Increased Histone Acetylation Levels Research Articles

Increased histone acetylation levels or a serotonin precursor reinstate the context memory impaired by the serotonin receptor blocker methiothepin

Increased histone acetylation levels or a serotonin precursor reinstate the context memory impaired by the serotonin receptor blocker methiothepin

HDAC2 in Primary Sensory Neurons Constitutively Restrains Chronic Pain by Repressing α2δ-1 Expression and Associated NMDA Receptor Activity.

α2δ-1 (encoded by the Cacna2d1 gene) is a newly discovered NMDA receptor-interacting protein and is the therapeutic target of gabapentinoids (e.g., gabapentin and pregabalin) frequently used for treating patients with neuropathic pain. Nerve injury causes sustained α2δ-1 upregulation in the dorsal root ganglion (DRG), which promotes NMDA receptor synaptic trafficking and activation in the spinal dorsal horn, a hallmark of chronic neuropathic pain. However, little is known about how nerve injury initiates and maintains the high expression level of α2δ-1 to sustain chronic pain. Here, we show that nerve injury caused histone hyperacetylation and diminished enrichment of histone deacetylase-2 (HDAC2), but not HDAC3, at the Cacna2d1 promoter in the DRG. Strikingly, Hdac2 knockdown or conditional knockout in DRG neurons in male and female mice consistently induced long-lasting mechanical pain hypersensitivity, which was readily reversed by blocking NMDA receptors, inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDA receptor interaction at the spinal cord level. Hdac2 deletion in DRG neurons increased histone acetylation levels at the Cacna2d1 promoter, upregulated α2δ-1 in the DRG, and potentiated α2δ-1-dependent NMDA receptor activity at primary afferent central terminals in the spinal dorsal horn. Correspondingly, Hdac2 knockdown-induced pain hypersensitivity was blunted in Cacna2d1 knockout mice. Thus, our findings reveal that HDAC2 functions as a pivotal transcriptional repressor of neuropathic pain via constitutively suppressing α2δ-1 expression and ensuing presynaptic NMDA receptor activity in the spinal cord. HDAC2 enrichment levels at the Cacna2d1 promoter in DRG neurons constitute a unique epigenetic mechanism that governs acute-to-chronic pain transition.SIGNIFICANCE STATEMENT Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 activity at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain.

Extracellular matrix derived from Wharton’s Jelly-derived mesenchymal stem cells promotes angiogenesis via integrin αVβ3/c-Myc/P300/VEGF

BackgroundAngiogenesis is required in many physiological conditions, including bone regeneration, wound healing, and tissue regeneration. Mesenchymal stem cells-derived extracellular matrix (MSCs-ECM) could guide intricate cellular and tissue processes such as homeostasis, healing and regeneration.MethodsThe purpose of this study is to explore the effect and mechanism of ECM derived from decellularized Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) on endothelial cell viability and angiogenesis. The human umbilical vein endothelial cells (HUVECs) were pretreated with WJ-MSCs ECM for 2d/7d/14d, respectively. After pretreatment, the angiogenesis ability of HUVECs was detected.ResultsIn this study, we found for the first time that WJ-MSCs ECM could improve the angiogenesis ability of HUVECs with a time-dependent manner in vitro. Mechanically, WJ-MSCs ECM activated the focal adhesion kinase (FAK)/P38 signaling pathway via integrin αVβ3, which further promoted the expression of the cellular (c)-Myc. Further, c-Myc increased histone acetylation levels of the vascular endothelial growth factor (VEGF) promoter by recruiting P300, which ultimately promoting VEGF expression.ConclusionsECM derived from Wharton’s Jelly-derived mesenchymal stem cells promotes angiogenesis via integrin αVβ3/c-Myc/P300/VEGF. This study is expected to provide a new approach to promote angiogenesis in bone and tissue regeneration.

