组蛋白化学修饰改变对c-Myb结合的影响及其在职业性苯中毒患者造血损伤中的作用

 目的:研究职业性苯中毒造血损伤患者中与拓扑异构酶Ⅱα启动子调控因子c-Myb结合的组蛋白化学修饰改变, 证实组蛋白乙酰化修饰水平改变在职业性苯中毒造血损伤中发挥一定的作用。方法:25例职业性苯中毒再生障碍性贫血患者为病例组,25例正常人为对照组,提取骨髓单个核细胞,用染色质免疫沉淀(ChIP)探讨与c-Myb结合的组蛋白乙酰化和甲基化水平的改变,RT-PCR法检测c-Myb的mRNA表达水平,组蛋白去乙酰化酶(HDAC)试剂盒检测HDAC活性的变化。结果:与正常对照组相比,职业性苯中毒再生障碍性贫血患者c-Myb与乙酰化组蛋白H4、H3结合的水平下降(P<0.01), 而与甲基化组蛋白H3K4和H3K9结合的水平无明显改变,差异无统计学显著性。与正常对照组相比,职业性苯中毒再生障碍性贫血患者c-Myb的mRNA表达水平降低,HDAC活性明显升高,差异均有统计学显著性(P<0.05)。结论:拓扑异构酶Ⅱα启动子调控因子c-Myb可能通过组蛋白乙酰化修饰的改变在职业性苯中毒造血损伤中发挥作用。     

Histone deacetylase 3 is required for centromeric H3K4 deacetylation and sister chromatid cohesion

We describe here the role of histone deacetylase 3 (HDAC3) in sister chromatid cohesion and the deacetylation of histone H3 Lys 4 (H3K4) at the centromere. HDAC3 knockdown induced spindle assembly checkpoint activation and sister chromatid dissociation. The depletion of Polo-like kinase 1 (Plk1) or Aurora B restored cohesion in HDAC3-depleted cells. HDAC3 was also required for Shugoshin localization at centromeres. Finally, we show that HDAC3 depletion results in the acetylation of centromeric H3K4, correlated with a loss of dimethylation at the same position. These findings provide a functional link between sister chromatid cohesion and the mitotic "histone code".     

细胞复制性衰老及早衰过程中组蛋白整体乙酰化改变

目的 检测人胚肺成纤维细胞复制性衰老及过氧化氢诱导细胞早衰过程中组蛋白整体乙酰化修饰的改变.方法 应用细胞免疫荧光实验观察组蛋白乙酰化水平变化,并基于ELISA样反应方法检测组蛋白总体去乙酰化酶的活性变化,荧光定量PCR检测乙酰化酶mRNA表达,荧光定量PCR和Western印迹检测去乙酰化酶表达变化及曲古霉素A对相应酶表达的影响.结果 在细胞复制性衰老及细胞早衰过程中,组蛋白H3和H4整体乙酰化水平逐渐下降;去乙酰化酶活性逐渐降低;与年轻细胞组相比,中年细胞与复制性衰老细胞组P300表达下降;中年细胞组PCAF稍升高,复制性衰老细胞组降低;早衰起始组P300和PCAF均升高,早衰持续组P300降低;中年细胞组HDAC1表达稍降低;复制性衰老细胞组HDAC2稍降低;而HDAC3均降低;早衰起始组HDAC1,HDAC3有不同程度升高;早衰持续组HDAC2,HDAC3降低显著,而HDAC1明显升高.曲古霉素A诱导P300,PCAF表达,而降低HDAC1,HDAC2和HDAC3表达.结论 组蛋白H3和H4整体低乙酰化是衰老细胞的伴随状态;细胞复制性衰老与过氧化氢诱导的早衰内在调控机制存在差别.     

苯中毒病人TOPOⅡα启动子调控因子Sp1组蛋白化学修饰改变

目的:研究拓扑异构酶Ⅱα(TOPOⅡα)启动子调控因子SP1组蛋白化学修饰在苯中毒病人中的改变。方法:25例临床慢性苯中毒患者骨髓单个核细胞为病例组,25例正常人骨髓单个核细胞为对照组,染色质免疫沉淀技术探讨TOPOⅡα启动子调控因子Sp1组蛋白乙酰化和甲基化水平的变化,RT-PCR法测定Sp1 mRNA的表达水平。结果:与对照组相比,临床苯中毒病例TOPOⅡα启动子调控因子Sp1组蛋白H4和H3乙酰化水平下降(P0.01),组蛋白H3K9甲基化水平升高(P0.01),组蛋白H3K4甲基化水平无明显改变。与正常对照组相比,临床苯中毒病例TOPOⅡα启动子调控因子Sp1的mRNA表达水平降低(P0.05)。结论:慢性苯中毒TOPOⅡα启动子调控因子Sp1组蛋白H4、H3乙酰化及H3K9甲基化修饰水平的改变伴随着mRNA水平的变化。TOPOⅡα启动子调控因子Sp1可能通过组蛋白H4、H3乙酰化及H3K9甲基化修饰改变在苯中毒所致的造血毒性中发挥作用。     

