A massive alteration of gene expression in undescended testicles of dogs and the association of KAT6A variants with cryptorchidism.

Cryptorchidism is the most common form of disorder of sex development in male dogs, but its hereditary predisposition is poorly elucidated. The gonadal transcriptome of nine unilaterally cryptorchid dogs and seven control dogs was analyzed using RNA-seq. Comparison between the scrotal and inguinal gonads of unilateral cryptorchid dogs revealed 8,028 differentially expressed genes (DEGs) (3,377 up-regulated and 4,651 down-regulated). A similar number of DEGs (7,619) was found by comparing the undescended testicles with the descended testicles of the control dogs. The methylation status of the selected DEGs was also analyzed, with three out of nine studied DEGs showing altered patterns. Bioinformatic analysis of the cDNA sequences revealed 20,366 SNP variants, six of which showed significant differences in allelic counts between cryptorchid and control dogs. Validation studies in larger cohorts of cryptorchid (n = 122) and control (n = 173) dogs showed that the TT genotype (rs850666472, p.Ala1230Val) and the AA genotype in 3'UTR (16:23716202G>A) in KATA6, responsible for acetylation of lysine 9 in histone H3, are associated with cryptorchidism (P = 0.0383). Both the transcript level of KAT6A and H3K9 acetylation were lower in undescended testes, and additionally, the acetylation depended on the genotypes in exon 17 and the 3'UTR. Our study showed that the massive alteration of the transcriptome in undescended testicles is not caused by germinal DNA variants in DEG regulatory sequences but is partly associated with an aberrant DNA methylation and H3K9 acetylation patterns. Moreover, variants of KAT6A can be considered markers associated with the risk of this disorder.

Sodium Valproate Is Effective Against Botrytis cinerea Infection of Tomato by Enhancing Histone H3 Acetylation-Directed Gene Transcription and Triggering Tomato Fruit Immune Response.

Botrytis cinerea causes gray mold resulting in enormous financial loss. Fungicide resistance of B. cinerea has become a serious issue in food safety and agricultural environmental protection. Sodium valproate (SV) has been used in clinical trials; thus, it is an excellent candidate for fungicide development, considering its safety. However, the antifungal activity remains unclear. SV was effective against B. cinerea by enhancing acetylation of histone H3, including H3K9ac, H3K14ac, and H3K56ac. A transcriptomics analysis revealed that the expression of 1,557 genes changed significantly in response to SV. A pathway enrichment analysis identified 16 significant GO terms, in which molecular functions were mainly involved. In addition, the expression levels of 13 genes involved in B. cinerea virulence and five genes involved in tomato immune response were altered by the SV treatment. These results indicate that SV inhibits B. cinerea by enhancing acetylation of histone H3 and modifying gene transcription. Thus, SV is an effective, safe, potential antifungal agent for control of both pre- and postharvest losses caused by B. cinerea.

New Approaches to Assess Mechanisms of Action of Selective Vitamin D Analogues.

Recent studies of transcription have revealed an advanced set of overarching principles that govern vitamin D action on a genome-wide scale. These tenets of vitamin D transcription have emerged as a result of the application of now well-established techniques of chromatin immunoprecipitation coupled to next-generation DNA sequencing that have now been linked directly to CRISPR-Cas9 genomic editing in culture cells and in mouse tissues in vivo. Accordingly, these techniques have established that the vitamin D hormone modulates sets of cell-type specific genes via an initial action that involves rapid binding of the VDR-ligand complex to multiple enhancer elements at open chromatin sites that drive the expression of individual genes. Importantly, a sequential set of downstream events follows this initial binding that results in rapid histone acetylation at these sites, the recruitment of additional histone modifiers across the gene locus, and in many cases, the appearance of H3K36me3 and RNA polymerase II across gene bodies. The measured recruitment of these factors and/or activities and their presence at specific regions in the gene locus correlate with the emerging presence of cognate transcripts, thereby highlighting sequential molecular events that occur during activation of most genes both in vitro and in vivo. These features provide a novel approach to the study of vitamin D analogs and their actions in vivo and suggest that they can be used for synthetic compound evaluation and to select for novel tissue- and gene-specific features. This may be particularly useful for ligand activation of nuclear receptors given the targeting of these factors directly to genetic sites in the nucleus.