Abstract 6060: Can the gut microbiota both radiosensitize tumors and spare normal tissue toxicity

Abstract Background: New non-toxic radiosensitizers are needed in the treatment of muscle-invasive bladder cancer because elderly patients are vulnerable to chemotherapy-related toxicity of currently available radiosensitizers. Our previous study showed that high fiber diets sensitized RT112 xenografts to ionizing radiation (IR) by modifying the gut microbiome and this phenotype was positively correlated with B.acidifaciens (BA) abundance. It is noted that gut microbiota can enhance anti-tumoral responses, so we hypothesise that gut microbiota including BA may radiosensitize tumors via secretion of metabolites and immunomodulation. Methods: UPPL1591 mouse bladder cancer cell line flank xenografts were implanted into C57BL/6 mice (an immune-competent setting) at the same time as starting low (0.2% cellulose) and high fiber (5% psyllium, 5% psyllium plus 10% resistant starch/10% inulin) diets for their lifetime (N=30). 16S rRNA sequencing, IC-MS, IHC and the Nanostring platform were used for gut microbiota, untargeted metabolomics, and local tumor immunity analyses. For normal tissue toxicity studies, mice received 10-14 Gy IR (N=84). We used an intestinal crypt assay and FACS analysis of spleen to assess acute normal effects in small intestine and systemic immunity. Cell viability, clonogenic assay and Western blotting were used to validate the cytotoxic and radiosensitizing effects of bacterial supernatants on RT112 human bladder cancer cells (N=3). Results: Psyllium plus either resistant starch (RS, p=0.007) or inulin (p<0.001) significantly decreased tumors size. Psyllium plus inulin raised inosine-producing bacteria (p<0.001) and faecal inosine levels (p<0.001) which has been shown to enhance immunotherapy efficacy. Increased systemic (p=0.189) and local (p=0.024) anti-tumoral T cell responses were observed in the psyllium plus inulin group. Furthermore, the two diets mitigated the radiation injury from 14 Gy in intestinal crypt assays (p=0.011) and increased systemic immunity, with higher populations of B (p=0.024), cytotoxic T (p=0.019) and helper T (p<0.001) cells. Psyllium plus RS or inulin significantly increased caecal size (p<0.001), implying higher fermentation levels. We previously found inulin can enhance the relative abundance of BA. The supernatants of co-cultures of BA and F.praunitzii (FP, butyrate-producer) conferred greater cytotoxity than BA alone (p<0.001) or co-cultures of Bifidobacterium and FP (p<0.001), and also increased histone acetylation levels. Bacterial supernatants of BA increased DNA damage with 5Gy IR (p<0.05) and radiosensitivity of bladder tumour cells (p=0.008 at 8Gy). Conclusions: B.acidifaciens is a potential radiosensitiser with enhanced efficacy in combination with butyrate-producing bacteria. Our in vivo experiments suggest a role for inosine and cytotoxic T cells generated by the gut microbiota in radiosensitisation of bladder tumors. Citation Format: Chee Kin Then, Salome Paillas, Xuedan Wang, Mariya Misheva, James McCullagh, Kevin R. Foster, Anne E. Kiltie. Can the gut microbiota both radiosensitize tumors and spare normal tissue toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6060.

Nuclear-translocation of ACLY induced by obesity-related factors enhances pyrimidine metabolism through regulating histone acetylation in endometrial cancer.

Endometrial cancer (EC) is becoming one of the most common gynecologic malignancies. Lipid metabolism is a hallmark feature of cancers. The molecular mechanisms underlying lipid metabolism in EC remain unclear. In this study, we revealed that many lipid metabolism-related genes were aberrantly expressed in endometrial cancer tissues, especially ACLY. Upregulated ACLY promoted EC cell proliferation and colony formation, and attenuated apoptosis. Mechanistically, cotreatment with obesity-related factors (estradiol, insulin and leptin) promoted nuclear translocation of ACLY through Akt-mediated phosphorylation of ACLY at Ser455. Nuclear-localized ACLY increased histone acetylation levels, thus resulting in upregulation of pyrimidine metabolism genes, such as DHODH. Moreover, STAT3 altered the ACLY expression at the transcriptional level via directly binding to its promoter region. In conclusion, our findings clarify the roles and mechanisms of ACLY in endometrial cancer and ACLY could link obesity risk factors to the regulation of histone acetylation. We believe that novel therapeutic strategies for EC can be designed by targeting the ACLY axis.