Histone deacetylase 3 inhibits expression of PUMA in gastric cancer cells

During cancer development, tumor suppressor genes were silenced by promoter methylation or histone deacetylation. Histone deacetylases (HDACs) are important to maintain histone deacetylation. HDAC inhibitors (HDACis) were thus proposed as a new therapeutic approach to cancer. The current study aims to understand the effect and molecular mechanisms of HDACis on gastric cancer cells. Trichostatin A (TSA) significantly inhibited the growth of gastric cancer cells by inducing apoptosis. Gene profiling results showed PUMA (p53 upregulated modulator of apoptosis) as one of 122 genes upregulated in TSA-treated gastric cancer cells. PUMA was downregulated in gastric cancer cell lines and primary gastric carcinoma tissues. Patients with low PUMA expression had significant decreases in overall survival (HR, 2.04; p?=?0.047). Ectopic PUMA expression inhibited the growth of gastric cancer cells while PUMA depletion promoted cellular growth. The knockdown of HDAC3 but not other HDACs upregulated PUMA expression. HDAC3 could bind to PUMA promoter, which was abrogated after TSA treatment. In contrast to TSA and SB, HDAC3 siRNA failed to upregulate p53 expression but promoted the interaction of p53 with PUMA promoter. In summary, proapoptotic PUMA was downregulated in gastric cancer and its mRNA expression level is a valuable prognosis factor for gastric cancer. HDAC3 is important to downregulate PUMA expression in gastric cancer and HDACis, like TSA, promoted PUMA expression through stabilizing p53 in addition to HDAC3 inhibition. In combination with chemotherapy, targeting HDAC3 might be a promising strategy to induce apoptosis of gastric cancer cells.     

曲古抑菌素A对核移植供体细胞组蛋白H3乙酰化水平的影响

目的探讨组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)对组蛋白H3乙酰化水平的影响,进而提高他们重编程的能力。方法用不同浓度的TSA处理胎儿成纤维细胞,应用免疫荧光和WesternBlot方法对细胞乙酰化水平进行量化分析。结果0.5nM,5nM,50nM和100nM浓度的TSA均引起供体成纤维细胞形态学的变化,对供体成纤维细胞组蛋白H3乙酰化有明显的剂量效应,随着TSA浓度增加,组蛋白H3乙酰化水平逐渐增加,在TSA浓度5nM时比0.5nM增加尤为明显。结论TSA能够提高胎儿成纤维细胞组蛋白H3乙酰化水平,提高核移植重编程的能力。     

同型半胱氨酸对小鼠卵母细胞成熟过程中组蛋白表观遗传修饰的影响

目的 探讨卵母细胞发育过程中同型半胱氨酸（HCY）对组蛋白表观遗传修饰的影响。 方法 首先使用10只2周ICR雌性小鼠建立完整卵泡的体外培养体系,在卵母细胞发育早期,利用免疫组织化学方法,观察HCY对甲基化组蛋白H3K4、H3K9以及乙酰化组蛋白H3K9分布的影响;其次使用10只4周ICR雌性小鼠,利用卵母细胞的体外成熟培养体系,观察HCY对卵母细胞成熟过程的影响,同时利用实时定量PCR方法,观察在此成熟过程中HCY对卵母细胞内组蛋白乙酰化水平的调控酶GCN5和HDAC表达的影响。 结果 HCY明显抑制卵母细胞的体外成熟,在HCY作用下,甲基化组蛋白H3K4、H3K9以及乙酰化组蛋白H3K9的分布没有变化,但是表达强度降低,核呈现去浓缩的趋势。成熟过程中HCY并不改变基因GCN5的表达水平,却明显抑制卵母细胞内HDAC基因的表达。 结论 卵母细胞发育过程中高水平的同型半胱氨酸影响组蛋白的表观遗传修饰,HCY对卵母细胞核内组蛋白甲基化和乙酰化修饰的影响有可能是造成核染色体稳定性下降的重要原因。     

p38MAPK通过上调组蛋白乙酰化水平影响APP表达

 摘要：目的 研究p38MAPK信号通路的激活对淀粉前体蛋白（β-Amyloid precursor protein, APP）表达的影响及其相关表观遗传学机制。方法 采用体外培养神经母细胞瘤细胞（SH-SY5Y）,Western blot方法检测p38MAPK信号通路激活后APP蛋白的表达及组蛋白乙酰化酶(Histone Acetyltranferase, HAT)和组蛋白去乙酰化酶(Histone Deacetyltranferase, HDAC)的表达情况;光密度值法检测组蛋白H3和H4整体乙酰化水平。结果 p38MAPK信号通路经特异性激动剂激活72小时后,APP表达明显增高至对照组1.5倍;同时组蛋白H3整体乙酰化水平增高,但H4乙酰化水平无明显改变;Western blot结果显示,组蛋白乙酰化酶 CBP表达增高至对照组2.5倍,而组蛋白去乙酰化酶HDAC3表达下降至对照组40%。结论 以上结果提示,p38MAPK信号通路可能通过对组蛋白乙酰化水平的调节影响APP蛋白的表达。     

