组蛋白去甲基化酶KDM5A调控H3K4me3参与神经管畸形发生的分子机制

神经管畸形(NTDs)的病因与防治是出生缺陷领域研究的重点,叶酸可以预防神经管畸形但其机制不明。本文借助低叶酸细胞模型和低叶酸NTDs小鼠模型通过染色质免疫共沉淀、Cut&Tag等技术,探讨了组蛋白去甲基化酶lysine demethylase 5A(KDM5A)及其调控的下游组蛋白H3K4me3修饰在叶酸缺乏导致的NTDs发生中的潜在分子机制。结果显示,低叶酸的细胞模型中,qRT-PCR、Western印迹结果显示,KDM5A分子表达明显下降(P<0.05)。作为组蛋白H3K4me3调控的上游关键酶,进一步通过染色质免疫共沉淀ChIP、ChIP-qPCR实验证实,叶酸缺乏下组蛋白H3K4me3在神经发育基因Axin 2和Atoh 1基因启动子区富集增加(P<0.05)。通过构建KDM5A基因敲除细胞模型,借助Cut&Tag试验证实,KDM5A基因敲除后H3K4me3主要富集在神经发育基因上。最后在低叶酸导致的NTDs小鼠模型的脑组织中,RT-qPCR、Western印迹以及ChIP-qPCR实验显示,E9.5 d的NTDs胎鼠脑组织中KDM5A表达下降(P<0.05),Axin2、Atoh1表达升高(P<0.05),Axin2、Atoh 1基因启动子区的H3K4me3富集增多(P<0.05)。综上所述,KDM5A蛋白在叶酸缺乏导致的NTDs中发挥重要作用,其可通过调控下游H3K4me3进而调控神经发育靶基因Axin2、Atoh 1异常表达,介导NTDs的发生。本研究从叶酸缺乏介导KDM5A调控组蛋白修饰来探讨NTDs的发病机制,为降低出生缺陷,促进生殖健康提供依据。

鸡卵泡颗粒层组蛋白H3K4me3和H3K27me3的表达研究

卵母细胞成熟过程受组蛋白H3K4me3(trimethylation of lysine 4 on histone 3)和H3K27me3(trimethylation of lysine 27 on histone 3)及其相关的甲基化和去甲基化酶的调控,因此考虑对鸡的卵泡发育也存在一定的影响。选取“苏禽3号”配套系第一母本为研究对象,采用Western blot法探究组蛋白H3K4me3和H3K27me3在鸡卵泡不同发育阶段颗粒层中蛋白的表达模式。结果表明:在苏禽3号卵泡颗粒层中,组蛋白H3K4me3在卵泡发育不同阶段表达模式呈降低→升高→降低→升高的波浪形趋势,波浪变化较为平缓,在F5、F2和F13个表达高点的表达量与SWF(small white follicle)、LWF(large white follicle)、SYF(small yellow follicle)和F34个表达低点的表达差异显著(P<0.05)。组蛋白H3K27me3在不同发育阶段表达模式亦呈波浪形表达趋势,波浪变化起伏较明显,在SWF、SYF和F33个表达高点的表达量与F5、F4、F1和F24个表达低点的表达差异显著(P<0.05)。相关性分析显示,组蛋白H3K4me3与H3K27me3在不同发育阶段卵泡颗粒细胞中的表达呈较强的负线性相关(R=-0.808,P=0.000)。结果提示:组蛋白H3K4me3和H3K27me3在不同发育阶段卵泡颗粒层中的表达具有组织差异性,呈负相关的动态修饰性,可能共同协调卵泡生长过程中各基因的表达与功能,研究结果为鸡繁殖性状调控机理提供了理论依据。

氮限制条件下链状亚历山大藻H3K4me3的调控机制解析

为探索链状亚历山大藻(Alexandrium pacificam)在低氮条件下的适应性反应机制,本研究采用结合位点分析法(ChIP-seq)比较了链状亚历山大藻氮限制和氮正常条件下组蛋白第三亚基四号赖氨酸的三甲基化(H3K4me3)修饰情况及调控机制,GO分析结果表明H3K4me3修饰在低氮条件下调控了跨膜转运蛋白活性,表明在低氮条件下链状亚历山大藻增强了转运体活性来获取外界营养物质。通过KEGG分析发现还富集了植物病原菌相互作用途径和谷胱甘肽代谢途径,表明链状亚历山大藻在低氮条件下,病原菌防御和抗氧化应激对其生存至关重要。此外还发现在低氮条件下H3K4me3调控了泛素介导的蛋白质降解和自噬途径来缓解氮的耗竭而响应氮胁迫。

