Histone Acetyltransferase Tip60 Research Articles

Abstract 1990: TIP60-mediated acetylation of ΔNp63α is associated with cisplatin resistance

Abstract The chemotherapeutic drug Cisplatin is often used to treat squamous cell carcinoma (SCC) patients, but low response rates and disease recurrence are common. About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. ΔNp63α, a member of the p53 family of transcription factors, is overexpressed and considered oncogenic in non-melanoma skin cancer where it regulates cell survival, promotes proliferation, and inhibits cell apoptosis. ΔNp63α has also been shown to promote resistance to cisplatin by orchestrating the transcriptional regulation of several DNA damage response (DDR) genes. Our previous study showed that the histone acetyltransferase (HAT) TIP60 promotes SCC proliferation by positively regulating ΔNp63α protein levels in a manner reliant on the catalytic activity of TIP60. This observation suggested that TIP60 may contribute to the failure of platinum-based drugs in SCC and led us to hypothesize that TIP60-mediated acetylation of ΔNp63α regulates its stability and transcriptional activity to promote chemoresistance. We found that TIP60 levels positively correlated with ΔNp63α stability, protein levels, and cisplatin resistance. Further, silencing endogenous TIP60 led to a decrease in ΔNp63α transcript and protein levels in multiple cisplatin resistant SCC cell lines. Further, stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α or TIP60 induced G2/M arrest, increased cell death, and sensitized cells to cisplatin. Additionally, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α, decreased colony formation, and sensitized resistant cells to cisplatin. Finally, we demonstrated that ΔNp63α and TIP60 levels positively correlated with DNA repair capacity and negatively correlated with cisplatin-DNA adduct formation. Silencing of either TIP60 or ΔNp63α intensified cisplatin-DNA adduct formation and significantly reduced the expression of drug efflux transporters. Taken together, our data indicate that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance in SCC. Our findings suggest that the inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC and other epithelial cancers. Citation Format: Akshay Hira, Jin Zhang, Mike Kemp, Madhavi P. Kadakia. TIP60-mediated acetylation of ΔNp63α is associated with cisplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1990.

Novel Tip60 HAT activators as neuroepigenetic therapeutic modulators for Alzheimer’s Disease

AbstractBackgroundAlzheimer’s Disease (AD) etiology involves a complex interplay of genetics, age, and environmental factors that influence neuroepigenetic mediated neural gene control. Reduced histone acetylation in the AD brain is a widespread hallmark underlying neural gene repression and cognitive impairment in AD etiology. As histone acetylation is dynamically regulated by antagonistic histone deacetylases (HDACs) and histone acetyltransferases (HATs), pharmacological treatments aiming to restore histone acetylation via HDAC inhibition are a research hotspot for developing AD cognitive enhancing drugs. Although promising, HDAC inhibitor treatments in the AD animal models are rarely translated into clinical trials due to non‐specific global hyperacetylation. Alternatively, enhancing activity of specific HATs with non‐redundant functions serves as an exciting new therapeutic strategy that remains unexplored. To this end, our lab has identified and published a neuroprotective role for HAT Tip60 in multiple neural circuits impaired in AD pathology. Human and Drosophila AD brains exhibit reduced Tip60 and enhanced repressor HDAC2 that triggers histone deacetylation with concomitant repression of cognition genes. All these deficits are prevented by genetically increasing Tip60 in the Drosophila AD brain. Here, we test that pharmacologically enhancing Tip60’s HAT function replicates this AD neuroprotective function.MethodWe used in silico structural modeling and a novel pharmacophore based virtual screening approach to design and synthesize novel small molecule compounds strongly predicted to target and enhance Tip60’s HAT activity.ResultOur screen produced 19,000 hits that we individually docked on Tip60’s HAT domain using GOLD docking software. 13 compounds were identified that exhibited significantly high Tip60 docking scores, and optimal ease of synthesis, drug like properties and blood brain barrier peneteration. These compounds were custom synthesized. We show that several of these compounds fully rescue locomotor neural based deficits and cognition gene expression alterations in both Tip60 knockdown and AD Drosophila in vivo models with no observable side effects, underscoring their therapeutic effectiveness. These compounds will ultimately be assessed for further specificity and for their therapeutic effectiveness in AD animal models in vivo.ConclusionWe propose our first‐in‐class small molecule Tip60 HAT activators will serve as powerful novel chemical entities for development of specific acetylation‐based cognition enhancing drugs for AD.

