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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.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/imunologia , Células Enteroendócrinas/metabolismo , Microbioma Gastrointestinal/imunologia , Histona Acetiltransferases/metabolismo , Intestinos/imunologia , Acetatos/imunologia , Acetilcoenzima A/metabolismo , Animais , Montagem e Desmontagem da Cromatina/fisiologia , Drosophila melanogaster/microbiologia , Ecdisona/metabolismo , Imunidade Inata/imunologia , Intestinos/microbiologia , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Interferência de RNA , Transdução de Sinais/imunologia , Taquicininas/metabolismoRESUMO
Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.
Assuntos
Cromatina , Histonas , Histonas/genética , Histonas/metabolismo , Acetilação , Ativação Transcricional , Cromatina/genética , Processamento de Proteína Pós-Traducional , NucleossomosRESUMO
In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.
Assuntos
Proteínas de Ligação a Telômeros , Telômero , Animais , Regulação da Expressão Gênica , Genoma , Mamíferos/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.
Assuntos
Neoplasias do Endométrio , Sarcoma do Estroma Endometrial , Sarcoma , Cromatina , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Histonas/metabolismo , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Sarcoma/genética , Sarcoma do Estroma Endometrial/genética , Sarcoma do Estroma Endometrial/metabolismo , Sarcoma do Estroma Endometrial/patologia , Translocação Genética/genéticaRESUMO
mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism.
Assuntos
Autofagossomos/enzimologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Quinase 3 da Glicogênio Sintase/metabolismo , Metabolismo dos Lipídeos , Fígado/enzimologia , Lisina Acetiltransferase 5/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Transativadores/metabolismo , Acetilação , Animais , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Quinase 3 da Glicogênio Sintase/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Gotículas Lipídicas/metabolismo , Fígado/patologia , Lisina Acetiltransferase 5/genética , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Fosfato/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Transdução de Sinais , Transativadores/genética , Proteínas Supressoras de TumorRESUMO
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.
RESUMO
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.
Assuntos
Neoplasias , Humanos , Neoplasias/genética , Transição Epitelial-Mesenquimal/genética , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization.
Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Histona Acetiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Reparo de DNA por Recombinação , ATPases Associadas a Diversas Atividades Celulares , Acetilação , Animais , Arginina , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/genética , DNA Helicases/genética , Instabilidade Genômica , Células HEK293 , Células HeLa , Histona Acetiltransferases/genética , Histonas/metabolismo , Humanos , Lisina Acetiltransferase 5 , Metilação , Camundongos , Camundongos Transgênicos , Proteína-Arginina N-Metiltransferases/genética , Interferência de RNA , Fatores de Tempo , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs, and Prms. Here, by studying spermatogenesis in the absence of a spermatid-specific histone variant, H2A.L.2, we discover a fundamental mechanism involved in the transformation of nucleosomes into nucleoprotamines. H2A.L.2 is synthesized at the same time as TPs and enables their loading onto the nucleosomes. TPs do not displace histones but rather drive the recruitment and processing of Prms, which are themselves responsible for histone eviction. Altogether, the incorporation of H2A.L.2 initiates and orchestrates a series of successive transitional states that ultimately shift to the fully compacted genome of the mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should be revisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Protaminas/metabolismo , Espermatogênese , Espermatozoides/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Biologia Computacional , Bases de Dados Genéticas , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Genoma , Histonas/deficiência , Histonas/genética , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Nucleossomos/genética , Fenótipo , Espermatogênese/genética , Espermatozoides/patologia , TransfecçãoRESUMO
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.
Assuntos
Doença de Alzheimer , Proteínas de Drosophila , Animais , Humanos , Doença de Alzheimer/metabolismo , Proteínas de Drosophila/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Alternativo/genética , DNA Recombinante/metabolismo , Drosophila/fisiologia , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , MamíferosRESUMO
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.
RESUMO
H2AX is a histone H2A variant that becomes phosphorylated upon genotoxic stress. The phosphorylated H2AX (γ-H2AX) plays an antioncogenic role in the DNA damage response and its foci patterns are highly variable, in terms of intensities and sizes. However, whether characteristic γ-H2AX foci patterns are associated with oncogenesis (oncogenic-specific γ-H2AX foci patterns) remains unknown. We previously reported that a defect in the acetyltransferase activity of TIP60 promotes cancer cell growth in human cell lines. In this study, we compared γ-H2AX foci patterns between TIP60 wild-type cells and TIP60 HAT mutant cells by using machine learning. When focused solely on the intensity and size of γ-H2AX foci, we extracted the TIP60 HAT mutant-like oncogenic-specific γ-H2AX foci pattern among all datasets of γ-H2AX foci patterns. Furthermore, by using the dimensionality reduction method UMAP, we also observed TIP60 HAT mutant-like oncogenic-specific γ-H2AX foci patterns in TIP60 wild-type cells. In summary, we propose the existence of an oncogenic-specific γ-H2AX foci pattern and the importance of a machine learning approach to extract oncogenic signaling among the γ-H2AX foci variations.