Decreased Histone Acetylation Levels at Th1 and Regulatory Loci after Induction of Food Allergy.

Immunoglobulin E (IgE)-mediated allergy against cow’s milk protein fractions such as whey is one of the most common food-related allergic disorders of early childhood. Histone acetylation is an important epigenetic mechanism, shown to be involved in the pathogenesis of allergies. However, its role in food allergy remains unknown. IgE-mediated cow’s milk allergy was successfully induced in a mouse model, as demonstrated by acute allergic symptoms, whey-specific IgE in serum, and the activation of mast cells upon a challenge with whey protein. The elicited allergic response coincided with reduced percentages of regulatory T (Treg) and T helper 17 (Th17) cells, matching decreased levels of H3 and/or H4 histone acetylation at pivotal Treg and Th17 loci, an epigenetic status favoring lower gene expression. In addition, histone acetylation levels at the crucial T helper 1 (Th1) loci were decreased, most probably preceding the expected reduction in Th1 cells after inducing an allergic response. No changes were observed for T helper 2 cells. However, increased histone acetylation levels, promoting gene expression, were observed at the signal transducer and activator of transcription 6 (Stat6) gene, a proallergic B cell locus, which was in line with the presence of whey-specific IgE. In conclusion, the observed histone acetylation changes are pathobiologically in line with the successful induction of cow’s milk allergy, to which they might have also contributed mechanistically.

Nuclear-Translocation of ACLY Induced by Obesity Related Factors Enhances Pyrimidine Metabolism Through Regulating Histone Acetylation in Endometrial Cancer

Background: Endometrial cancer (EC) is a common female reproductive system cancer, and obesity is a major risk factor for the disease. ATP-citrate lyase (ACLY), a vital enzyme linking both glycolytic and lipid metabolism, plays important roles in cancer progression. However, there is little evidence of ACLY involvement in EC development. Methods: The ACLY expression in EC was evaluated by using TCGA and GEO databases and verified it using clinical samples by IHC and RT-PCR methods. The functions of ACLY was determined by both the vitro and vivo studies. Gene expression array, co-immunoprecipitation, TUNEL assay, EdU assay, chromatin immunoprecipitation and luciferase reporter assay were applied for mechanism dissection. Findings: ACLY was significantly upregulated in EC patients, particularly in the nucleus. Upregulated ACLY promoted EC cell proliferation and colony formation, and attenuated apoptosis. Mechanistically, cotreatment with obesity-related factors (estradiol, insulin and leptin) promoted nuclear translocation of ACLY through Akt-mediated phosphorylation of ACLY at Ser455 . Nuclear-localized ACLY increased histone acetylation levels, thus resulting in upregulation of pyrimidine metabolism genes, such as DHODH. Moreover, STAT3 altered the ACLY expression at the transcriptional level via directly binding to its promoter region. Interpretation: ACLY nuclear-translocation induced by obesity-related factors promotes EC progression by modulating histone acetylation levels, The ACLY axis could be served as potential therapeutic targets for the treatment of EC. Funding Statement: This research was supported by The National Natural Science Foundation of China (Grant number: 81974406) and The Sailing Program Foundation of Hunan Cancer Hospital. Declaration of Interests: The authors declare that they have no competing interests with the contents of this article. Ethics Approval Statement: The study was approved by the Research Ethics Committee of the Sixth People's Hospital, and informed consent was provided by all patients before this study.

Histone hyperacetylation mediates enhanced IL-1β production in LPS/IFN-γ-stimulated macrophages.

SummaryUnder the condition of lipopolysaccharide (LPS)/interferon (IFN)‐γ activation, macrophage metabolism is converted from oxidative phosphorylation to glycolysis. In the present work, we analysed whether glycolysis could affect interleukin (IL)‐1β expression through altering histone acetylation levels in mouse bone marrow‐derived macrophages. Immunocytochemistry and Western blot analysis are used to characterize histone acetylation in macrophages stimulated by LPS/IFN‐γ. Real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay were used to determine IL‐1β production. The metabolism of macrophages was monitored in real‐time by the Seahorse test. Our results showed that glycolytic metabolism could enhance histone acetylation and promote IL‐1β production in LPS/IFN‐γ‐activated macrophages. Moreover, increased production of IL‐1β by glycolysis was mediated through enhanced H3K9 acetylation. Importantly, it was found that a high dose of histone deacetylase inhibitor could also significantly increase the expression of IL‐1β in the absence of glycolytic metabolism. In conclusion, this study demonstrates that glycolytic metabolism could regulate IL‐1β expression by increasing histone acetylation levels in LPS/IFN‐γ‐stimulated macrophages.

