大鼠基底外侧杏仁核组蛋白乙酰化对吗啡成瘾记忆的调控作用

为了探索组蛋白乙酰化对吗啡成瘾记忆相关分子表达调控机制,文章选取健康成年雄性SD大鼠34只,随机分为正常对照组(n = 6)及基底外侧杏仁核(Basolateral amygdala, BLA)颅内定位手术组(n =28)。在条件性位置偏爱(Conditioned place preference, CPP)训练阶段,大鼠BLA内给予组蛋白去乙酰化酶抑制剂曲古抑菌素A(Trichostafin A, TSA)并且腹腔注射吗啡溶液(10.0 mg/kg),对照组给予相同体积的10%二甲基亚砜(Dimethyl sulfoxide,DMSO)或盐水。应用蛋白质印记方法,检测吗啡诱导大鼠CPP建立后BLA内组蛋白H3K14乙酰化和脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)蛋白表达水平。结果显示,腹腔注射10 mg/kg吗啡能成功建立CPP。吗啡、TSA联合给药组大鼠比单纯吗啡给药组大鼠表现出更强烈的CPP(P<0.0001)。吗啡和TSA都能使BLA内的组蛋白H3乙酰化水平和BDNF的表达显著增高(P < 0.0001),同时二者之间具有协同作用。结果表明,大鼠BLA内组蛋白乙酰化水平与吗啡成瘾记忆形成有关,抑制BLA内组蛋白去乙酰化酶(Histone deacetylases, HDACs)的活性可强化吗啡诱导的线索记忆的形成;大鼠BLA内BDNF参与了吗啡诱导的线索记忆的形成并可能受到组蛋白乙酰化的调控。     

CD1d induction in solid tumor cells by histone deacetylase inhibitors through inhibition of HDAC1/2 and activation of Sp1

CD1d is a MHC class-like molecule that presents glycolipids to natural killer T (NKT) cells, then regulates innate and adaptive immunity. The regulation of CD1d gene expression in solid tumors is still largely unknown. Gene expression can be epigenetically regulated by DNA methylation and histone acetylation. We found that histone deacetylase inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), induced CD1d gene expression in human (A549 and NCI-H292) and mouse (TC-1 and B16/F0) cancer cells. Simultaneous knockdown of HDAC1 and 2 induced CD1d gene expression. Sp1 inhibitor mitramycin A (MTM) blocked TSA- and SAHA-induced CD1d mRNA expression and Sp1 luciferase activity. Co-transfection of GAL4-Sp1 and Fc-luciferase reporters demonstrated that TSA and SAHA induced Sp1 luciferase reporter activity by enhancing Sp1 transactivation activity. The binding of Sp1 to CD1d promoter and histone H3 acetylation on Sp1 sites were increased by TSA and SAHA. These results indicate that TSA and SAHA could up-regulate CD1d expression in tumor cells through inhibition of HDAC1/2 and activation of Sp1.     

CD1d induction in solid tumor cells by histone deacetylase inhibitors through inhibition of HDAC1/2 and activation of Sp1

CD1d is a MHC class-like molecule that presents glycolipids to natural killer T (NKT) cells, then regulates innate and adaptive immunity. The regulation of CD1d gene expression in solid tumors is still largely unknown. Gene expression can be epigenetically regulated by DNA methylation and histone acetylation. We found that histone deacetylase inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), induced CD1d gene expression in human (A549 and NCI-H292) and mouse (TC-1 and B16/F0) cancer cells. Simultaneous knockdown of HDAC1 and 2 induced CD1d gene expression. Sp1 inhibitor mitramycin A (MTM) blocked TSA- and SAHA-induced CD1d mRNA expression and Sp1 luciferase activity. Co-transfection of GAL4-Sp1 and Fc-luciferase reporters demonstrated that TSA and SAHA induced Sp1 luciferase reporter activity by enhancing Sp1 transactivation activity. The binding of Sp1 to CD1d promoter and histone H3 acetylation on Sp1 sites were increased by TSA and SAHA. These results indicate that TSA and SAHA could up-regulate CD1d expression in tumor cells through inhibition of HDAC1/2 and activation of Sp1.     