PGE2 alters chromatin through H2A.Z-variant enhancer nucleosome modification to promote hematopoietic stem cell fate

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122, 3074-3081(2013); W. Goessling et al. Cell Stem Cell 8, 445-458 (2011); W. Goessling et al. Cell 136, 1136-1147 (2009)]. Here, we report that PGE2-induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE2, lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

Abstract 400: TIP60-mediated regulation of DNp63a is associated with cisplatin resistance

Abstract About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. The chemotherapeutic drug, Cisplatin is often used to treat squamous cell carcinoma (SCC) patients, but low response rates and disease recurrence is common. ΔNp63α, a member of the p53 family of transcription factors, is overexpressed and considered oncogenic in non-melanoma skin cancer where it regulates cell survival, promotes proliferation and inhibits cell apoptosis. ΔNp63α has also been shown to promote resistance to cisplatin by transcriptionally regulating several DNA damage response (DDR) genes. A previous study from our lab showed that the histone acetyltransferase (HAT) TIP60 promotes SCC proliferation by positively regulating ΔNp63α protein levels in manner dependent on the catalytic activity of TIP60. This finding suggested that TIP60 may contribute to the failure of platinum-based drugs in SCC and led us to hypothesize that TIP60-mediated acetylation of ΔNp63α regulates its stability and transcriptional activity to promote chemoresistance. Silencing endogenous TIP60 led to a decrease in ΔNp63α transcript and protein levels in multiple SCC cell lines, indicating the positive regulation of ΔNp63α by TIP60 is not cell-line specific. Further, TIP60 levels positively correlated with ΔNp63α stability, protein levels and cisplatin resistance. Stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α and TIP60 induced G2/M arrest, increased cell-death and sensitized cells to cisplatin. Moreover, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α and sensitized resistant cells to cisplatin. Finally, we demonstrated that ΔNp63α and TIP60 levels positively correlated with DNA repair capacity and negatively correlated with cisplatin-DNA adduct formation. Silencing of either TIP60 or ΔNp63α enhanced cisplatin-DNA adduct formation and significantly reduced expression of genes involved in DDR. Taken together, our data indicate that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance through regulation of genes involved in DNA damage repair. Our findings suggest that inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC other epithelial cancers. Citation Format: Akshay Hira, Andrew J. Stacy, Jin Zhang, Mike Kemp, Madhavi P. Kadakia. TIP60-mediated regulation of DNp63a is associated with cisplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 400.

Tip60's Novel RNA-Binding Function Modulates Alternative Splicing of Pre-mRNA Targets Implicated in Alzheimer's Disease.

The severity of Alzheimer's disease (AD) progression involves a complex interplay of genetics, age, and environmental factors orchestrated by histone acetyltransferase (HAT)-mediated neuroepigenetic mechanisms. While disruption of Tip60 HAT action in neural gene control is implicated in AD, alternative mechanisms underlying Tip60 function remain unexplored. Here, we report a novel RNA binding function for Tip60 in addition to its HAT function. We show that Tip60 preferentially interacts with pre-mRNAs emanating from its chromatin neural gene targets in the Drosophila brain and this RNA binding function is conserved in human hippocampus and disrupted in Drosophila brains that model AD pathology and in AD patient hippocampus of either sex. Since RNA splicing occurs co-transcriptionally and alternative splicing (AS) defects are implicated in AD, we investigated whether Tip60-RNA targeting modulates splicing decisions and whether this function is altered in AD. Replicate multivariate analysis of transcript splicing (rMATS) analysis of RNA-Seq datasets from wild-type and AD fly brains revealed a multitude of mammalian-like AS defects. Strikingly, over half of these altered RNAs are identified as bona-fide Tip60-RNA targets that are enriched for in the AD-gene curated database, with some of these AS alterations prevented against by increasing Tip60 in the fly brain. Further, human orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brains, implicating disruption of Tip60's splicing function in AD pathogenesis. Our results support a novel RNA interaction and splicing regulatory function for Tip60 that may underly AS impairments that hallmark AD etiology.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) has recently emerged as a hotbed for RNA alternative splicing (AS) defects that alter protein function in the brain yet causes remain unclear. Although recent findings suggest convergence of epigenetics with co-transcriptional AS, whether epigenetic dysregulation in AD pathology underlies AS defects remains unknown. Here, we identify a novel RNA interaction and splicing regulatory function for Tip60 histone acetyltransferase (HAT) that is disrupted in Drosophila brains modeling AD pathology and in human AD hippocampus. Importantly, mammalian orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brain. We propose that Tip60-mediated AS modulation is a conserved critical posttranscriptional step that may underlie AS defects now characterized as hallmarks of AD.