DNA methylation and histone acetylation are involved in Wnt10b expression during the secondary hair follicle cycle in Angora rabbits.

Secondary hair follicles (SHFs) in the Angora rabbit exhibit classic cyclic hair development, but the multiple molecular signals involved in hair cycling are yet to be explored in detail. In the present study, we investigated the expression pattern, methylation and histone H3 acetylation status of Wnt10b, as a molecular signal participating in hair cycling, during the SHF cycle in the Angora rabbit. Expression of Wnt10b at the anagen phase was significantly higher than that at both the telogen and catagen phases, suggesting that Wnt10b might serve as a critical activator during cyclic transition of SHFs. Methylation frequency of the fifth CpG site (CpG5-175 bp) in CpG islands at the anagen phase was lower than that at both the catagen and telogen phases. The methylation status of the CpG5 site was negatively correlated with Wnt10b expression. This indicated that the methylation of CpG5 might participate in Wnt10b transcriptional suppression in SHFs. Furthermore, histone H3 acetylation status in the regions-256~-11 bp and 98 ~ 361 bp were significantly lower at both the catagen and telogen phases than at the anagen phase. The histone H3 acetylation level was significantly positively correlated with Wnt10b expression. This confirmed that histone acetylation was likely involved in upregulating Wnt10b transcription in SHFs. Additionally, potential binding to the transcription factors ZF57 and HDBP was predicted within the CpG5 site. In conclusion, our findings reveal the epigenetic mechanism of Wnt10b transcription and provide a new insight into epigenetic regulation during the SHF cycle in the Angora rabbit.

Adolescent Alcohol Exposure-Induced Changes in Alpha-Melanocyte Stimulating Hormone and Neuropeptide Y Pathways via Histone Acetylation in the Brain During Adulthood.

Background: Adolescent intermittent ethanol exposure causes long-lasting alterations in brain epigenetic mechanisms. Melanocortin and neuropeptide Y signaling interact and are affected by ethanol exposure in the brain. Here, the persistent effects of adolescent intermittent ethanol on alpha-melanocyte stimulating hormone, melanocortin 4 receptor, and neuropeptide Y expression and their regulation by histone acetylation mechanisms were investigated in adulthood. Methods: Male rats were exposed to adolescent intermittent ethanol (2 g/kg, i.p.) or volume-matched adolescent intermittent saline from postnatal days 28 to 41 and allowed to grow to postnatal day 92. Anxiety-like behaviors were measured by the elevated plus-maze test. Brain regions from adult rats were used to examine changes in alpha-melanocyte stimulating hormone, melanocortin 4 receptor, and neuropeptide Y expression and the histone acetylation status of their promoters. Results: Adolescent intermittent ethanol-exposed adult rats displayed anxiety-like behaviors and showed increased pro-opiomelanocortin mRNA levels in the hypothalamus and increased melanocortin 4 receptor mRNA levels in both the amygdala and hypothalamus compared with adolescent intermittent saline-exposed adult rats. The alpha-Melanocyte stimulating hormone and melanocortin 4 receptor protein levels were increased in the central and medial nucleus of the amygdala, paraventricular nucleus, and arcuate nucleus of the hypothalamus in adolescent intermittent ethanol-exposed compared with adolescent intermittent saline-exposed adult rats. Neuropeptide Y protein levels were decreased in the central and medial nucleus of the amygdala of adolescent intermittent ethanol-exposed compared with adolescent intermittent saline-exposed adult rats. Histone H3K9/14 acetylation was decreased in the neuropeptide Y promoter in the amygdala but increased in the melanocortin 4 receptor gene promoter in the amygdala and the melanocortin 4 receptor and pro-opiomelanocortin promoters in the hypothalamus of adolescent intermittent ethanol-exposed adult rats compared with controls. Conclusions: Increased melanocortin and decreased neuropeptide Y activity due to changes in histone acetylation in emotional brain circuitry may play a role in adolescent intermittent ethanol-induced anxiety phenotypes in adulthood.

Dysregulation of Sirtuin 2 (SIRT2) and histone H3K18 acetylation pathways associates with adverse prostate cancer outcomes.

