RNA modifications have been known for many years, but their function has not been fully elucidated yet. For instance, the regulatory role of acetylation on N4-cytidine (ac4C) in RNA can be explored not only in terms of RNA stability and mRNA translation but also in DNA repair. Here, we observe a high level of ac4C RNA at DNA lesions in interphase cells and irradiated cells in telophase. Ac4C RNA appears in the damaged genome from 2 to 45 min after microirradiation. However, RNA cytidine acetyltransferase NAT10 did not accumulate to damaged sites, and NAT10 depletion did not affect the pronounced recruitment of ac4C RNA to DNA lesions. This process was not dependent on the G1, S, and G2 cell cycle phases. In addition, we observed that the PARP inhibitor, olaparib, prevents the recruitment of ac4C RNA to damaged chromatin. Our data imply that the acetylation of N4-cytidine, especially in small RNAs, has an important role in mediating DNA damage repair. Ac4C RNA likely causes de-condensation of chromatin in the vicinity of DNA lesions, making it accessible for other DNA repair factors involved in the DNA damage response. Alternatively, RNA modifications, including ac4C, could be direct markers of damaged RNAs.
- MeSH
- acetylace MeSH
- chromatin MeSH
- cytidin * genetika metabolismus MeSH
- PARP inhibitory MeSH
- RNA * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.
- MeSH
- acetylace MeSH
- acetylkoenzym A metabolismus MeSH
- karnitin-O-palmitoyltransferasa metabolismus MeSH
- lysinové acetyltransferasy * metabolismus MeSH
- myši MeSH
- NF-kappa B * metabolismus MeSH
- palmitany MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
During the preclinical development of small molecule inhibitors, compounds or compound libraries are typically first screened using purified target enzymes in vitro to select candidates with high potency. In the later stages of the development, however, functional cell-based assays may provide biologically more relevant data. In this chapter, we describe a detailed protocol for determining the potency of inhibitors targeting human histone deacetylase 6 in complex cellular environments. Cells are first treated with a dilution series of tested compounds, cell lysates separated by SDS-PAGE, and electrotransferred to a blotting membrane. The inhibitor potency is then determined indirectly by quantifying the levels of acetylated tubulin as a surrogate readout.
Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.
- MeSH
- acetylace MeSH
- chromatin * metabolismus MeSH
- histony metabolismus MeSH
- lidé MeSH
- myši MeSH
- nukleozomy * metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- protaminy metabolismus MeSH
- restrukturace chromatinu MeSH
- sperma metabolismus MeSH
- spermatidy metabolismus MeSH
- spermatogeneze fyziologie MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Histone deacetylases (HDACs) target acetylated lysine residues in histone and non-histone proteins. HDACs are implicated in the regulation of genomic stability, cell cycle, cell death and differentiation and thus critically involved in tumorigenesis. Further, HDACs regulate T-cell development and HDAC inhibitors (HDACis) have been approved for clinical use in some T-cell malignancies. Still, the exact targets and mechanisms of HDAC inhibition in cancer are understudied. We isolated tumor cell lines from a transgenic mouse model of anaplastic large cell lymphoma (ALCL), a rare T-cell lymphoma, and abrogated HDAC activity by treatment with the HDACis Vorinostat and Entinostat or Cre-mediated deletion of Hdac1. Changes in overall protein expression as well as histone and protein acetylation were measured following Hdac1 deletion or pharmacological inhibition using label-free liquid chromatography mass spectrometry (LC-MS/MS). We found changes in overall protein abundance and increased acetylation of histones and non-histone proteins, many of which were newly discovered and associated with major metabolic and DNA damage pathways. For non-histone acetylation, we mapped a total of 1204 acetylated peptides corresponding to 603 proteins, including chromatin modifying proteins and transcription factors. Hyperacetylated proteins were involved in processes such as transcription, RNA metabolism and DNA damage repair (DDR). The DDR pathway was majorly affected by hyperacetylation following HDAC inhibition. This included acetylation of H2AX, PARP1 and previously unrecognized acetylation sites in TP53BP1. Our data provide a comprehensive view of the targets of HDAC inhibition in malignant T cells with general applicability and could have translational impact for the treatment of ALCL with HDACis alone or in combination therapies.
