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1.
Cancer Lett ; 553: 215971, 2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36257380

RESUMO

Ovarian cancer (OC) is a malignant tumor that seriously threatens women's health. Due to the difficulty of early diagnosis, most patients exhibit advanced disease or peritoneal metastasis at diagnosis. We discovered that IFFO1 is a novel tumor suppressor, but its role in tumorigenesis, development and chemoresistance is unknown. In this study, IFFO1 levels were downregulated across cancers, leading to the acceleration of tumor development, metastasis and/or cisplatin resistance. Overexpression of IFFO1 inhibited the translocation of ß-catenin to the nucleus and decreased tumor metastasis and cisplatin resistance. Furthermore, we demonstrated that IFFO1 was regulated at both the transcriptional and posttranscriptional levels. At the transcriptional level, the recruitment of HDAC5 inhibited IFFO1 expression, which is mediated by the transcription factor YY1, and the METTL3/YTHDF2 axis regulated the mRNA stability of IFFO1 in an m6A-dependent manner. Mice injected with IFFO1-overexpressing cells had lower ascites volumes and tumor weights throughout the peritoneal cavity than those injected with parental cells expressing the vector control. In conclusion, we demonstrated that IFFO1 is a novel tumor suppressor that inhibits tumor metastasis and reverses drug resistance in ovarian cancer. IFFO1 was downregulated at both the transcriptional level and posttranscriptional level by histone deacetylase and RNA methylation, respectively, and the IFFO1 signaling pathway was identified as a potential therapeutic target for cancer.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas de Filamentos Intermediários , Metiltransferases , Neoplasias Ovarianas , Animais , Feminino , Humanos , Camundongos , Adenosina/farmacologia , Carcinogênese , Cisplatino/farmacologia , Regulação para Baixo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-36029930

RESUMO

Deterioration of inhibitory synapse may be an essential neurological basis underlying abnormal social behaviours. Manipulations that regulate GABAergic transmission are associated with improved behavioural phenotypes in sociability. The synaptic protein, Ephrin-B2 (EB2), plays an important role in the maintenance and reconfiguration of inhibitory synapses in the medial prefrontal cortex (mPFC). However, the inhibitory cell-type specific role of EB2 in the pathophysiology and treatment of social deficits remains unknown. As expected, we revealed that tdTomato-expressing cells were only found in GABAergic neurons instead of excitatory neurons in transgenic EB2-vGATCre mice. This result indicated that depletion of EB2 would occur in those neurons, which further contribute to social deficits. In addition, specific over-expression of mPFC EB2 restored the defective social behaviour abnormalities. These results suggest that the effect of EB2 on social deficits is anatomically and cell-type specific. More importantly, the global upregulation of HDAC4 expression was found in EB2-vGATCre mice. Significant subcellular nuclear shuttling of HDAC4 in vGAT+ neurons was examined and quantified, suggesting a role of nuclear HDAC4 in mediating the mechanism underlying EB2 impairment in vGAT+ neurons. Treatment with LMK235 led to a remarkable rescue of social deficits, thus our data revealed a new domain for the potential utility of HDAC targeting agents to treat social deficits. In conclusion, these results not only revealed a novel molecular mechanism underlying the pathophysiology of social deficits, but also suggested a potential intervention avenue for the treatment of social deficits.


Assuntos
Efrina-B2 , Histona Desacetilases , Animais , Camundongos , Proteínas de Transporte , Efrina-B2/metabolismo , Neurônios GABAérgicos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Camundongos Transgênicos , Mutação , Sinapses/metabolismo
3.
Methods Mol Biol ; 2589: 27-49, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255616