BACKGROUND: Histones undergo extensive post-translational modifications and this epigenetic regulation plays an important role in modulating transcriptional programs capable of driving cancer progression. Acetylation of histone H3K18, associated with gene activation, is enhanced by P300 and opposed by the deacetylase Sirtuin2 (SIRT2). As these enzymes represent an important target for cancer therapy, we sought to determine whether the underlying genes are altered during prostate cancer (PCa) progression. METHODS: Tissue microarrays generated from 71 radical prostatectomy patients were initially immunostained for H3K18Ac, P300 and SIRT2. Protein levels were quantified using VECTRA automation and correlated with clinicopathologic parameters. The Cancer Genome Atlas (TGCA, n = 499) and Gene Expression Omnibus (n = 504) databases were queried for expression, genomic and clinical data. Statistics were performed using SPSSv23. RESULTS: Nuclear histone H3K18Ac staining increases in primary cancer (p = 0.05) and further in metastases (p < 0.01) compared to benign on tissue arrays. P300 protein expression increases in cancer (p = 0.04) and metastases (p < 0.001). A progressive decrease in nuclear SIRT2 staining occurs comparing benign to cancer or metastases (p = 0.04 and p = 0.03 respectively). Decreased SIRT2 correlates with higher grade cancer (p = 0.02). Time to Prostate Specific Antigen (PSA) recurrence is shorter in patients exhibiting high compared to low H3K18Ac expression (350 vs. 1542 days respectively, P = 0.03). In GEO, SIRT2 mRNA levels are lower in primary and metastatic tumors (p = 0.01 and 0.001, respectively). TGCA analysis demonstrates SIRT2 deletion in 6% and increasing clinical stage, positive margins and lower PSA recurrence-free survival in patients with SIRT2 loss/deletion (p = 0.01, 0.04 and 0.04  respectively). In this dataset, a correlation between decreasing SIRT2 and increasing P300 mRNA expression occurs in tumor samples (R = -0.46). CONCLUSIONS: In multiple datasets, decreases in SIRT2 expression portend worse clinicopathologic outcomes. Alterations in SIRT2-H3K18Ac suggest altered P300 activity and identify a subset of tumors that could benefit from histone deacetylation inhibition.

The cell- and tissue-specific transcription mechanism of the TATA-less syntaxin 1A gene.

Syntaxin 1A (Stx1a) plays an important role in regulation of neuronal synaptic function. To clarify the mechanism of basic transcriptional regulation and neuron-specific transcription of Stx1a we cloned the Stx1a gene from rat, in which knowledge of the expression profile was accumulated, and elucidated that Stx1a consisting of 10 exons, possesses multiple transcription initiation sites and a 204-bp core promoter region (CPR) essential for transcription in PC12 cells. The TATA-less, conserved, GC-rich CPR has 2 specific protein (SP) sites that bind SP1 and are responsible for 65% of promoter activity. The endogenous CPR, including 23 CpG sites, is not methylated in PC12 cells, which express Stx1a and fetal rat skin keratinocyte (FRSK) cells, which do not, although an exogenous methylated CPR suppresses reporter activity in both lines. Trichostatin A (TSA) and class I histone deacetylase (HDAC) inhibitors, but not 5-azacytidine, induce Stx1a in FRSK cells. Acetylated histone H3 only associates to the CPR in FRSK cells after TSA addition, whereas the high acetylated histone H3-CPR association in PC12 cells was unchanged following treatment. HDAC inhibitor induction of Stx1a was negated by mithramycin A and deletion/mutation of 2 SP sites. HDAC1, HDAC2, and HDAC8 detach from the CPR when treated with TSA in FRSK cells and are associated with the CPR in lungs, and acetylated histone H3 associates to this region in the brain. In the first study characterizing a syntaxin promoter, we show that association of SP1 and acetylated histone H3 to CPR is important for Stx1a transcription and that HDAC1, HDAC2, and HDAC8 decide cell/tissue specificity in a suppressive manner.

Alcohol Exposure Causes Overexpression of Heart Development-Related Genes by Affecting the Histone H3 Acetylation via BMP Signaling Pathway in Cardiomyoblast Cells.