H3K4me3高表达与肝癌患者较差生存预后相关

目的:探讨检测肝癌组织中组蛋白第三亚基四号赖氨酸的三甲基化(H3K4me3)蛋白的表达与肿瘤病理特点和肝癌患者生存预后的相关性。方法:免疫组化和Western-blot检测H3K4me3和组蛋白甲基转移酶(SET and MYND domain-containing protein 3,SMYD3)在肝癌组织(n=168)和细胞株中的表达。此外,实验结果还在另外一个肝癌组织芯片(n=147)中进行验证。H3K4me3表达的最佳分界点(optimal cut-point)由X-tile程序确定,患者的预后由Kaplan-meier生存曲线描述。结果:H3K4me3高表达于肝癌细胞系和肝癌组织,其高表达与肝癌尤其是早期TNM1/2期患者的较差总体生存显著相关。单因素和多因素分析均提示H3K4me3表达水平是患者预后的独立危险因素。此外,H3K4me3和SMYD3在两组肝癌组织中均存在正相关表达。结论:H3K4me3表达水平能成为肝癌患者术后生存的预测因子,其高表达可能与SMYD3有关。

H3K4me3对猪卵母细胞体外成熟和早期胚胎发育的影响

哺乳动物卵母细胞的成熟与组蛋白甲基化修饰密切相关.CPI-455是一种组蛋白去甲基化酶抑制剂,可特异性抑制组蛋白去甲基转移酶KDM5A/B的活性,提高H3K4me3(组蛋白H3第4位赖氨酸三甲基化)的表达水平.本实验在猪卵母细胞体外成熟过程中添加CPI-455,探究H3K4me3组蛋白甲基化对猪卵母细胞体外成熟和早期胚胎发育的影响.首先,用不同浓度CPI-455在不同时间处理猪卵母细胞,发现在0~22 h添加5μmol CPI-455的处理方法效果最佳,卵母细胞成熟率与囊胚形成率均显著高于未处理组(p<0.05).0~22 h添加5μmol CPI-455处理卵母细胞显著提高了囊胚中H3K4me3的表达(p<0.05),降低了卵母细胞中KDM5A和KDM5B的表达(p<0.05),并且显著提高了囊胚中Nanog,Oct4,CDX2等多能性基因的表达(p<0.05).CPI-455处理可以通过降低卵母细胞中相关基因的表达,上调囊胚中多能性基因的表达,调控H3K4me3的甲基化水平,从而促进猪卵母细胞体外成熟和早期胚胎发育.

CXXC finger protein 1 (CFP1) bridges the reshaping of genomic H3K4me3 signature to the advancement of lung adenocarcinoma

Histone H3 lysine 4 trimethylation(H3K4me3)is a canonical chromatin modification associated with active gene transcription,playing a pivotal role in regulating various cellular functions.Components of the H3K4me3 methyltransferase complex,known as the proteins associated with SET1(COMPASS),have been implicated in exerting cancer-protective or cancer-inhibitory effects through inducive H3K4me3 modification.However,the role of the indispensable non-catalytic component of COMPASS CXXC-type zinc finger protein 1(CFP1)in malignant progression remains unclear.