Assuntos
Dano ao DNA , Histonas , Humanos , Linhagem Celular , Histonas/metabolismo , Aprendizado de Máquina , FosforilaçãoRESUMO
The long noncoding RNA LINC00839 has been shown to be involved in the progression of some cancer types, such as bladder cancer, prostate cancer, breast cancer, and neuroblastoma. However, if LINC00839 has roles in colorectal cancer (CRC), it has not been elucidated so far. Here, we focus on the biological role and involved mechanisms of LINC00839 in CRC. We show that LINC00839 is selectively upregulated in CRC and locates to the nucleus. High expression of LINC00839 is associated with poor outcomes in CRC patients. Functional experiments show that LINC00839 promotes CRC proliferation, invasion, and metastasis in vitro and in vivo. Mechanistically, LINC00839 recruits Ruvb1 to the Tip60 complex and increases its acetylase activity. LINC00839 guides the complex to the NRF1 promoter and promotes acetylation of lysines 5 and 8 of histones H4, thereby upregulating the expression of NRF1. Subsequently, NRF1 activates mitochondrial metabolism and biogenesis, thereby promoting CRC progression. In summary, our study reports on a mechanism by which LINC00839 positively regulates NRF1, thus promoting mitochondrial metabolism and biogenesis, as well as CRC progression.
Assuntos
Neoplasias Colorretais , RNA Longo não Codificante , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Neoplasias Colorretais/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Lisina Acetiltransferase 5/metabolismo , Masculino , Fator 1 Nuclear Respiratório/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
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.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sítios de Ligação , Ligação Competitiva , Sistemas CRISPR-Cas , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histona Acetiltransferases/genética , Histonas/genética , Humanos , Células K562 , Lisina Acetiltransferase 5 , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , UbiquitinaçãoRESUMO
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.
Assuntos
Doença de Alzheimer , Proteínas de Drosophila , Animais , Ratos , Transporte Ativo do Núcleo Celular , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Regulação da Expressão Gênica , Drosophila/metabolismo , Doença de Alzheimer/metabolismo , Plasticidade Neuronal/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Histona AcetiltransferasesRESUMO
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.
Assuntos
Apoptose , Células-Tronco Mesenquimais , Poliploidia , Transcriptoma , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Apoptose/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Cílios/metabolismo , Cílios/genética , Simulação por Computador , Feminino , Perfilação da Expressão Gênica , Epigênese Genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Biologia Computacional/métodosRESUMO
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.
Assuntos
Apoptose , Proteínas de Drosophila , Fatores de Transcrição Forkhead , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Drosophila/genética , Drosophila/metabolismo , Sistema de Sinalização das MAP Quinases , Humanos , Transdução de Sinais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genéticaRESUMO
Wound healing is a complex phenomenon that requires coordination of numerous molecular and cellular changes to facilitate timely and efficient repair of the damaged tissue. Although many of these molecular pathways have been detailed, others remain to be elucidated. In the present work, we show for the first time, roles for the acetyltransferase TIP60 and nuclear receptor transcription factor PXR in this process, participating in wound healing by altering actin dynamics and cellular motility. We found that in response to wound-injury, TIP60 induces rapid formation of filopodia at the wounded cell front, leading to enhanced cell migration and faster closure of the wound. Further, qPCR analysis revealed heightened expression of Cdc42 and ROCK1 genes, key regulators involved in filopodia formation and actin reorganization, exclusively in TIP60-PXR-expressing cells upon wound-induction. We also performed ChIP assays to confirm the context-specific binding of TIP60 on the ROCK1 promoter and demonstrated that the TIP60 chromodomain is essential for loading of the TIP60-PXR complex onto the chromatin. Results from immunoprecipitation assays revealed that during the wounded condition, TIP60 alters the chromatin microenvironment by specifically acetylating histones H2B and H4, thereby modulating the expression of target genes. Overall, findings of this study show that TIP60 is a novel regulator of the wound healing process by regulating the expression of wound repair-related genes.
Assuntos
Actinas , Lisina Acetiltransferase 5 , Pseudópodes , Acetilação , Actinas/metabolismo , Movimento Celular , Cromatina/metabolismo , Células Hep G2 , Humanos , Lisina Acetiltransferase 5/genética , Lisina Acetiltransferase 5/metabolismo , Pseudópodes/genética , Pseudópodes/metabolismo , Cicatrização , Proteína cdc42 de Ligação ao GTP , Quinases Associadas a rhoRESUMO
Tat-interactive protein 60 kDa (TIP60, also known as lysine acetyltransferase 5 [KAT5]) is a member of the MYST protein family with histone acetyltransferase activity. Recent studies have reported that TIP60 has multiple functions in many signal transduction mechanisms, especially p53-mediated apoptosis. Although the activation of apoptosis signaling pathways requires the presence of cellular reactive oxygen species (ROS) at a certain level, an imbalance between the production and consumption of ROS in cells results in oxidative stress (OS). In this study, we investigated for the first time how the absence of the Tip60 gene in the liver affects gene expression, enzyme activity, and protein expression of the hepatic antioxidant members localized in the cytoplasm, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). First, we successfully generated liver-specific Tip60 knockout mice (mutants) using Cre/LoxP recombination. The reduced glutathione level and nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, a marker of OS, increased significantly in the Tip60 mutant liver. Gene expression, activity, and protein expression of the enzymatic antioxidant system, including SOD, CAT, GR, GPx, and GST were investigated in mutants and control groups. Despite a significant correlation between the gene, enzyme activity, and protein content for CAT and GR, this was not true for SOD and GPx. The overall results suggest that TIP60 acts on the hepatic antioxidant system both at the gene and protein levels, but the actual effect of the deletion of Tip60 is observed at the protein level, especially for SOD and GPx.
Assuntos
Antioxidantes , Fígado , Lisina Acetiltransferase 5 , Estresse Oxidativo , Transativadores , Animais , Camundongos , Antioxidantes/metabolismo , Catalase/genética , Catalase/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Redutase/genética , Lisina Acetiltransferase 5/genética , Lisina Acetiltransferase 5/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fígado/enzimologiaRESUMO
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.