- MeSH
- acetylace MeSH
- anaplastický velkobuněčný lymfom * farmakoterapie MeSH
- chromatografie kapalinová MeSH
- histondeacetylasy * metabolismus MeSH
- histony metabolismus MeSH
- kyseliny hydroxamové farmakologie MeSH
- myši MeSH
- tandemová hmotnostní spektrometrie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Aging is the most critical factor that influences the quality of post-ovulatory oocytes. Age-related molecular pathways remain poorly understood in fish oocytes. In this study, we examined the effect of oocyte aging on specific histone acetylation in common carp Cyprinus carpio. The capacity to progress to the larval stage in oocytes that were aged for 28 h in vivo and in vitro was evaluated. Global histone modifications and specific histone acetylation (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac) were investigated during oocyte aging. Furthermore, the activity of histone acetyltransferase (HAT) was assessed in fresh and aged oocytes. Global histone modifications did not exhibit significant alterations during 8 h of oocyte aging. Among the selected modifications, H4K12ac increased significantly at 28 h post-stripping (HPS). Although not significantly different, HAT activity exhibited an upward trend during oocyte aging. Results of our current study indicate that aging of common carp oocytes for 12 h results in complete loss of egg viability rates without any consequence in global and specific histone modifications. However, aging oocytes for 28 h led to increased H4K12ac. Thus, histone acetylation modification as a crucial epigenetic mediator may be associated with age-related defects, particularly in oocytes of a more advanced age.
Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.
- MeSH
- acetylace MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- biotest metody MeSH
- buněčné jádro genetika metabolismus MeSH
- fosforylace MeSH
- interakční proteinové domény a motivy MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- nádory kostí genetika metabolismus patologie MeSH
- oprava DNA MeSH
- osteosarkom genetika metabolismus patologie MeSH
- poškození DNA MeSH
- proteinfosfatasa 2C genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Gamma-glutamyltransferase (GGT), a marker of liver disease, has been shown to be associated with increased risk of diabetes and relative insulin secretion deficiency. However, the mechanism of hepatic Ggt regulation has not been explored fully. In this study, we made a concerted effort to understand the mechanism by investigating the effects of acetylation of histones H3 and H4, and bindings of histone acetyltransferases, CREB binding protein (CBP) and p300, at the Ggt promoter on the regulation of the expression of Ggt gene in the livers of streptozotocin (STZ)-induced moderate hypoinsulinemia rat model. The rats treated with STZ showed remarkably higher serum GGT level and hepatic Ggt/GGT expression than the untreated control rats. Furthermore, the acetylation of histones H3 and H4, and the binding of CBP not p300 at the Ggt promoter regions were significantly higher in the livers of STZ rats than those of the control rats. These results suggest that an enhanced hepatic expression of Ggt is associated with increased acetylation of histones H3 and H4 and CBP binding at the Ggt promoter in STZ-induced moderate hypoinsulinemic rats.
- MeSH
- acetylace MeSH
- experimentální diabetes mellitus enzymologie genetika MeSH
- gama-glutamyltransferasa biosyntéza genetika MeSH
- histonacetyltransferasy metabolismus MeSH
- histony metabolismus MeSH
- játra enzymologie MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- promotorové oblasti (genetika) MeSH
- protein p300 asociovaný s E1A metabolismus MeSH
- protein vázající CREB metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of neurodegenerative disorders. SW-100 (1a), a phenylhydroxamate-based HDAC6 inhibitor (HDAC6i) bearing a tetrahydroquinoline (THQ) capping group, is a highly potent and selective HDAC6i that was shown to be effective in mouse models of Fragile X syndrome and Charcot-Marie-Tooth disease type 2A (CMT2A). In this study, we report the discovery of a new THQ-capped HDAC6i, termed SW-101 (1s), that possesses excellent HDAC6 potency and selectivity, together with markedly improved metabolic stability and druglike properties compared to SW-100 (1a). X-ray crystallography data reveal the molecular basis of HDAC6 inhibition by SW-101 (1s). Importantly, we demonstrate that SW-101 (1s) treatment elevates the impaired level of acetylated α-tubulin in the distal sciatic nerve, counteracts progressive motor dysfunction, and ameliorates neuropathic symptoms in a CMT2A mouse model bearing mutant MFN2. Taken together, these results bode well for the further development of SW-101 (1s) as a disease-modifying HDAC6i.
- MeSH
- acetylace MeSH
- benzamidy chemie metabolismus MeSH
- Charcotova-Marieova-Toothova nemoc farmakoterapie metabolismus patologie MeSH
- chinoliny chemie metabolismus terapeutické užití MeSH
- fenotyp MeSH
- histondeacetylasa 6 antagonisté a inhibitory metabolismus MeSH
- inhibitory histondeacetylas chemie metabolismus terapeutické užití MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- poločas MeSH
- protein - isoformy antagonisté a inhibitory metabolismus MeSH
- simulace molekulového dockingu MeSH
- tubulin metabolismus MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.
- MeSH
- acetylace MeSH
- buněčná inkluze metabolismus MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- chromatin metabolismus MeSH
- DNA metabolismus MeSH
- epigeneze genetická MeSH
- G-kvadruplexy * MeSH
- genetická transkripce * MeSH
- histony metabolismus MeSH
- lidé MeSH
- metylace MeSH
- myši MeSH
- oprava DNA MeSH
- zastoupení bazí genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