The roles of ING5 in cancer: A tumor suppressor.

As a Class II tumor suppressor, ING5 contains nuclear localization signal, plant homeodomain, novel conserved region, and leucine zipper-like domains. ING5 proteins form homodimer into a coil-coil structure, and heterodimers with ING4, histone H3K4me3, histone acetyltransferase (HAT) complex, Tip60, Cyclin A1/CDK2, INCA1 and EBNA3C for the transcription of target genes. The acetylated proteins up-regulated by ING5 are preferentially located in nucleus and act as transcription cofactors, chromatin and DNA binding functions, while those down-regulated by ING5 mostly in cytoplasm and contribute to metabolism. ING5 promotes the autoacetylation of HAT p300, p53, histone H3 and H4 for the transcription of downstream genes (Bax, GADD45, p21, p27 and so forth). Transcriptionally, YY1 and SRF up-regulate ING5 mRNA expression by the interaction of YY1-SRF-p53-ING5 complex with ING5 promoter. Translationally, ING5 is targeted by miR-196, miR-196a, miR-196b-5p, miR-193a-3p, miR-27-3p, miR-200b/200a/429, miR-1307, miR-193, miR-222, miR-331-3p, miR-181b, miR-543 and miR-196-b. ING5 suppresses proliferation, migration, invasion and tumor growth of various cancer cells via the suppression of EGFR/PI3K/Akt, IL-6/STAT3, Akt/NF-κB/NF-κB/MMP-9 or IL-6/CXCL12 pathway. ING5-mediated chemoresistance is closely linked to anti-apoptosis, overexpression of chemoresistant genes, the activation of PI3K/Akt/NF-κB and Wnt/β-catenin signal pathways. Histologically, ING5 abrogation in gastric stem-like and pdx1-positive cells causes gastric dysplasia and cancer, and conditional ING5 knockout in pdx1-positive and gastric chief cells increases MNU-induced gastric carcinogenesis. Intestinal ING5 deletion increases AOM/DSS- induced colorectal carcinogenesis and decreases high-fat-diet weight. The overexpression and nucleocytoplasmic translocation of ING5 are seen during carcinogenesis, and ING5 expression was inversely associated with aggressive behaviors and poor prognosis in a variety of cancers. These findings indicated that ING5 might be used for a molecular marker for carcinogenesis and following progression, and as a target for gene therapy if its chemoresistant function might be ameliorated.

EP400NL is involved in PD-L1 gene activation by forming a transcriptional coactivator complex

EP400 is an ATP-dependent chromatin remodelling enzyme that regulates DNA double-strand break repair and transcription, including cMyc-dependent gene expression. We previously showed that the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. As the EP400 N-terminal-Like (EP400NL) gene resides next to the EP400 gene locus, this prompted us to investigate whether EP400NL plays a similar role in transcriptional regulation to the full-length EP400 protein. We found that EP400NL forms a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, this EP400NL complex displays H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. We demonstrated that the transcriptional coactivator function of EP400NL is required for serum and IFNγ-induced PD-L1 gene activation. Furthermore, transcriptome analysis indicates that EP400NL contributes to cMyc-responsive mitochondrial biogenesis. Taken together, our studies show that EP400NL plays a role as a transcription coactivator of PD-L1 gene regulation and provides a potential target to modulate cMyc functions in cancer therapy.