Wheat Elongator subunit 4 is required for epigenetic regulation of host immune response to Rhizoctonia cerealis

Wheat (Triticum aestivum) is necessary for global food security. The necrotrophic fungus Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of wheat. Although the Elongator complex, composed of six subunits, has been implicated in growth, development, and innate immunity in Arabidopsis, little is known about its functions in wheat or the involvement of Elongator subunit 4 in histone acetylation. In this study, we identified the Elongator subunit 4-encoding gene TaELP4 in wheat resistance response to R. cerealis, and verified that TaELP4 increased histone acetylation in regions of defense-associated genes and regulated immune response to R. cerealis. TaELP4 was more highly expressed in resistant than in susceptible wheat cultivars and was induced in resistant wheat after infection by R. cerealis. Silencing of TaELP4 in wheat not only impaired resistance to R. cerealis, but also repressed both histone acetylation levels and the expression of a subset of defense-associated genes, including TaAGC1, TaCPK7-D, TaPAL5, Defensin, and Chitinase2. Ectopic expression of TaELP4 in Arabidopsis increased histone acetylation levels in coding and promoter regions of defense genes and increased their transcription, leading to increased resistance to infection by the pathogen Botrytis cinerea. These results suggest that TaELP4 positively regulates innate immune responses of wheat and Arabidopsis to R. cerealis and B. cinerea by increasing histone acetylation levels of defense-associated genes and increasing their transcription. This study has shed light on the involvement of TaELP4 in histone acetylation and resistance response against R. cerealis. TaELP4 may potentially be used to improve wheat resistance against sharp eyespot.

Heat-shock transcription factor 2 promotes sodium butyrate-induced autophagy by inhibiting mTOR in ulcerative colitis

Butyrate-induced autophagy and anti-inflammatory effects of IECs plays an important role in UC. HSP has been proved to be associated with autophagy. HSF2, as an important regulator of HSP, has been determined to be highly expressed in UC. This study was designed to elucidate the relationship between HSF2, butyrate and epithelial autophagy and the potential mechanism of HSF2-related autophagy in UC. The autophagy levels and HSF2 expression in intestinal mucosa were increased in UC patients compared to controls. In DSS colitis models, hsf2-/- mice exhibited more severe intestinal inflammation and lower autophagy levels than wild-type mice. HSF2 expression could be induced by sodium butyrate and LPS as a dose-response relationship in HT-29 cells, epigenetically via increasing histone acetylation levels at the promoter region by sodium butyrate. Autophagy induced by sodium butyrate was promoted by overexpression HSF2 in HT-29 cells. Moreover, overexpression HSF2 decreased the expression and phosphorylation levels of PI3K, Akt and mTOR induced by sodium butyrate. HSF2 might induced by sodium butyrate and inflammation and played protective roles in UC by enhancing autophagy of IECs. This indicated that HSF2 may be a critical target for autophagy modulation and a new potential therapeutic target in UC.

PROTAC-mediated degradation of class I histone deacetylase enzymes in corepressor complexes.

We have identified a proteolysis targeting chimera (PROTAC) of class I HDACs 1, 2 and 3. The most active degrader consists of a benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-Lindau E3 ligand. Our PROTAC increased histone acetylation levels and compromised colon cancer HCT116 cell viability, establishing a degradation strategy as an alternative to class I HDAC inhibition.