镧暴露对子代大鼠学习记忆及海马组蛋白H3K4me3表达的影响

目的探讨镧暴露对子代大鼠学习记忆能力及海马组蛋白H3赖氨酸4三甲基化(H3K4me3)水平的影响。方法将24只SPF级雌性Wistar孕鼠随机分为对照组,2.5、5.0和10.0 g/L氯化镧(LaCl_(3))染毒组,其子代大鼠断乳后通过自由饮水方式按原浓度继续镧暴露。而后在不同时间采用Morris水迷宫实验检测子代大鼠的学习记忆能力,ELISA法测定海马中组蛋白甲基转移酶(HMT)的活性,Western blot法检测海马中H3K4me3和脑源性神经营养因子(BDNF)的蛋白表达水平。结果与对照组比较,出生后第14、21、28、35和49天LaCl_(3)染毒组子代大鼠体质量明显下降(P<0.05)。LaCl_(3)染毒组子代大鼠寻找逃逸平台的潜伏期延长(P<0.05),穿越平台次数和目标象限停留时间均减少(P<0.05),提示子代大鼠空间学习记忆能力受损;与对照组相比,LaCl_(3)染毒组子代大鼠海马HMT活性降低(P<0.05),H3K4me3和BDNF蛋白表达水平均降低(P<0.05),并呈剂量-反应关系。结论镧暴露导致子代大鼠学习记忆能力损伤可能与海马组蛋白H3K4me3表达下降有关。

雷公藤内酯醇对多发性骨髓瘤KM3细胞增殖与凋亡和H3K4me3蛋白表达的影响

目的探讨雷公藤内酯醇对多发性骨髓瘤KM3细胞增殖、凋亡和组蛋白3第4位赖氨酸的三甲基化(H3K4me3)蛋白表达的影响。方法以人多发性骨髓瘤细胞株KM3为研究对象,用噻唑蓝(MTT)法检测细胞增殖活性;Annexin V-FITC/PI双标流式细胞术和共聚焦显微镜检测细胞凋亡;蛋白质印迹法和共聚焦显微镜检测雷公藤内酯醇对KM3细胞H3K4me3表达的影响。结果雷公藤内酯醇对KM3细胞的增殖有明显抑制作用,呈现剂量和时间依赖关系(P<0.05)。雷公藤内酯醇对KM3细胞有明显诱导凋亡的作用,并且随着雷公藤内酯醇作用浓度的增加,细胞凋亡比例逐渐增加,其中总凋亡率分别为(48.97±1.78)%,(53.72±2.21)%和(60.75±2.43)%。H3K4me3集中分布于细胞核内,80 nmol·L-1雷公藤内酯醇干预48 h后,KM3细胞H3K4me3的平均荧光强度值(21.96±0.34)显著低于对照组(39.86±0.47)(P<0.05)。结论雷公藤内酯醇可抑制多发性骨髓瘤KM3细胞增殖,诱导KM3细胞凋亡,降低H3K4me3蛋白表达。

H3K27me3 and H3K4me3 Chromatin Environment at Super-Induced Dehydration Stress Memory Genes of Arabidopsis thaliana

Pre-exposure to a stress may alter the plant＇s cellular, biochemical, and/or transcriptional responses during future encounters as a ＂memory＇ from the previous stress. Genes increasing transcription in response to a first dehydra- tion stress, but producing much higher transcript levels in a subsequent stress, represent the super-induced ＇transcription memory＇ genes in Arabidopsis thaliana. The chromatin environment （histone H3 tri-methylations of Lys 4 and Lys 27, H3K4me3, and H3K27me3） studied at five dehydration stress memory genes revealed existence of distinct memory- response subclasses that responded differently to CLF deficiency and displayed different transcriptional activities dur- ing the watered recovery periods. Among the most important findings is the novel aspect of the H3K27me3 function observed at specific dehydration stress memory genes. In contrast to its well-known role as a chromatin repressive mechanism at developmentally regulated genes, H3K27me3 did not prevent transcription from the dehydration stress- responding genes. The high H3K27me3 levels present during transcriptionally inactive states did not interfere with the transition to active transcription and with H3K4me3 accumulation. H3K4me3 and H3K27me3 marks function indepen- dently and are not mutually exclusive at the dehydration stress-responding memory genes.

The Association Between H3K4me3 and Antisense Transcription

Histone H3 lysine 4 trimethylation （H3K4me3） is well known to occur in the promoter region of genes for transcription activation. How- ever, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3＇ end of actively transcribed （sense） genes, named as 3＇-H3K4me3. 3＇-H3K4me3 is associated with ~15% of pro- tein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II （RNAPII） binding sites and Y-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3＇-H3K4me3 is associated with the ini- tiation of antisense transcription. Furthermore, 3＇-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3＇-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3＇ end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3＇-H3K4me3 modification. More importantly, we observed the 3＇-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3＇-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in ver- tebrate. Therefore, we speculate that these 3＇-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3＇-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.