BACKGROUND: Abusive alcohol utilization of pregnant woman may cause congenital heart disease (CHD) of fetus, where alcohol ignites histone H3 hyperacetylation leading to abnormal development of heart morphogenesis and associated genes. Knowledge about the regularized upstream genes is little, but bone morphogenetic protein (BMP) signaling may actively and prominently take part in alteration in acetylation of histone H3. The supreme objective of this study was to unearth the involvement of BMP signaling pathway in alcohol-driven hyperacetylation of histone H3 in cardiomyoblast cells. METHODS: Cardiomyoblast cells (H9c2 cells) were addicted with alcohol (100 mM) for 24 hours. Dorsomorphin (5 µM) was used for the inhibition of BMP signaling pathway. We detected the phosphorylation activity of SMAD1/5/8, mRNA expression, histone acetyltransferases (HAT)/histone deacetylase (HDAC) activity, and acetylation of histone H3. RESULTS: Following alcohol exposure, phosphorylation of SMAD1/5/8 and HAT activities was increased to a significant extent, while histone H3 acetylation and expression of heart development-related genes were also increased. The said phenomenon influenced by alcohol was reverted upon dorsomorphin treatment to the cells without effecting HDAC activity. CONCLUSIONS: The data clearly identified that BMP-mediated histone H3 acetylation of heart development-related genes might be one of the possible cellular mechanisms to control alcohol-induced expression of heart development-related genes. Dorsomorphin, on the other hand, may modulate alcohol-induced hyperacetylation of histone H3 through BMP targeting, which could be a potential way to block CHD.

Inhibition of MDA-MB-231 breast cancer cell proliferation and tumor growth by apigenin through induction of G2/M arrest and histone H3 acetylation-mediated p21WAF1/CIP1 expression.

Apigenin (4',5,7-trihydroxyflavone), a flavonoid commonly found in fruits and vegetables, has anticancer properties in various malignant cancer cells. However, the molecular basis of the anticancer effect remains to be elucidated. In this study, we investigated the cellular mechanisms underlying the induction of cell cycle arrest by apigenin. Our results showed that apigenin at the nonapoptotic induction concentration inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in the MDA-MB-231 breast cancer cell line. Immunoblot analysis indicated that apigenin suppressed the expression of cyclin A, cyclin B, and cyclin-dependent kinase-1 (CDK1), which control the G2-to-M phase transition in the cell cycle. In addition, apigenin upregulated p21WAF1/CIP1 and increased the interaction of p21WAF1/CIP1 with proliferating cell nuclear antigen (PCNA), which inhibits cell cycle progression. Furthermore, apigenin significantly inhibited histone deacetylase (HDAC) activity and induced histone H3 acetylation. The subsequent chromatin immunoprecipitation (ChIP) assay indicated that apigenin increased acetylation of histone H3 in the p21WAF1/CIP1 promoter region, resulting in the increase of p21WAF1/CIP1 transcription. In a tumor xenograft model, apigenin effectively delayed tumor growth. In these apigenin-treated tumors, we also observed reductions in the levels of cyclin A and cyclin B and increases in the levels of p21WAF1/CIP1 and acetylated histone H3. These findings demonstrate for the first time that apigenin can be used in breast cancer prevention and treatment through epigenetic regulation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 434-444, 2017.

6-Mercaptopurine attenuates tumor necrosis factor-α production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation.