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.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling.

FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated in both epithelial and natural killer (NK) cells. The histone acetyltransferase TIP60 contributes to the acetylation of SUPT16H middle domain (MD) at lysine 674 (K674). Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H in both epithelial and NK cells. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (HDAC1, EZH2), and further regulates their activation status and/or promoter association as well as affects the relevant histone marks (H3ac, H3K9me3 and H3K27me3). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, depletion or inhibition of SUPT16H is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that depletion or inhibition of SUPT16H also causes the remarkable activation of IFN signaling in NK cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in coordinating with BRD4 and regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of the FACT inhibitor CBL0137 to treat viral infections.

RGS7-ATF3-Tip60 Complex Promotes Hepatic Steatosis and Fibrosis by Directly Inducing TNFα.

Aims: The pathophysiological mechanism(s) underlying non-alcoholic fatty liver disease (NAFLD) have yet to be fully delineated and only a single drug, peroxisome proliferator-activated receptor (PPAR) α/γ agonist saroglitazar, has been approved. Here, we sought to investigate the role of Regulator of G Protein Signaling 7 (RGS7) in hyperlipidemia-dependent hepatic dysfunction. Results: RGS7 is elevated in the livers of NAFLD patients, particularly those with severe hepatic damage, pronounced insulin resistance, and high inflammation. In the liver, RGS7 forms a unique complex with transcription factor ATF3 and histone acetyltransferase Tip60, which is implicated in NAFLD. The removal of domains is necessary for ATF3/Tip60 binding compromises RGS7-dependent reactive oxygen species generation and cell death. Hepatic RGS7 knockdown (KD) prevented ATF3/Tip60 induction, and it provided protection against fibrotic remodeling and inflammation in high-fat diet-fed mice translating to improvements in liver function. Hyperlipidemia-dependent oxidative stress and metabolic dysfunction were largely reversed in RGS7 KD mice. Interestingly, saroglitazar failed to prevent RGS7/ATF3 upregulation but it did partially restore Tip60 levels. RGS7 drives the release of particularly tumor necrosis factor α (TNFα) from isolated hepatocytes, stellate cells and its depletion reverses steatosis, oxidative stress by direct TNFα exposure. Conversely, RGS7 overexpression in the liver is sufficient to trigger oxidative stress in hepatocytes that can be mitigated via TNFα inhibition. Innovation: We discovered a novel non-canonical function for an R7RGS protein, which usually functions to regulate G protein coupled receptor (GPCR) signaling. This is the first demonstration for a functional role of RGS7 outside the retina and central nervous system. Conclusion: RGS7 represents a potential novel target for the amelioration of NAFLD. Antioxid. Redox Signal. 38, 137-159.

Retraction Note: Stabilization of the histone acetyltransferase Tip60 by deubiquitinating enzyme USP7 stimulates the release of pro-inflammatory mediators in acute lung injury.

Retraction Note: Stabilization of the histone acetyltransferase Tip60 by deubiquitinating enzyme USP7 stimulates the release of pro-inflammatory mediators in acute lung injury.

HAUSP Is a Key Epigenetic Regulator of the Chromatin Effector Proteins.

HAUSP (herpes virus-associated ubiquitin-specific protease), also known as Ubiquitin Specific Protease 7, plays critical roles in cellular processes, such as chromatin biology and epigenetics, through the regulation of different signaling pathways. HAUSP is a main partner of the “Epigenetic Code Replication Machinery,” ECREM, a large protein complex that includes several epigenetic players, such as the ubiquitin-like containing plant homeodomain (PHD) and an interesting new gene (RING), finger domains 1 (UHRF1), as well as DNA methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), histone methyltransferase G9a, and histone acetyltransferase TIP60. Due to its deubiquitinase activity and its ability to team up through direct interactions with several epigenetic regulators, mainly UHRF1, DNMT1, TIP60, the histone lysine methyltransferase EZH2, and the lysine-specific histone demethylase LSD1, HAUSP positions itself at the top of the regulatory hierarchies involved in epigenetic silencing of tumor suppressor genes in cancer. This review highlights the increasing role of HAUSP as an epigenetic master regulator that governs a set of epigenetic players involved in both the maintenance of DNA methylation and histone post-translational modifications.