Dynamic Histone Acetylation of Late Embryonic Genes during Seed Germination

Histone acetylation is involved in the regulation of gene expression in plants and eukaryotes. Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from histones, which is associated with the repression of gene expression. To study the role of histone acetylation in the regulation of gene expression during seed germination, trichostatin A (TSA), a specific inhibitor of histone deacetylase, was used to treat imbibing Arabidopsis thaliana seeds. GeneChip arrays were used to show that TSA induces up-regulation of 45 genes and down-regulation of 27 genes during seed germination. Eight TSA-up-regulated genes were selected for further analysis – RAB18, RD29B, ATEM1, HSP70 and four late embryogenesis abundant protein genes (LEA). A gene expression time course shows that these eight genes are expressed at high levels in the dry seed and repressed upon seed imbibition at an exponential rate. In the presence of TSA, the onset of repression of the eight genes is not affected but the final level of repressed expression is elevated. Chromatin immunoprecipitation and HDAC assays show that there is a transient histone deacetylation event during seed germination at 1 day after imbibition, which serves as a key developmental signal that affects the repression of the eight genes. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Regulation of RANKL promoter activity is associated with histone remodeling in murine bone stromal cells

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)2D3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)2D3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.     

Histone deacetylase inhibitors decrease reelin promoter methylation in vitro

We investigated the effects of agents that induce reelin mRNA expression in vitro on the methylation status of the human reelin promoter in neural progenitor cells (NT2). NT2 cells were treated with the histone deacetylase inhibitors, trichostatin A (TSA) and valproic acid (VPA), and the methylation inhibitor aza-2'-deoxycytidine (AZA) for various times. All three drugs reduced the methylation profile of the reelin promoter relative to untreated cells. The acetylation status of histones H3 and H4 increased following treatment with VPA and TSA at times as short as 15 min following treatment; a result consistent with the reported mode of action of these drugs. Chromatin immunoprecipitation experiments showed that these changes were accompanied by changes occurring at the level of the reelin promoter as well. Interestingly, AZA decreased reelin promoter methylation without concomittantly increasing histone acetylation. In fact, after prolonged treatments with AZA, the acetylation status of histones H3 and H4 decreased relative to untreated cells. We also observed a trend towards reduced methylated H3 after 18 h treatment with TSA and VPA. Our data indicate that while TSA and VPA act to increase histone acetylation and reduce promoter methylation, AZA acts only to decrease the amount of reelin promoter methylation.     

Changes in histone modifications during in vitro maturation of porcine oocytes

Nuclear core histone modifications influence chromosome structures and functions. Recently, the involvement of histone acetylations in the cell memory of gene expression has been suggested in mouse oocyte maturation. At present, there is little available data on histone modifications in mammalian oocyte maturation. In the present study, we examined changes in the acetylation of histone H3 lysines 9 (H3K9) and 14 (H3K14), and histone H4 lysines 5 (H4K5), 8 (H4K8) and 12 (H4K12), and trimethylation of H3K9 during in vitro maturation of porcine oocytes. Immunocytochemical analyses revealed that the all of the lysines examined were highly acetylated in the germinal vesicle stage, and this level of acetylation was maintained until the first prometaphase. In the first metaphase, the lysines near the N-terminal end, H3K9 and H4K5, were completely deacetylated. The acetylation of the lysines far from the N-terminal end, H3K14, H4K8, and H4K12, was markedly decreased but still present. The acetylations were increased transiently at the first anaphase and telophase, and then decreased again at the second metaphase to the same level as the first metaphase. Since effective concentrations of trichostatin A (TSA) to inhibit the deacetylation were different in various lysine residues, multiple histone deacetylases (HDACs) were suggested to function during meiotic maturation. The trimethylation of H3K9 was maintained in a high level throughout maturation. These results suggest that the histone acetylation during porcine oocyte maturation is precisely controlled by the cell cycle.     

Inhibition of histone deacetylation protects wild-type but not gelsolin-deficient neurons from oxygen/glucose deprivation

Histone acetylation and deacetylation participate in the epigenetic regulation of gene expression. In this paper, we demonstrate that pre-treatment with the histone deacetylation inhibitor trichostatin A (TSA) enhances histone acetylation in primary cortical neurons and protects against oxygen/glucose deprivation, a model for ischaemic cell death in vitro. The actin-binding protein gelsolin was identified as a mediator of neuroprotection by TSA. TSA enhanced histone acetylation of the gelsolin promoter region, and up-regulated gelsolin messenger RNA and protein expression in a dose- and time-dependent manner. Double-label confocal immunocytochemistry visualized the up-regulation of gelsolin and histone acetylation within the same neuron. Together with gelsolin up-regulation, TSA pre-treatment decreased levels of filamentous actin. The neuroprotective effect of TSA was completely abolished in neurons lacking gelsolin gene expression. In conclusion, we demonstrate that the enhancement of gelsolin gene expression correlates with neuroprotection induced by the inhibition of histone deacetylation.