BACKGROUND: The pathogenesis of several neurodegenerative diseases often involves the microglial activation and associated inflammatory processes. Activated microglia release pro-inflammatory factors that may be neurotoxic. 6-Mercaptopurine (6-MP) is a well-established immunosuppressive drug. Common understanding of their immunosuppressive properties is largely limited to peripheral immune cells. However, the effect of 6-MP in the central nervous system, especially in microglia in the context of neuroinflammation is, as yet, unclear. Tumor necrosis factor-α (TNF-α) is a key cytokine of the immune system that initiates and promotes neuroinflammation. The present study aimed to investigate the effect of 6-MP on TNF-α production by microglia to discern the molecular mechanisms of this modulation. METHODS: Lipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Released TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Signaling molecules were analyzed by western blotting, and activation of NF-κB was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Chromatin immunoprecipitation (ChIP) analysis was performed to examine NF-κB p65 and coactivator p300 enrichments and histone modifications at the endogenous TNF-α promoter. RESULTS: Treatment of LPS-activated microglia with 6-MP significantly attenuated TNF-α production. In 6-MP pretreated microglia, LPS-induced MAPK signaling, IκB-α degradation, NF-κB p65 nuclear translocation, and in vitro p65 DNA binding activity were not impaired. However, 6-MP suppressed transactivation activity of NF-κB and TNF-α promoter by inhibiting phosphorylation and acetylation of p65 on Ser276 and Lys310, respectively. ChIP analyses revealed that 6-MP dampened LPS-induced histone H3 acetylation of chromatin surrounding the TNF-α promoter, ultimately leading to a decrease in p65/coactivator-mediated transcription of TNF-α gene. Furthermore, 6-MP enhanced orphan nuclear receptor Nur77 expression. Using RNA interference approach, we further demonstrated that Nur77 upregulation contribute to 6-MP-mediated inhibitory effect on TNF-α production. Additionally, 6-MP also impeded TNF-α mRNA translation through prevention of LPS-activated PI3K/Akt/mTOR signaling cascades. CONCLUSIONS: These results suggest that 6-MP might have a therapeutic potential in neuroinflammation-related neurodegenerative disorders through downregulation of microglia-mediated inflammatory processes.

P/CAF-mediated spermidine acetylation regulates histone acetyltransferase activity.

Histones and polyamines are important determinants of the chromatin structure. Histones form the core of nucleosome particles and their modification by acetylation of N-terminal tails is involved in chromatin structural changes and transcriptional regulation. Polyamines, including spermidine, are also targets of both cytoplasmic and nuclear acetylation, which in turn alters their affinity for DNA and nucleosomes. Previous studies report the interplay between polyamines metabolism and levels of histone acetylation, but the molecular basis of this effect is still unclear. In this work, we have analyzed the in vitro effect of spermidine on histone H3 acetylation catalyzed by P/CAF, a highly conserved histone acetyltransferase (HAT) (E.C. 2.3.1.48). We have observed that spermidine at very low concentrations activates P/CAF, while it has an inhibitory effect at concentrations higher than 4 µM. In addition, the in vitro bimodal effect of spermidine on histone H3 acetylation was also distinctly observed in vivo on polytene chromosomes of Drosophila melanogaster. We also performed kinetic studies indicating that the activating effect of low spermidine concentrations on P/CAF-HAT activity is based on its involvement as a substrate for P/CAF to produce N8-acetylspermidine that is able in turn to increase the enzyme activity up to four fold.

Regulation of Retinal Development via the Epigenetic Modification of Histone H3.

We are interested in the roles of epigenetic mechanisms in retinal development. By ChIP-qPCR using whole retinal extracts at various developmental stages, we found that the levels of methylation of histones H3K27 and H3K4 and acetylation of histone H3 at specific loci in various genes, which play critical roles in retinal proliferation and differentiation, changed dramatically during retinal development. We next focused on the roles of H3K27 trimethylation in retinal development. Ezh1 and Ezh2 are methyltransferases that act on H3K27, while Jmjd3 and Utx are demethylases. We found that Ezh2 and Jmjd3 were mainly expressed during retinal development, and a loss-of-function of these genes revealed a role for H3K27me3 in the maturation of subsets of bipolar cells. Furthermore, Ezh2 and Jmjd3 regulate H3K27 trimethylation at specific loci within Bhlhb4 and Vsx1, which play critical roles in the differentiation of subsets of bipolar cells. Utx is expressed weakly in retina, and the down-regulation of Utx by sh-RNA in retinal explants suggested that Utx also participates in the maturation of bipolar cells. Ezh1 is expressed weakly in postnatal retina, and the phenotype of Ezh2-knockout retina suggested that Ezh1 plays a role in the methylation of H3K27 in the late phase of retinal differentiation. Taken together, we found that these four genes, which exhibit temporally and spatially unique expression patterns during retinal development, play critical roles in the differentiation of retinal subsets through the regulation of histone H3K27 methylation at critical genetic loci.

Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

Extracellular cold-inducible RNA-binding protein mediated neuroinflammation and neuronal apoptosis after traumatic brain injury.