RESUMO

Defined human primary cell model systems with growth dependence on oncogenes are highly requested to investigate tumor pathogenesis and to validate pharmacological inhibitors that specifically target oncoproteins and their executing protein complex partners. In acute myeloid leukemia (AML), transcription factors such as RUNX1 and MLL1, which are important for normal blood cell development, frequently harbor mutations including chromosomal translocations with other coding genes, resulting in tumor-promoting gain-of-function fusion proteins. These oncoproteins completely modify transcriptional programs, thereby inducing malignant cell phenotypes. A common theme of the chimeric gene products is their physical interaction with a variety of chromatin-modifying effector molecules, including histone acetyltransferases (HATs) and histone deacetylases (HDACs). These aberrant multiprotein machineries disturb gene expression and promote malignant cell growth. In this chapter, we briefly summarize the current understanding regarding AML-associated oncogene-driven human CD34+ blood progenitor cell expansion in ex vivo liquid cultures. We provide a step-by-step protocol to establish oncogene-induced human CD34+ blood progenitor cell cultures suitable to analyze the impact of transcriptional repressor/HDAC activity in these human AML cell models.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Leucemia Mieloide Aguda , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Fusão Oncogênica/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Leucemia Mieloide Aguda/genética , Antígenos CD34 , Moléculas de Adesão Celular/genética , Cromatina , Histona Acetiltransferases/genética
4.
Methods Mol Biol ; 2589: 51-73, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255617

RESUMO

Class I histone deacetylases (HDACs) are important regulators of cellular functions in health and disease. HDAC1, HDAC2, HDAC3, and HDAC8 are promising targets for the treatment of cancer, neurological, and immunological disorders. These enzymes have both catalytic and non-catalytic functions in the regulation of gene expression. We here describe the generation of a genetic toolbox by the CRISPR/Cas9 methodology in nearly haploid human tumor cells. This novel model system allows to discriminate between catalytic and structural functions of class I HDAC enzymes and to mimic the treatment with specific HDAC inhibitors.


Assuntos
Inibidores de Histona Desacetilases , Neoplasias , Humanos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteínas Repressoras
5.
Methods Mol Biol ; 2589: 145-155, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255623

RESUMO

Class I histone deacetylase (HDAC) enzymes are key regulators of cell proliferation and are frequently dysregulated in cancer cells. Here we describe the synthesis of a novel series of class-I selective HDAC inhibitors containing anilinobenzamide moieties as ZBG connected with a central (piperazin-1-yl)pyrazine moiety. Compounds were tested in vitro against class-I HDAC1, 2, and 3 isoforms. Some highly potent HDAC inhibitors were obtained and were tested in pancreatic cancer cells and showed promising activity. Moreover, we summarize how the growth-inhibitory effects of these compounds can be determined in murine pancreatic cancer cell lines.


Assuntos
Inibidores de Histona Desacetilases , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Inibidores de Histona Desacetilases/farmacologia , Pirazinas/farmacologia , Linhagem Celular Tumoral , Histona Desacetilases/metabolismo , Proliferação de Células , Isoformas de Proteínas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Relação Estrutura-Atividade , Histona Desacetilase 1/metabolismo
6.
Methods Mol Biol ; 2589: 157-177, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255624

RESUMO

The aberrant activity of histone deacetylases (HDACs) across a broad range of cancers and other disease indications has led to the development of small-molecule inhibitors that target one or more members of the HDAC protein family. Emerging HDAC inhibitors that show promise in drug discovery programs must be assessed across a range of in vitro assays to establish an inhibitor profile for potency and cellular selectivity towards target HDAC(s) as well as preliminary absorption, distribution, metabolism, and excretion (ADME) features. Here we provide an overview of methods to determine a subset of pivotal in vitro drug-like parameters for HDAC inhibitors (HDACi). We initially describe protocols for parallel artificial membrane permeability assays (PAMPA) to evaluate the passive permeability of small molecules across lipid membranes. Subsequently, we elaborate on cytotoxicity assays using CellTiter-Blue to determine HDACi-induced cell death in healthy/diseased cellular models. We next focus on assessing the target engagement of inhibitors with the appropriate HDAC isoforms in a cellular environment via Western blotting of acetylated HDAC substrates. Finally, we provide detailed guidelines on how to assess the metabolic stability of HDACi through whole blood stability assays. Collectively, these assays provide an overview of the permeability, selectivity, and stability of the HDAC inhibitor under development.


Assuntos
Inibidores de Histona Desacetilases , Histona Desacetilases , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Isoformas de Proteínas/metabolismo , Membranas Artificiais , Lipídeos
7.
Methods Mol Biol ; 2589: 179-193, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255625

RESUMO

Histone deacetylases are considered promising epigenetic targets for chemical protein degradation due to their diverse roles in physiological cellular functions and in the diseased state. Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that hijack the cell's ubiquitin-proteasome system (UPS). One of the promising targets for this approach is histone deacetylase 6 (HDAC6), which is highly expressed in several types of cancers and is linked to the aggressiveness of tumors. In the present work, we describe the synthesis of HDAC6 targeting PROTACs based on previously synthesized benzohydroxamates selectively inhibiting HDAC6 and how to assess their activities in different biochemical in vitro assays and in cellular assays. HDAC inhibition was determined using fluorometric assays, while the degradation ability of the PROTACs was assessed using western blot analysis.