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

Amyloid precursor protein elevates fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load

The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates consisting of its adapter protein FE65, the histone acetyltransferase TIP60 and the tumour suppressor proteins p53 and PML. APP C-terminal (APP-CT50) complexes co-localize and co-precipitate with p53 and PML. The PML nuclear body generation is induced and fusion occurs over time depending on APP signalling and STED imaging revealed active gene expression within the complex. We further show that the nuclear aggregates of APP-CT50 fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimer’s disease brains reveal a highly significant reduction of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT50 signalling to the nucleus takes place in the aged human brain and is involved in the pathophysiology of AD.

JAZF1, A Novel p400/TIP60/NuA4 Complex Member, Regulates H2A.Z Acetylation at Regulatory Regions.

Histone variants differ in amino acid sequence, expression timing and genomic localization sites from canonical histones and convey unique functions to eukaryotic cells. Their tightly controlled spatial and temporal deposition into specific chromatin regions is accomplished by dedicated chaperone and/or remodeling complexes. While quantitatively identifying the chaperone complexes of many human H2A variants by using mass spectrometry, we also found additional members of the known H2A.Z chaperone complexes p400/TIP60/NuA4 and SRCAP. We discovered JAZF1, a nuclear/nucleolar protein, as a member of a p400 sub-complex containing MBTD1 but excluding ANP32E. Depletion of JAZF1 results in transcriptome changes that affect, among other pathways, ribosome biogenesis. To identify the underlying molecular mechanism contributing to JAZF1’s function in gene regulation, we performed genome-wide ChIP-seq analyses. Interestingly, depletion of JAZF1 leads to reduced H2A.Z acetylation levels at > 1000 regulatory sites without affecting H2A.Z nucleosome positioning. Since JAZF1 associates with the histone acetyltransferase TIP60, whose depletion causes a correlated H2A.Z deacetylation of several JAZF1-targeted enhancer regions, we speculate that JAZF1 acts as chromatin modulator by recruiting TIP60’s enzymatic activity. Altogether, this study uncovers JAZF1 as a member of a TIP60-containing p400 chaperone complex orchestrating H2A.Z acetylation at regulatory regions controlling the expression of genes, many of which are involved in ribosome biogenesis.

Pontin arginine methylation by CARM1 is crucial for epigenetic regulation of autophagy

Autophagy is a catabolic process through which cytoplasmic components are degraded and recycled in response to various stresses including starvation. Recently, transcriptional and epigenetic regulations of autophagy have emerged as essential mechanisms for maintaining homeostasis. Here, we identify that coactivator-associated arginine methyltransferase 1 (CARM1) methylates Pontin chromatin-remodeling factor under glucose starvation, and methylated Pontin binds Forkhead Box O 3a (FOXO3a). Genome-wide analyses and biochemical studies reveal that methylated Pontin functions as a platform for recruiting Tip60 histone acetyltransferase with increased H4 acetylation and subsequent activation of autophagy genes regulated by FOXO3a. Surprisingly, CARM1-Pontin-FOXO3a signaling axis can work in the distal regions and activate autophagy genes through enhancer activation. Together, our findings provide a signaling axis of CARM1-Pontin-FOXO3a and further expand the role of CARM1 in nuclear regulation of autophagy.

Tip60 protects against amyloid-β-induced transcriptomic alterations via different modes of action in early versus late stages of neurodegeneration