Interactive effects of histone deacetylase inhibitors and TRAIL on apoptosis in human leukemia cells: involvement of both death receptor and mitochondrial pathways

In the present study, we aimed to elucidate the mechanism responsible for the interactive effects of histone deacetylase (HDAC) inhibitors [suberoylanilide hydroxamic acid (SAHA), MS-275, m-carboxycinnamic acid bishydroxamide (CBHA), and trichostatin-A (TSA)] and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on apoptosis in leukemia cells. HDAC inhibitors enhance the apoptosis-inducing potential of TRAIL in leukemia cells (HL60, Jurkat, K562, and U937) through multiple mechanisms; up-regulation of DR4, DR5, Bak, Bax, Bim, Noxa and PUMA, down-regulation of IAPs, Mcl-1, Bcl-2, Bcl-XL and cFLIP, release of mitochondrial proteins (cytochrome c, Smac/DIABLO and Omi/Htr2) to the cytosol, induction of p21WAF1/CIP1 and p27KIP1, activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). The sequential treatment of cells with HDAC inhibitors followed by TRAIL was more effective in inducing apoptosis than the concurrent treatment or single agent alone. The up-regulation of death receptors and inhibition of cFLIP by HDAC inhibitors will increase the ability of TRAIL to induce apoptosis, due to enhance activation of caspase-8, cleavage of Bid, and release of mitochondrial proteins to the cytosol, and subsequent activation of caspase-9 and caspase-3. Thus, the combination of HDAC inhibitors and TRAIL can be used as a new therapeutic approach for the treatment of leukemia.     

Histone Acetylation Level and Histone Acetyltransferase/Deacetylase Activity in Ejaculated Sperm from Normozoospermic Men

Purpose

The aim of this work was to evaluate nuclear histone acetylation level and total histone acetyltransferase (HAT) and deacetylase (HDAC) activity in ejaculated sperm and their relevance to conventional sperm parameters.

Materials and Methods

Thirty-three normozoospermic men were included in this study. Semen samples were processed by swim-up and then immunostained by six acetylation antibodies (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac). Our preliminary study verified the expression of HAT/HDAC1 in mature human sperm. From vitrified-warmed sperm samples, total HAT/HDAC activity was measured by commercially available kits. Nuclear DNA integrity was also measured by TUNEL assay.

Results

The levels of six acetylation marks were not related with conventional sperm parameters including sperm DNA fragmentation index (DFI) as well as HAT/HDAC activity. However, sperm DFI was positively correlated with HAT activity (r=0.038 after adjustment, p<0.02). HAT activity showed a negative relationship with HDAC activity (r=-0.51, p<0.01). Strict morphology was negatively correlated with acetylation enzyme index (=HAT activity/HDAC activity) (r=-0.53, p<0.01).

Conclusion

Our works demonstrated a significant relationship of acetylation-associated enzyme activity and strict morphology or sperm DFI.     

组蛋白去乙酰化酶抑制剂的研究进展

组蛋白乙酰化和去乙酰化可调节染色体的多种功能,如基因表达和染色体分离等。研究发现,组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors, HDACIs)可诱导肿瘤细胞及干细胞分化、生长阻断和凋亡。现综述HDAC抑制剂的种类、生物学作用、抗肿瘤作用机制的研究进展及应用前景等。     

氧化应激上调人神经母细胞瘤细胞内β-裂解酶的表达

目的研究氧化应激对人神经母细胞瘤细胞(SH-SY5Y)β-裂解酶(BACE1)表达的影响及组蛋白乙酰化、DNA甲基化的改变。方法采用H2O2处理体外培养的SH-SY5Y,Western blot法检测细胞的BACE1表达及DNA甲基转移酶(DNMTs)和组蛋白去乙酰化酶(HDAC)的表达;实时定量PCR检测BACE1 mRNA的表达;吸光度值法检测组蛋白3(H3)和组蛋白4(H4)整体乙酰化水平。结果 SH-SY5Y细胞经H2O2处理1和72 h后BACE1 mRNA和蛋白表达均明显增多;H2O2处理72 h后DNMT1、DNMT3A表达均下降,分别是对照组的75%和65%(P<0.01);而组蛋白去乙酰化酶HDAC3的表达增高至对照组的1.6倍(P<0.01);同时,组蛋白H3整体乙酰化水平下降,但H4乙酰化水平无明显改变。结论氧化应激可能通过改变SH-SY5Y细胞内DNA甲基化水平及组蛋白乙酰化状态调节BACE1的表达,在阿尔茨海默病发病过程中发挥作用。