Background: Extracellular cold-inducible RNA-binding protein (eCIRP) plays a vital role in the inflammatory response during cerebral ischaemia. However, the potential role and regulatory mechanism of eCIRP in traumatic brain injury (TBI) remain unclear. Here, we explored the effect of eCIRP on the development of TBI using a neural-specific CIRP knockout (KO) mouse model to determine the contribution of eCIRP to TBI-induced neuronal injury and to discover novel therapeutic targets for TBI. Methods: TBI animal models were generated in mice using the fluid percussion injury method. Microglia or neuron lines were subjected to different drug interventions. Histological and functional changes were observed by immunofluorescence and neurobehavioural testing. Apoptosis was examined by a TdT-mediated dUTP nick end labelling assay in vivo or by an annexin-V assay in vitro. Ultrastructural alterations in the cells were examined via electron microscopy. Tissue acetylation alterations were identified by non-labelled quantitative acetylation via proteomics. Protein or mRNA expression in cells and tissues was determined by western blot analysis or real-time quantitative polymerase chain reaction. The levels of inflammatory cytokines and mediators in the serum and supernatants were measured via enzyme-linked immunoassay. Results: There were closely positive correlations between eCIRP and inflammatory mediators, and between eCIRP and TBI markers in human and mouse serum. Neural-specific eCIRP KO decreased hemispheric volume loss and neuronal apoptosis and alleviated glial cell activation and neurological function damage after TBI. In contrast, eCIRP treatment resulted in endoplasmic reticulum disruption and ER stress (ERS)-related death of neurons and enhanced inflammatory mediators by glial cells. Mechanistically, we noted that eCIRP-induced neural apoptosis was associated with the activation of the protein kinase RNA-like ER kinase-activating transcription factor 4 (ATF4)-C/EBP homologous protein signalling pathway, and that eCIRP-induced microglial inflammation was associated with histone H3 acetylation and the α7 nicotinic acetylcholine receptor. Conclusions: These results suggest that TBI obviously enhances the secretion of eCIRP, thereby resulting in neural damage and inflammation in TBI. eCIRP may be a biomarker of TBI that can mediate the apoptosis of neuronal cells through the ERS apoptotic pathway and regulate the inflammatory response of microglia via histone modification.

Lithium-Containing Biomaterials Stimulate Cartilage Repair through Bone Marrow Stromal Cells-Derived Exosomal miR-455-3p and Histone H3 Acetylation.

The repair of damaged cartilage still remains a great challenge in clinic. It is demonstrated that bone marrow stromal cells (BMSCs)-chondrocytes communication is of great significance for cartilage repair. Moreover, BMSCs have been confirmed to enhance biological function of chondrocytes via exosome-mediated paracrine pathway. Lithium-containing scaffolds have been reported to effectively promote cartilage regeneration; however, whether lithium-containing biomaterial could facilitate cartilage regeneration through regulating BMSCs-derived exosomes has not been illustrated. In the study, the model lithium-substituted bioglass ceramic (Li-BGC) is selected and regulatory effects of BMSCs-derived exosomes after Li-BGC treatment (Li-BGC-Exo) are systemically evaluated. The data reveal that Li-BGC-Exo notably promotes chondrogenesis, which attributes to the upregulated exosomal miR-455-3p transfer, consequently leads to suppression of histone deacetylase 2 (HDAC2) and enhanced histone H3 acetylation in chondrocytes. Notably, BMSCs-derived exosomes after LiCl treatment (LiCl-Exo) exhibits the similar regulatory effect with Li-BGC-Exo, indicating that the pro-chondrogenesis capability of them is mainly owing to the lithium ions. Furthermore, the in vivo study proves that LiCl-Exo remarkably facilitates cartilage regeneration. The research may provide novel possibility for the intrinsic mechanism of chondrogenesis trigged by lithium-containing biomaterials, and suggests that application of lithium-containing scaffolds may be a promising strategy for cartilage regeneration.

Tributyrin Mitigates Ethanol-Induced Lysine Acetylation of Histone-H3 and p65-NFκB Downregulating CCL2 Expression and Consequent Liver Inflammation and Injury.