Assuntos
Neoplasias , Complexo de Endopeptidases do Proteassoma , Humanos , Desacetilase 6 de Histona/metabolismo , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Quimera/metabolismo , Ubiquitina/metabolismo , Histona Desacetilases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
8.
Methods Mol Biol ; 2589: 303-316, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255633

RESUMO

The class III histone deacetylase (HDACs) also known as sirtuins (SIRTs 1-7) are ubiquitously expressed, but SIRT7 mainly resides as nucleolar protein. In this chapter a couple of methods are described that are used to detect modulation of SIRT7 in response to DNA damage. SIRT7 is localized in the nucleoli and binds to the chromatin after DNA damage. Therefore, a protocol was optimized by our lab for chromatin fractionation. By this method, the movement of SIRT7 can be detected from the soluble part (cytosol+nucleoplasm) to the solid part (chromatin) of the cell. Change of SIRT7 expression levels, in different cells or after different treatment, can be detected by isolating whole-cell lysate followed by Western blotting. For analyzing binding of SIRT7 to other substrates, we have also optimized manual immunoprecipitation assays by using 1% NP40 buffer. This protocol is very helpful to pull down SIRT7 and associated proteins by using a single buffer. SIRT7 is a deacetylase, and its deacetylation activity can be checked both inside the cell by in vivo deacetylation assay and outside the cell by in vitro deacetylation assays. Recently it was also discovered that SIRT7 has desuccinylase activity which can be detected by histone desuccinylation assay. This chapter provides the methodology of SIRT7 detection in the whole cell lysate, binding of SIRT7 to the chromatin and other proteins for performing deacetylation and desuccinylation activity.


Assuntos
Histonas , Sirtuínas , Histonas/metabolismo , Cromatina , Dano ao DNA , Sirtuínas/metabolismo , Histona Desacetilases/metabolismo
9.
Methods Mol Biol ; 2589: 269-291, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255631

RESUMO

Posttranslational modifications are important for protein functions and cellular signaling pathways. The acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), with the latter being grouped into four phylogenetic classes. The class III of the HDAC family, the sirtuins (SIRTs), contributes to gene expression, genomic stability, cell metabolism, and tumorigenesis. Thus, several specific SIRT inhibitors (SIRTi) have been developed to target cancer cell proliferation. Here we provide an overview of methods to study SIRT-dependent cell metabolism and mitochondrial functionality. The chapter describes metabolic flux analysis using Seahorse analyzers, methods for normalization of Seahorse data, flow cytometry and fluorescence microscopy to determine the mitochondrial membrane potential, mitochondrial content per cell and mitochondrial network structures, and Western blot analysis to measure mitochondrial proteins.


Assuntos
Sirtuínas , Sirtuínas/metabolismo , Lisina/metabolismo , Filogenia , Acetilação , Histona Desacetilases/metabolismo , Histona Acetiltransferases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Inibidores de Histona Desacetilases/farmacologia
10.
Methods Mol Biol ; 2589: 401-409, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255639

RESUMO

Dynamic deacetylation of non-histone proteins by histone deacetylases (HDACs) is a key regulator of protein functions, interactions, and turnover. Among class I HDACs, human HDAC1 and HDAC2 share more than 80% global homology at the amino acid level. However, despite the high redundancy, there are examples for differential substrate specificities of HDAC1 and HDAC2. Until now it remains quite unclear how specific and overlapping functions of HDAC1/HDAC2 are regulated in different contexts. Here, we describe molecular cloning techniques for the generation of HDAC1/HDAC2 hybrid proteins, HDAC1/HDAC2 mutants lacking known interaction domains, and HDAC1/HDAC2 hybrid proteins with interchanged N-terminal domains. These proteins are tools for the analysis of specific protein interactions and functions in mammalian cells.