Elevated histone H3 acetylation and loss of the Sp1–HDAC1 complex de-repress the GM2-synthase gene in renal cell carcinoma

GM2-synthase produces sialic acid-containing glycosphingolipids called gangliosides, and its mRNA overexpression and the gangliosides it generates are linked to tumor progression, migration, and suppression of tumor-specific host immune responses. However, the mechanism underlying GM2-synthase de-repression in renal cell carcinoma (RCC) is poorly understood. Here, we demonstrate that higher GM2-synthase mRNA expression levels in various cancer cells and in human RCC tumors correlate with higher histone acetylation levels (H3K9, H3K14, or both) at region +38/+187 relative to the transcription start site (TSS) of the GM2-synthase gene than in normal kidney epithelial (NKE) cells or healthy adjacent tissues. An increase in GM2-synthase mRNA expression in cells treated with a histone deacetylase (HDAC) inhibitor was accompanied by increased histone acetylation levels at this promoter region. DNA methylation around the TSS was absent in both RCC cell lines and NKE cells. Of note, both the transcription factor Sp1 and corepressor HDAC1 associated with the +38/+187 region when the GM2-synthase gene was repressed in NKE and tumor-adjacent tissues, indicating plausible site-specific repressive roles of HDAC1 and Sp1 in GM2-synthase mRNA expression. Site-directed mutagenesis of the Sp1-binding site within the +38/+187 region relieved repressed luciferase activity of this region by limiting HDAC1 recruitment. Moreover, Sp1 or HDAC1 knock down increased GM2-synthase transcription, and butyrate-mediated activation of GM2-synthase mRNA expression in SK-RC-45 cells was accompanied by Sp1 and HDAC1 loss from the +38/+187 region. Taken together, we have identified an epigenetic mechanism for the de-repression of the GM2-synthase gene in RCC.

The Tumor Suppressor CIC Directly Regulates MAPK Pathway Genes via Histone Deacetylation.

Oligodendrogliomas are brain tumors accounting for approximately 10% of all central nervous system cancers. CIC is a transcription factor that is mutated in most patients with oligodendrogliomas; these mutations are believed to be a key oncogenic event in such cancers. Analysis of the Drosophila melanogaster ortholog of CIC, Capicua, indicates that CIC loss phenocopies activation of the EGFR/RAS/MAPK pathway, and studies in mammalian cells have demonstrated a role for CIC in repressing the transcription of the PEA3 subfamily of ETS transcription factors. Here, we address the mechanism by which CIC represses transcription and assess the functional consequences of CIC inactivation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched for MAPK effector genes involved in cell-cycle regulation and proliferation. CIC binding to target genes was abolished by high MAPK activity, which led to their transcriptional activation. CIC interacted with the SIN3 deacetylation complex and, based on our results, we suggest that CIC functions as a transcriptional repressor through the recruitment of histone deacetylases. Independent single amino acid substitutions found in oligodendrogliomas prevented CIC from binding its target genes. Taken together, our results show that CIC is a transcriptional repressor of genes regulated by MAPK signaling, and that ablation of CIC function leads to increased histone acetylation levels and transcription at these genes, ultimately fueling mitogen-independent tumor growth.Significance: Inactivation of CIC inhibits its direct repression of MAPK pathway genes, leading to their increased expression and mitogen-independent growth.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4114/F1.large.jpg Cancer Res; 78(15); 4114-25. ©2018 AACR.

HISTONE DEACETYLASE 6 represses pathogen defence responses in Arabidopsis thaliana.

Plant defence mechanisms are suppressed in the absence of pathogen attack to prevent wasted energy and growth inhibition. However, how defence responses are repressed is not well understood. Histone deacetylase 6 (HDA6) is a negative regulator of gene expression, and its role in pathogen defence response in plants is not known. In this study, a novel allele of hda6 (designated as shi5) with spontaneous defence response was isolated from a forward genetics screening in Arabidopsis. The shi5 mutant exhibited increased resistance to hemibiotrophic bacterial pathogen Pst DC3000, constitutively activated expression of pathogen-responsive genes including PR1, PR2, etc. and increased histone acetylation levels at the promoters of most tested genes that were upregulated in shi5. In both wild type and shi5 plants, the expression and histone acetylation of these genes were upregulated by pathogen infection. HDA6 was found to bind to the promoters of these genes under both normal growth conditions and pathogen infection. Our research suggests that HDA6 is a general repressor of pathogen defence response and plays important roles in inhibiting and modulating the expression of pathogen-responsive genes in Arabidopsis.