PURPOSE: Chemokine-driven leukocyte infiltration and sustained inflammation contribute to alcohol-associated liver disease (ALD). Elevated hepatic CCL2 expression, seen in ALD, is associated with disease severity. However, mechanisms of CCL2 regulation are not completely elucidated. Post-translational modifications (PTMs) of proteins, particularly acetylation, modulate gene expression. This study examined the acetylation changes of promoter-associated histone-H3 and key transcription factor-NFκB in regulating hepatic CCL2 expression and subsequent inflammation and injury. Further, the effect of therapeutic modulation of the acetylation state by tributyrin (TB), a butyrate prodrug, was assessed. METHODS: Hepatic CCL2 expression was assessed in mice fed control (PF) or an ethanol-containing Lieber-DeCarli (5% v/v, EF) diet for 7 weeks with or without oral administration of tributyrin (TB, 2 g/kg, 5 days/week). A chromatin immunoprecipitation (ChIP) assay evaluated promoter-associated modifications. Nuclear association between SIRT1, p300, and NFκB-p65 and acetylation changes of p65 were determined using immunoprecipitation and Western blot analyses. A Student's t-test and one-way ANOVA determined the significance. RESULTS: Ethanol significantly increased promoter-associated histone-H3-lysine-9 acetylation (H3K9Ac), reflecting a transcriptionally permissive state with a resultant increase in hepatic CCL2 mRNA and protein expression. Moreover, increased lysine-310-acetylation of nuclear RelA/p65 decreased its association with SIRT1, a class III HDAC, but concomitantly increased with p300, a histone acetyltransferase. This further led to enhanced recruitment of NF-κB/p65 and RNA polymerase-II to the CCL2 promoter. Oral TB administration prevented ethanol-associated acetylation changes, thus downregulating CCL2 expression, hepatic neutrophil infiltration, and inflammation/ injury. CONCLUSION: The modulation of a protein acetylation state via ethanol or TB mechanistically regulates hepatic CCL2 upregulation in ALD.

AIMP1 promotes multiple myeloma malignancy through interacting with ANP32A to mediate histone H3 acetylation.

BACKGROUND: Multiple myeloma (MM) is the second most common hematological malignancy. An overwhelming majority of patients with MM progress to serious osteolytic bone disease. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) participates in several steps during cancer development and osteoclast differentiation. This study aimed to explore its role in MM. METHODS: The gene expression profiling cohorts of MM were applied to determine the expression of AIMP1 and its association with MM patient prognosis. Enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting were used to detect AIMP1 expression. Protein chip analysis, RNA-sequencing, and chromatin immunoprecipitation and next-generation sequencing were employed to screen the interacting proteins and key downstream targets of AIMP1. The impact of AIMP1 on cellular proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro and a xenograft model in vivo. Bone lesions were evaluated using tartrate-resistant acid phosphatase staining in vitro. A NOD/SCID-TIBIA mouse model was used to evaluate the effect of siAIMP1-loaded exosomes on bone lesion formation in vivo. RESULTS: AIMP1 expression was increased in MM patients and strongly associated with unfavorable outcomes. Increased AIMP1 expression promoted MM cell proliferation in vitro and in vivo via activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Protein chip assays and subsequent experiments revealed that AIMP1 interacted with acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) to regulate histone H3 acetylation. In addition, AIMP1 increased histone H3 acetylation enrichment function of GRB2-associated and regulator of MAPK protein 2 (GAREM2) to increase the phosphorylation of extracellular-regulated kinase 1/2 (p-ERK1/2). Furthermore, AIMP1 promoted osteoclast differentiation by activating nuclear factor of activated T cells c1 (NFATc1) in vitro. In contrast, exosome-coated small interfering RNA of AIMP1 effectively suppressed MM progression and osteoclast differentiation in vitro and in vivo. CONCLUSIONS: Our data demonstrate that AIMP1 is a novel regulator of histone H3 acetylation interacting with ANP32A in MM, which accelerates MM malignancy via activation of the MAPK signaling pathway.

LACTB promotes metastasis of nasopharyngeal carcinoma via activation of ERBB3/EGFR-ERK signaling resulting in unfavorable patient survival.