Assuntos
Histona Desacetilase 1 , Histona Desacetilase 2 , Animais , Humanos , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilases/metabolismo , Aminoácidos , Clonagem Molecular , Mamíferos/metabolismo
11.
Methods Mol Biol ; 2589: 411-428, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255640

RESUMO

Protein lysine acylation represents one of the most common post-translational modifications. Obviously, highly reactive metabolic intermediates, like thioesters and mixed anhydrides between phosphoric acid and organic acids, modify lysine residues spontaneously. Additionally, enzymes using acyl-CoAs as co-substrates transfer the acyl residue specifically to defined sequences within proteins. The counteracting enzymes are called histone deacetylases (HDACs), releasing the free lysine side chain. Such enzymatic activities are involved in different cellular processes like tumor progression, immune response, regulation of metabolism, and aging. Modulators of such enzymatic activities represent valuable tools in drug discovery. Therefore, direct and continuous assays to monitor enzymatic activity of HDACs are needed. Here we describe different assay formats allowing both monitoring of Zn2+-dependent HDACs via UV-Vis-spectroscopy and NAD+-dependent HDACs (sirtuins) by fluorescence-based assay formats. Additionally, we describe methods enabling efficient screening of HDAC-inhibitors via fluorescence displacement assays.


Assuntos
Histonas , Sirtuínas , Lisina/metabolismo , NAD/metabolismo , Histona Desacetilases/metabolismo , Sirtuínas/metabolismo , Ácidos Fosfóricos/metabolismo , Anidridos
12.
Methods Mol Biol ; 2589: 361-376, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255637

RESUMO

Experiments determining the chromatin association of histone acetylases (HATs) and deacetylases (HDACs) at the genome-wide level provide precise maps of locus occupancy, but do not allow conclusions on the functional consequences of this locus-specific enrichment. Here we describe a protocol that allows tethering of HATs or HDACs to specific genomic loci upon fusion with a fluorescent protein and a DNA-binding protein such as the E. coli Lac repressor (LacI), which binds to genomically inserted lac operon sequences (lacO) via DNA/protein interactions. Integration of these lacO sequences into a genomic region of interest allows to monitor the functional consequences of HAT/HDAC targeting on chromatin (de)compaction, histone modification, and interaction with other proteins by quantitative light microscopy, as described here. As DNA-binding of LacI can be tightly controlled by the addition of galactose-derivatives, this method also allows to monitor the effects of locus-specific recruitment in a time-resolved manner.


Assuntos
Cromatina , Histona Acetiltransferases , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Cromatina/genética , Repressores Lac/genética , Histonas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Galactose , Histona Desacetilases/metabolismo , DNA/genética , DNA/metabolismo , Acetilação , Acetiltransferases/metabolismo
13.
Methods Mol Biol ; 2589: 493-508, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255645

RESUMO

The lysine deacetylase HDAC6 has unique structural and functional properties: It contains tandem catalytic domains that can deacetylate a variety of proteins and a zinc finger domain that binds ubiquitin. HDAC6 has been implicated in a variety of biological processes, normal or pathological, such as cellular motility, stress response, cancer, neurodegeneration, or viral infection. Due to this, HDAC6 is considered an attractive therapeutic target, and there is a major interest to identify small molecule inhibitors. To gain a mechanistic understanding of how HDAC6 impacts these different biological processes, there is a continued need to discover additional substrates as well as interacting proteins in different paradigms. One approach to achieve this is to perform HDAC6 immunoprecipitations to identify partner proteins. We describe here our optimized protocols to immunoprecipitate HDAC6 with the goal to identify or validate interacting proteins.


Assuntos
Histona Desacetilases , Lisina , Desacetilase 6 de Histona/metabolismo , Histona Desacetilases/metabolismo , Ubiquitina/metabolismo , Imunoprecipitação , Inibidores de Histona Desacetilases
14.
J Mol Cell Cardiol ; 162: 53-61, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34416247

RESUMO

Epigenetic modifications, such as histone or DNA modifications are key regulators of gene transcription and changes are often associated with maladaptive processes underlying cardiovascular disease. Epigenetic regulators therefore likely play a crucial role in cardiomyocyte homeostasis and facilitate the cellular adaption to various internal and external stimuli, responding to different intercellular and extracellular cues. Class IIa histone deacetylases are a class of epigenetic regulators that possess a myriad of post-transcriptional modification sites that modulate their activity in response to oxidative stress, altered catecholamine signalling or changes in the cellular metabolism. This review summaries the known reversible, post-translational modifications (PTMs) of class IIa histone deacetylases (HDACs) that ultimately drive transcriptional changes in homeostasis and disease. We also highlight the idea of a crosstalk of various PTMs on class IIa HDACs potentially leading to compensatory or synergistic effects on the class IIa HDAC-regulated cell behavior.