Broad H3K4me3 as A Novel Epigenetic Signature for Normal Development and Disease

The breadth of the enrichment site for post-translational trimethylation of histone H3 at lysine 4 （H3K4me3） on chromatin has attracted great attention recently. H3K4me3, an extensively-studied histone modification, is reported to promote gene transcription by directing preinitiation complex assembly through interaction with effector proteins, e.g.,

Transgenerational analysis of H3K4me3 and H3K27me3 by ChIP-Seq links epigenetic inheritance to metabolism

Histone methylation is a kind of important epigenetic modification which occurs on the lysine residue or arginine residue of histone tails（Zhang and Reinberg,2001）.It takes part in multiple biological processes,including gene expression,genomic stability,stem cell maturity,genetic imprinting,mitosis and development（Fischle et al.,2005）.

A study on the relationship between arsenic exposure and H3K4me3 and H3K79me3 in human peripheral leukocyte histone

Objective To observe the effect of arsenic exposure to drinking water on the level of histone 3 lysine 4 trimethylation(H3K4me3)and histone 3 lysine 79 trimethylation(H3K79me3)in peripheral blood leukocytes of human,and to analyze the relationship between arsenic exposure and H3K4me3,H3K79me3 modification levels.Methods A cluster sampling survey was carried out in typical endemic arsenicosis areas of Shanxi and Jilin provinces.Two hundred eighty-one local residents with a drinking water age of≥10 years were selected as the survey subjects.According to the arsenic content of drinking water,the tested population was divided into control group(water arsenic content≤0.01 mg/L,60 cases),low water arsenic exposure group(>0.01-0.05 mg/L,61 cases),medium water arsenic exposure group(>0.05-0.10 mg/L,50 cases),and 110 cases of high water arsenic exposure group(>0.10 mg/L).Drinking water samples,immediate urine samples and peripheral blood samples were collected from the subjects.Arsenic content in drinking water and urinary arsenic content were determined via the atomic fluorescence method;histone H3K4me3 and H3K79me3 in peripheral blood leukocytes were determined by dot blot hybridization(Dot Blotting).Results There were no statistically significant differences in age(61.50,60.00,59.50,59.50 years old),different gender(male:20,27,17,40 cases,female:40,34,33,70 cases),body mass index(BMI),smoking and drinking status between the control group,low,medium and high water arsenic exposure groups.Water arsenic content in the control group,low,medium and high water arsenic exposure groups(median:0.005,0.024,0.076,0.150 mg/L),urinary arsenic content(0.011,0.018,0.061,0.134 mg/L),and water arsenic cumulative exposure levels(0.342,1.641,5.273,7.716 mg)were compared between the groups,the differences were statistically significant(H=256.041,88.615,218.610,P<0.01).In the control group,low,medium and high water arsenic exposure groups,the modification levels of H3K4me3(0.100,0.059,0.083,0.083)and H3K79me3(0.049,0.036,0.055,0.052)in peripheral blood leukocytes were not significantly different(H=1.488,2.097,P>0.05).The levels of H3K4me3 and H3K79me3 in peripheral blood leukocytes were positively correlated with water arsenic content,urinary arsenic content,water arsenic cumulative exposure levels(r=0.245,0.221;0.299,0.318;0.149,0.149;P<0.01 or<0.05);there was a positive correlation between H3K4me3 and H3K79me3 modification levels(r=0.811,P<0.01).Conclusion There is a positive correlation between arsenic exposure through drinking water and the levels of H3K4me3 and H3K79me3 in the peripheral blood leukocytes of the population,but it is necessary to expand the sample size for further study.

