suppression of TIP60 activity shows tumor-specific efficacy against lung cancer, with no overt effect on normal tissues. Our work suggests that targeting TIP60 could be a promising approach to treating lung cancer.