Nasopharyngeal carcinoma (NPC) originates in the nasopharyngeal epithelium and has the highest metastatic rate among head and neck cancers. Distant metastasis is the main reason for treatment failure with the underlying mechanisms remaining unclear. By comparing the expression profiling of NPCs versus non-cancerous nasopharyngeal tissues, we found LACTB was highly expressed in the tumor tissues. We found that elevated expression of the LACTB protein in primary NPCs correlated with poorer patient survival. LACTB is known to be a serine protease and a ubiquitous mitochondrial protein localized in the intermembrane space. Its role in tumor biology remains controversial. We found that the different methylation pattern of LACTB promoter led to its differential expression in NPC cells. Overexpressing LACTB in NPC cells promoted their motility in vitro and metastasis in vivo. While knocking down LACTB reduced the metastasis capability of NPC cells. However, LACTB did not influence cellular proliferation. We further found the role of LACTB in promoting NPC metastasis depended on the activation of ERBB3/EGFR-ERK signaling, which in turn, affected the stability and the following acetylation of histone H3. These findings may shed light on unveiling the mechanisms of NPC metastasis.

Remodeling the homeostasis of pro- and anti-angiogenic factors by Shenmai injection to normalize tumor vasculature for enhanced cancer chemotherapy.

ETHNOPHARMACOLOGICAL RELEVANCE: Normalization of the tumor vasculature can enhance tumor perfusion and the microenvironment, leading to chemotherapy potentiation. Shenmai injection (SMI) is a widely used traditional Chinese herbal medicine for the combination treatment of cancer in China. AIM OF THIS STUDY: This study aimed to investigate whether SMI can regulate tumor vasculature to improve chemotherapy efficacy and identify the underlying mechanism. MATERIALS AND METHODS: The antitumor effect of SMI combined with 5-florouracil (5-FU) was investigated in xenograft tumor mice. Two-photon microscopy, laser speckle contrast imaging and immunofluorescence staining were used to investigate the effects of SMI on tumor vasculature in vivo. The mRNA and protein expression of pro- and anti-angiogenic factors were measured by Q-PCR and ELISA. Histone acetylation and transcriptional regulation were detected by Western blot and ChIP assay. RESULTS: SMI promoted normalization of tumor microvessels within a certain time window, which was accompanied by enhanced blood perfusion and 5-FU distribution in tumors. SMI significantly increased the expression of antiangiogenic factor angiostatin and decreased the pro-angiogenic factors VEGF, FGF and PAI-1 by day 10. SMI combined with neoadjuvant chemotherapy in colorectal cancer patients also showed a significant increase in angiostatin and decrease in VEGF and FGF in surgically resected tumors when compared to the neoadjuvant chemotherapy group. Further in vitro and in vivo studies revealed that SMI downregulated VEGF, FGF and PAI-1 mRNA expression by inhibiting histone H3 acetylation at the promoter regions. The enhanced production of angiostatin was attributed to the regulation of the plasminogen proteolysis system via SMI-induced PAI-1 inhibition. CONCLUSION: SMI can remodel the homeostasis of pro- and anti-angiogenic factors to promote tumor vessel normalization, and thus enhance drug delivery and anti-tumor effect. This study provides additional insights into the pharmacological mechanisms of SMI on tumors from the perspective of vascular regulation.

Development and Validation of High-Content Analysis for Screening HDAC6-Selective Inhibitors.

Throughout recent decades, histone deacetylase (HDAC) inhibitors have shown encouraging potential in cancer treatment, and several pan-HDAC inhibitors have been approved for treating malignant cancers. Numerous adverse effects of pan-HDAC inhibitors have been reported, however, during preclinical and clinical evaluations. To avoid undesirable responses, an increasing number of investigations are focusing on the development of isotype-selective HDAC inhibitors. In this study, we present an effective and quantitative cellular assay using high-content analysis (HCA) to determine compounds' inhibition of the activity of HDAC6 and Class I HDAC isoforms, by detecting the acetylation of their corresponding substrates (i.e., α-tubulin and histone H3). Several conditions that are critical for HCA assays, such as cell seeding number, fixation and permeabilization reagent, and antibody dilution, have been fully validated in this study. We used selective HDAC6 inhibitors and inhibitors targeting different HDAC isoforms to optimize and validate the capability of the HCA assay. The results indicated that the HCA assay is a robust assay for quantifying compounds' selectivity of HDAC6 and Class I HDAC isoforms in cells. Moreover, we screened a panel of compounds for HDAC6 selectivity using this HCA assay, which provided valuable information for the structure-activity relationship (SAR). In summary, our results suggest that the HCA assay is a powerful tool for screening selective HDAC6 inhibitors.