基于高通量测序的梅花鹿卵巢衰老的转录组分析

【目的】获取青年梅花鹿和老年梅花鹿卵巢组织的转录组信息,为深入研究梅花鹿卵巢衰老的潜在机制提供依据。【方法】以4岁龄青年梅花鹿和10岁龄老年梅花鹿卵巢作为试验样本,对样本进行石蜡切片、苏木精-伊红染色和卵泡计数;用Trizol法提取卵巢样本RNA构建转录组文库,应用Illumina HiSeq TM 2000测序平台测序,对测序结果进行差异表达基因、GO功能、KEGG通路和蛋白互作网络分析,并挑选部分差异表达基因进行实时荧光定量PCR验证。通过RIPA裂解液提取卵巢样本蛋白并对组蛋白H3赖氨酸4三甲基化(H3K4me3)蛋白在青年和老年梅花鹿中的表达情况进行Western blotting检测。【结果】组织学分析结果表明,老年梅花鹿的卵巢卵泡数显著低于青年梅花鹿(P<0.05),而闭锁卵泡数极显著高于青年梅花鹿(P<0.01)。转录组学分析结果表明,老年和青年梅花鹿卵巢间共有1477个差异表达基因,其中表达上调基因503个,表达下调基因974个。GO功能和KEGG通路富集结果表明,免疫系统过程、转录、DNA损伤修复、炎症反应、凋亡等生物事件与卵巢衰老有关。实时荧光定量PCR结果显示,共有17个基因在老年和青年梅花鹿卵巢中表达量差异达显著或极显著水平(P<0.05;P<0.01),表明RNA-Seq结果可靠。Western blotting结果表明,H3K4me3蛋白在老年和青年梅花鹿卵巢中表达量存在显著差异(P<0.05)。【结论】青年和老年梅花鹿卵巢在卵泡数方面存在显著差异,通过RNA-Seq筛选出PI3K-Akt信号通路影响DNA损伤修复效率,参与梅花鹿卵巢衰老。H3K4me3在老年梅花鹿卵巢中高表达与转录激活有关。

Long-read genome assemblies reveal a cis-regulatory landscape associated with phenotypic divergence in two sister Siniperca fish species

Due to the difficulty in accurately identifying structural variants(SVs) across genomes,their impact on cisregulato ry diverge n ce of closely related species,especially fish,remains to be explored.Recently identified broad H3K4me3 domains are essential for the regulation of genes involved in several biological processes.However,the role of broad H3K4me3 domains in phenotypic divergence remains poorly understood.Siniperca chuatsi and S.scherzeri are closely related but divergent in several phenotypic traits,making them an ideal model to study cis-regulatory evolution in sister species.Here,we generated chromosome-level genomes of S.chuatsi and S.scherzeri,with assembled genome sizes of 716.35 and740.54 Mb,respectively.The evolutionary histories of S.chuatsi and S.scherzeri were studied by inferring dynamic changes in ancestral population sizes.To explore the genetic basis of adaptation in S.chuatsi and S.scherzeri,we performed gene family expansion and contraction analysis and identified positively selected genes(PSGs).To investigate the role of SVs in cis-regulatory divergence of closely related fish species,we identified high-quality SVs as well as divergent H3K27ac and H3K4me3 domains in the genomes of S.chuatsi and S.scherzeri.Integrated analysis revealed that cis-regulatory divergence caused by SVs played an essential role in phenotypic divergence between S.chuatsi and S.scherzeri.Additionally,divergent broad H3K4me3 domains were mostly associated with cancer-related genes in S.chuatsi and S.scherzeri and contributed to their phenotypic divergence.

骨髓间充质干细胞机械刺激成骨的记忆效应

背景:骨髓间充质干细胞作为组织工程的理想种子细胞,已证实力学刺激有引导其分化的作用。研究表明,干细胞存在着机械记忆,即力学刺激所引起的改变可能会长期影响干细胞的命运。目的:观察骨髓间充质干细胞对力学刺激的记忆效应,解析其发生的表观遗传学机制。方法:(1)分离原代小鼠骨髓间充质干细胞,检测其三系分化能力,传至第3代待用;(2)利用拉伸仪,体外给予小鼠骨髓间充质干细胞机械刺激,检测细胞拉伸前后的成骨分化情况;(3)流式细胞仪检测拉伸后细胞干性;(4)将拉伸处理与未处理的细胞消化后铺种在常规12孔板,继续培养14 d后检测细胞的成骨分化情况;(5)免疫荧光染色检测并筛选造成干细胞机械记忆的关键组蛋白修饰变化,初步明确表观遗传调控的可能机制。结果与结论:(1)5%形变,0.5 Hz,6 h/d,为期7 d的机械刺激可以引导小鼠骨髓间充质干细胞向成骨方向分化;(2)重新铺种培养皿后,拉伸处理后的细胞仍能保持干性且继续向成骨方向分化;(3)结果显示,骨髓间充质干细胞对机械刺激存在记忆效应,这种效应可能与力学刺激导致的组蛋白H3K4me3修饰有关。