Assuntos
Doenças Cardiovasculares , Histona Desacetilases , Doenças Cardiovasculares/metabolismo , Epigenômica , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Processamento de Proteína Pós-Traducional
15.
PLoS Genet ; 18(11): e1010473, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36413574

RESUMO

Histone acetylation has been shown to involve in stress responses. However, the detailed molecular mechanisms that how histone deacetylases and transcription factors function in drought stress response remain to be understood. In this research, we show that ENAP1 and ENAP2 are positive regulators of drought tolerance in plants, and the enap1enap2 double mutant is more sensitive to drought stress. Both ENAP1 and ENAP2 interact with MYB44, a transcription factor that interacts with histone deacetylase HDT4. Genetics data show that myb44 null mutation enhances the sensitivity of enap1enap2 to drought stress. Whereas, HDT4 negatively regulates plant drought response, the hdt4 mutant represses enap1enap2myb44 drought sensitive phenotype. In the normal condition, ENAP1/2 and MYB44 counteract the HDT4 function for the regulation of H3K27ac. Upon drought stress, the accumulation of MYB44 and reduction of HDT4 leads to the enrichment of H3K27ac and the activation of target gene expression. Overall, this research provides a novel molecular mechanism by which ENAP1, ENAP2 and MYB44 form a complex to restrict the function of HDT4 in the normal condition; under drought condition, accumulated MYB44 and reduced HDT4 lead to the elevation of H3K27ac and the expression of drought responsive genes, as a result, plants are drought tolerant.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Secas , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo
16.
SAR QSAR Environ Res ; 33(11): 861-883, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36412121

RESUMO

Alteration and abnormal epigenetic mechanisms can lead to the aberration of normal biological functions and the occurrence of several diseases. The histone deacetylase (HDAC) family of enzymes is one of the prime regulators of epigenetic functions modifying the histone proteins, and thus, regulating epigenetics directly. HDAC1 is one of those HDACs which have important contributions to cellular epigenetics. The abnormality of HDAC is correlated to the occurrence, progression, and poor prognosis in several disease conditions namely neurodegenerative disorders, cancer cell proliferation, metastasis, chemotherapy resistance, and survival in various cancers. Therefore, the progress of potent and effective HDAC1 inhibitors is one of the prime approaches to combat such diseases. In this study, both regression and classification-based molecular modelling studies were conducted on some AR-42 derivatives as HDAC1 inhibitors to elucidate the crucial structural aspects that are responsible for regulating their biological responses. This study revealed that the molecular polarizability, van der Waals volume, the presence of aromatic rings as well as the higher number of hydrogen bond acceptors might affect prominently their inhibitory activity and might be responsible for proper fitting and interactions at the HDAC1 active site to pertain effective inhibition.


Assuntos
Histona Desacetilases , Relação Quantitativa Estrutura-Atividade , Histona Desacetilases/metabolismo , Fenilbutiratos , Proliferação de Células
17.
J Exp Clin Cancer Res ; 41(1): 321, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36357906

RESUMO

BACKGROUND: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor clinical outcomes and respond poorly to current therapies. Epigenetic deregulation is very common in MYC-driven MB. The bromodomain extra-terminal (BET) proteins and histone deacetylases (HDACs) are epigenetic regulators of MYC transcription and its associated tumorigenic programs. This study aimed to investigate the therapeutic potential of inhibiting the BET proteins and HDACs together in MB. METHODS: Using clinically relevant BET inhibitors (JQ1 or OTX015) and a pan-HDAC inhibitor (panobinostat), we evaluated the effects of combined inhibition on cell growth/survival in MYC-amplified MB cell lines and xenografts and examined underlying molecular mechanism(s). RESULTS: Co-treatment of JQ1 or OTX015 with panobinostat synergistically suppressed growth/survival of MYC-amplified MB cells by inducing G2 cell cycle arrest and apoptosis. Mechanistic investigation using RNA-seq revealed that co-treatment of JQ1 with panobinostat synergistically modulated global gene expression including MYC/HDAC targets. SYK and MSI1 oncogenes were among the top 50 genes synergistically downregulated by JQ1 and panobinostat. RT-PCR and western blot analyses confirmed that JQ1 and panobinostat synergistically inhibited the mRNA and protein expression of MSI1/SYK along with MYC expression. Reduced SYK/MSI expression after BET (specifically, BRD4) gene-knockdown further confirmed the epigenetic regulation of SYK and MSI1 genes. In addition, the combination of OTX015 and panobinostat significantly inhibited tumor growth in MYC-amplified MB xenografted mice by downregulating expression of MYC, compared to single-agent therapy. CONCLUSIONS: Together, our findings demonstrated that dual-inhibition of BET and HDAC proteins of the epigenetic pathway can be a novel therapeutic approach against MYC-driven MB.