Chemosensitivity enhancement toward arsenic trioxide by inhibition of histone deacetylase in NB4 cell line.

ObjectiveTo investigate the cytotoxic effects of suberanilohydroxamic acid (vorinostat) in combination with arsenic trioxide (ATO) on the human NB4 cell line in vitro.MethodsThe rates of cell proliferation following treatment with vorinostat with or without ATO were measured. Flow cytometry of Annexin-V/propidium iodide double-stained cells was used to measure apoptosis. Acridine Orange and ethidium bromide staining was used to observe morphological changes characteristic of apoptosis. Western blot analysis was used to measure protein levels.ResultsVorinostat and ATO, alone and in combination, inhibited the proliferation of NB4 cells in a time- and dose-dependent manner and the effect was additive. NB4 cells treated with vorinostat + ATO demonstrated greater levels of apoptosis compared with cells treated with either drug alone. Both vorinostat and ATO alone and in combination resulted in lower levels of promyelocytic leukaemia/retinoic acid receptor alpha fusion protein and increased levels of acetyl-histone H3 and acetyl-histone H4 proteins compared with controls. Vorinostat + ATO resulted in lower levels of Akt protein compared with either drug alone.ConclusionThe combination of vorinostat and ATO inhibited cell proliferation, induced apoptosis, and enhanced the chemosensitivity of NB4 cells. The mechanism might be associated with increasing histone acetylation levels as well as downregulation of the Akt signalling pathway.

Safety and efficacy of valproic acid treatment in SCA3/MJD patients

BackgroundSpinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is one of 10 known polyglutamine (polyQ) diseases. In Drosophila and rat models of polyQ diseases, histone deacetylation (HDAC) inhibitors improved locomotor function and survival time by increasing histone acetylation levels and modulating gene expression. Valproic acid (VPA) is a pan-HDAC inhibitor used clinically to treat bipolar and seizure disorders. We evaluated the clinical safety and efficacy of VPA treatment for SCA3/MJD patients. MethodsFirst, a randomized, open-label, dose-escalation method was used to evaluate tolerance to single-dose VPA administration in 12 SCA3/MJD patients. Patients were randomly assigned to three groups of four subjects, each with an oral dosage of 400 mg, 600 mg, or 800 mg (twice daily (bid) for one day). VPA was well-tolerated for one-dose by all patient groups. Second, a randomized, double-blind, placebo-controlled, dose-controlled study evaluated the safety and efficacy of multi-dose VPA (oral administration, twice daily (bid) for 12 weeks) in 36 SCA3/MJD patients. Patients received either low-dose VPA (800 mg/day), high-dose VPA (1200 mg/day), or placebo (n = 12 subjects per group). Symptoms were evaluated using the Scale for Assessment and Rating of Ataxia (SARA). ResultsMulti-dose VPA treatment improved SARA measures of locomotor function. Major adverse effects included dizziness and loss of appetite. ConclusionsVPA is a potentially beneficial agent for the treatment of SCA3/MJD. These results also provide insight into possible future therapeutics for polyQ diseases.

Vorinostat enhances chemosensitivity to arsenic trioxide in K562 cell line.

Objective. This study aimed to investigate the chemosensitive augmentation effect and mechanism of HDAC inhibitor Vorinostat (SAHA) in combination with arsenic trioxide (ATO) on proliferation and apoptosis of K562 cells.Methods. The CCK-8 assay was used to compare proliferation of the cells. Annexin-V and PI staining by flow cytometry and acridine orange/ethidium bromide stains were used to detect and quantify apoptosis. Western blot was used to detect expression of p21, Akt, pAkt, p210, Acetyl-Histone H3, and Acetyl-Histone H4 proteins.Results. SAHA and ATO inhibited proliferation of K562 cells in an additive and time- and dose-dependent manner. SAHA in combination with ATO showed significant apoptosis of K562 cells in comparison to the single drugs alone (p < 0.01). Both SAHA and ATO alone and in combination showed lower levels of p210 expression. SAHA and SAHA and ATO combined treatment showed increased levels of Acetyl-Histone H3 and Acetyl-Histone H4 protein expression. SAHA alone showed increased expression of p21, while ATO alone and in combination with SAHA showed no significant change. SAHA and ATO combined therapy showed lower levels of Akt and pAkt protein expression than SAHA or ATO alone.Conclusion. SAHA and ATO combined treatment inhibited proliferation, induced apoptosis, and showed a chemosensitive augmentation effect on K562 cells. The mechanism might be associated with increasing histone acetylation levels as well as regulating the Akt signaling pathway.