Alzheimer's disease (AD) is an age-related neurodegenerative disorder hallmarked by amyloid-β (Aβ) plaque accumulation, neuronal cell death, and cognitive deficits that worsen during disease progression. Histone acetylation dysregulation, caused by an imbalance between reduced histone acetyltransferases (HAT) Tip60 and increased histone deacetylase 2 (HDAC2) levels, can directly contribute to AD pathology. However, whether such AD-associated neuroepigenetic alterations occur in response to Aβ peptide production and can be protected against by increasing Tip60 levels over the course of neurodegenerative progression remains unknown. Here we profile Tip60 HAT/HDAC2 dynamics and transcriptome-wide changes across early and late stage AD pathology in the Drosophila brain produced solely by human amyloid-β42. We show that early Aβ42 induction leads to disruption of Tip60 HAT/HDAC2 balance during early neurodegenerative stages preceding Aβ plaque accumulation that persists into late AD stages. Correlative transcriptome-wide studies reveal alterations in biological processes we classified as transient (early-stage only), late-onset (late-stage only), and constant (both). Increasing Tip60 HAT levels in the Aβ42 fly brain protects against AD functional pathologies that include Aβ plaque accumulation, neural cell death, cognitive deficits, and shorter life-span. Strikingly, Tip60 protects against Aβ42-induced transcriptomic alterations via distinct mechanisms during early and late stages of neurodegeneration. Our findings reveal distinct modes of neuroepigenetic gene changes and Tip60 neuroprotection in early versus late stages in AD that can serve as early biomarkers for AD, and support the therapeutic potential of Tip60 over the course of AD progression.

Digging Deeper into Breast Cancer Epigenetics: Insights from Chemical Inhibition of Histone Acetyltransferase TIP60 In Vitro.

Breast cancer is often sporadic due to several factors. Among them, the deregulation of epigenetic proteins may be involved. TIP60 or KAT5 is an acetyltransferase that regulates gene transcription through the chromatin structure. This pleiotropic protein acts in several cellular pathways by acetylating proteins. RNA and protein expressions of TIP60 were shown to decrease in some breast cancer subtypes, particularly in triple-negative breast cancer (TNBC), where a low expression of TIP60 was exhibited compared with luminal subtypes. In this study, the inhibition of the residual activity of TIP60 in breast cancer cell lines was investigated by using two chemical inhibitors, TH1834 and NU9056, first on the acetylation of the specific target, lysine 4 of histone 3 (H3K4) by immunoblotting, and second, by chromatin immunoprecipitation (ChIP)-qPCR (-quantitative Polymerase Chain Reaction). Subsequently, significant decreases or a trend toward decrease of H3K4ac in the different chromatin compartments were observed. In addition, the expression of 48 human nuclear receptors was studied with TaqMan Low-Density Array in these breast cancer cell lines treated with TIP60 inhibitors. The statistical analysis allowed us to comprehensively characterize the androgen receptor and NR3C2 receptors in TNBC cell lines after TH1834 or NU9056 treatment. The understanding of the residual activity of TIP60 in the evolution of breast cancer might be a major asset in the fight against this disease, and could allow TIP60 to be used as a biomarker or therapeutic target for breast cancer progression in the future.

Tip60 Phosphorylation at Ser 99 Is Essential for Autophagy Induction in Bombyx mori.

Tip60, a key histone acetyltransferase of the MYST family and member of the nuclear multimeric protein complex (NuA4), regulates the activity and stability of proteins involved in the cell cycle, DNA damage responses, autophagy, etc. However, the function and regulatory mechanism of Tip60 homolog in Bombyx mori are not elucidated. In the present study, Bombyx Tip60 (BmTip60) was functionally identified. Developmental profiles showed that the protein levels and nuclear localization of BmTip60 peaked in fat body during the larval–pupal metamorphosis when autophagy was intensive; simultaneously, the BmTip60 protein migrated to form an upper band as detected by Western blot. Interestingly, the upper band of BmTip60 was reduced by λ-phosphatase treatment, indicating that it was a phosphorylated form of BmTip60. Results showed that BmTip60 was promoted by starvation but not 20-hydroxyecdysone treatment. Transcription factor AMP-activated protein kinase (AMPK) affected by starvation was pivotal for BmTip60 protein migration. In addition, one mammalian phosphorylation site was identified in BmTip60 at Ser99, the constitutive-activation mutation of Ser99 to Asp99 but not its inactive mutation to Ala99 significantly upregulated autophagy, showing the critical role of phosphorylation at Ser99 for BmTip60-mediated autophagy. In conclusion, the starvation-AMPK axis promotes BmTip60 in B. mori, which was requisite for autophagy induction. These results reveal a regulatory mechanism of histone acetyltransferase Tip60 homologs by phosphorylation in insects, and sheds light on further related studies of acetylation regulation.

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.