Assuntos
Neoplasias Cerebelares , Meduloblastoma , Humanos , Camundongos , Animais , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Panobinostat/farmacologia , Panobinostat/uso terapêutico , Azepinas/farmacologia , Epigênese Genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Apoptose , Proliferação de Células , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
18.
Proc Natl Acad Sci U S A ; 119(45): e2206846119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322735

RESUMO

Heat stress limits plant growth, development, and crop yield, but how plant cells precisely sense and transduce heat stress signals remains elusive. Here, we identified a conserved heat stress response mechanism to elucidate how heat stress signal is transmitted from the cytoplasm into the nucleus for epigenetic modifiers. We demonstrate that HISTONE DEACETYLASE 9 (HDA9) transduces heat signals from the cytoplasm to the nucleus to play a positive regulatory role in heat responses in Arabidopsis. Heat specifically induces HDA9 accumulation in the nucleus. Under heat stress, the phosphatase PP2AB'ß directly interacts with and dephosphorylates HDA9 to protect HDA9 from 26S proteasome-mediated degradation, leading to the translocation of nonphosphorylated HDA9 to the nucleus. This heat-induced enrichment of HDA9 in the nucleus depends on the nucleoporin HOS1. In the nucleus, HDA9 binds and deacetylates the target genes related to signaling transduction and plant development to repress gene expression in a transcription factor YIN YANG 1-dependent and -independent manner, resulting in rebalance of plant development and heat response. Therefore, we uncover an HDA9-mediated positive regulatory module in the heat shock signal transduction pathway. More important, this cytoplasm-to-nucleus translocation of HDA9 in response to heat stress is conserved in wheat and rice, which confers the mechanism significant implication potential for crop breeding to cope with global climate warming.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Células Vegetais/metabolismo , Melhoramento Vegetal , Arabidopsis/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo
19.
Front Endocrinol (Lausanne) ; 13: 989305, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36339432

RESUMO

Histone deacetylase 11 (HDAC11) is the only member of the class IV HDAC, and the latest member identified. It is highly expressed in brain, heart, kidney and some other organs, and located in mitochondria, cytoplasm and nuclei, depending on the tissue and cell types. Although studies in HDAC11 total knockout mice suggest its dispensable features for tissue development and life, it participates in diverse pathophysiological processes, such as DNA replication, tumor growth, immune regulation, oxidant stress injury and neurological function of cocaine. Recent studies have shown that HDAC11 is also critically involved in the pathogenesis of some metabolic diseases, including obesity, diabetes and complications of diabetes. In this review, we summarize the recent progress on the role and mechanism of HDAC11 in the regulation of metabolic disorders, with the focus on its regulation on adipogenesis, lipid metabolism, metabolic inflammation, glucose tolerance, immune responses and energy consumption. We also discuss the property and selectivity of HDAC11 inhibitors and their applications in a variety of in vitro and in vivo models of metabolic disorders. Given that pharmacological and genetic inhibition of HDAC11 exerts a beneficial effect on various metabolic disorders, HDAC11 may be a potential therapeutic target to treat chronic metabolic diseases.


Assuntos
Histona Desacetilases , Doenças Metabólicas , Camundongos , Animais , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Doenças Metabólicas/tratamento farmacológico , Camundongos Knockout , Obesidade/genética , Inflamação/genética
20.
Int J Mol Sci ; 23(22)2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36430635

RESUMO

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.


Assuntos
Clonagem de Organismos , Histona Desacetilases , Suínos , Gravidez , Animais , Feminino , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Clonagem de Organismos/métodos , Histonas/metabolismo , Cromatina , RNA
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