Sirtuins in epigenetic regulation.

1.1. Discovery of Sirtuins The founding member of sirtuin is the yeast-silencing information regulator 2 (SIR2) protein, one of four proteins (SIR1–4) required for silencing the mating-type information loci in yeast.1 SIR2–4, but not SIR1, are also required for gene silencing at telomeres.2 SIR2 also mediates gene silencing at the rDNA (rDNA) loci, which was shown to be independent of other SIR proteins.3,4 Immunofluorescence imaging showed that SIR2 is mainly in the nucleolus and telomeres in yeast.5 It was demonstrated that the silenced genetic loci have low histone acetylation levels compared to loci that are not silenced. Mutation in Sir2–4 increased histone acetylation levels and overexpression of SIR2 but not other SIR proteins led to decreases in histone H4, H2B, and H3 acetylation.6 Analyzing the sensitivity of yeast chromatin to micrococcal nuclease and dam methyltransferase indicated that Sir2 mutation affects the chromatin structure in the rDNA and mating-type loci.7 These interesting discoveries on SIR2 were further elevated by two discoveries made by Guarente and co-workers. The first one was that SIR2 is important for the replicative life span of yeast cells,8 a finding that was later extended to higher eukaryotic species,9,10 although the role of sirtuins in life span is highly controversial11 and may depend on genetic background and diet conditions.12,13 The second discovery was that SIR2 is an NAD-dependent histone deacetylase (Figure ​(Figure11),14 which established SIR2 as a mechanistically novel lysine deacetylase and revealed the connection between gene silencing and cellular metabolism. Indeed, it was later found that the life span extension effect of SIR2 is dependent on NAD level and NAD metabolism.15−17 Several other groups similarly reported the enzymatic activity of SIR2.18 These landmark discoveries established sirtuins as important players in epigenetics and triggered the explosion of research interest in sirtuins. Figure 1 Enzymatic function of sirtuins. (A) NAD-dependent protein lysine deacylation activity of different sirtuins. (B) Enzymatic reaction mechanism of sirtuins. (C) Structure of a ternary sirtuin-NAD-acetyl peptide complex (PDB ID 2H4F). NAD, acetyl lysine, ...

Epigenetics of hyper-responsiveness to allergen challenge following intrauterine growth retardation rat.

BackgroundEpidemiological studies have revealed that intrauterine growth retardation (IUGR) or low birth weight is linked to the later development of asthma. Epigenetic regulatory mechanisms play an important role in the fetal origins of adult disease. However, little is known regarding the correlation between epigenetic regulation and the development of asthma following IUGR.MethodsAn IUGR and ovalbumin (OVA)-sensitization/challenge rat model was used to study whether epigenetic mechanisms play a role in the development of asthma following IUGR.ResultsMaternal nutrient restriction increased histone acetylation levels of the endothelin-1 (ET-1) gene promoter in lung tissue of offspring, but did not cause significant alterations of DNA methylation. The effect was maintained until 10 weeks after birth. Furthermore, these epigenetic changes may have induced IUGR individuals to be highly sensitive to OVA challenge later in life, resulting in more significant changes related to asthma.ConclusionsThese findings suggest that epigenetic mechanisms might be closely associated with the development of asthma following IUGR, providing further insight for improved prevention of asthma induced by environmental factors.

Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT) and deacetylase activities (HDAC). Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB)–binding protein (CBP) as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD) in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min) subthreshold occlusion of the middle cerebral artery (MCA), followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.