醒脑净注射液改善新生儿缺氧缺血性脑损伤的机制研究

目的:探讨醒脑静注射液治疗新生儿缺氧缺血性脑损伤的机制。方法:建立新生儿缺氧缺血性损伤新生大鼠模型,将动物随机分为3组:假手术组、模型组和醒脑静组,每组24只。在不同时间点,收集各组脑组织,采用干湿质量法测定大鼠脑组织含水量,通过染色质免疫共沉淀及实时荧光定量PCR检测和比较各组脑组织HIF-1α在VEGF基因启动子的结合水平,以及VEGF启动子组蛋白H3第27位赖氨酸三甲基化(H3K27me3)和组蛋白H3赖氨酸4三甲基化(H3K4me3)的富集水平。结果:在各时间点,醒脑静治疗组HIF-1α结合在VEGF启动子上的水平较模型组均显著提高(P<0.05);醒脑静组VEGF启动子上H3K27me3富集水平较模型组显著降低(P<0.05),而H3K4me3富集水平显著升高(P<0.05)。结论:醒脑静注射液治疗HIBD的机制可能与促进HIF-1α结合到VEGF启动子上,促进VEGF的表达,同时改变VEGF启动子上H3K27me3和H3K4me3富集水平有关。

CUT&Tag产物回收和建库方法的优化

新兴的染色质靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用转座酶在目标蛋白结合的DNA附近进行切割并对切割下的DNA片段进行标签化,通过后续的二代测序可以快速鉴定蛋白质-DNA相互作用,极大的简化了染色质免疫共沉淀测序(chromatin immunoprecipitation sequencing,ChIP-seq)的实验过程。CUT&Tag中转座酶完成标签化后需要DNA回收或其他后处理才能进行建库PCR,不同的回收方法对CUT&Tag结果有着显著的影响。通过建立生物素化转座体-链霉亲和素磁珠体系(streptavidin beads recovery CUT&Tag,srCUT&Tag),可以快速便捷地完成CUT&Tag的产物回收。本文在K562细胞中展开H3K4me3、RNA聚合酶Ⅱ(RNA polymeraseⅡ,RNAPⅡ)、转录因子CTCF和HMGA1的CUT&Tag实验,并利用现有的乙醇沉淀、片段分选(solid-phase reversible immobilization,SPRI)磁珠回收和直接PCR法,以及本研究建立的srCUT&Tag方法对产物进行回收。结果表明,从整体上看,SPRI磁珠回收和srCUT&Tag方法着较高的回收效率,而乙醇沉淀法则回收效率低下。在全部4种CUT&Tag产物回收过程中,SPRI磁珠回收均会损失大部分小于150 bp的产物片段。在CTCF和HMGA1 CUT&Tag产物的回收中,直接PCR法则损失了大部分大于300 bp的片段并与其他回收方法的结果有较大的差别。因此,srCUT&Tag能够比其他三种回收方法提供更多更完整的测序信息。综上所述,新建立srCUT&Tag回收方法相比现有的CUT&Tag产物回收方法能提高建库效率并得到更好的数据质量,为表观遗传学研究提供了更好的技术选择。

Ash2l-1/Ash2l-2在小鼠胚胎干细胞中的表达特异性及互补效应

H3K4me3是一种重要的表观遗传修饰,主要由MLL(mixed lineage leukemia)甲基转移酶复合体催化,对小鼠胚胎干细胞(mouse embryonic stem cells,m ESCs)自我更新能力的维持具有重要作用。ASH2L是MLL复合体中一个重要的核心亚单位,参与调控m ESCs中染色质的开放状态。ASH2L在m ESCs中有2个异构体:ASH2L-1(80 k Da)和ASH2L-2(65 k Da),且以ASH2L-2的表达为主;而在小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)中,只有ASH2L-1表达。目前,Ash2l-1和Ash2l-2在m ESCs中的作用尚不清楚。本文利用CRISPR/Cas9基因组编辑技术,建立了Ash2l-1^(–/–)和Ash2l-2^(–/–)m ESCs。通过碱性磷酸酶染色、免疫荧光染色和q RT-PCR发现,Ash2l-1^(–/–)和Ash2l-2^(–/–)m ESCs在碱性磷酸酶、多能性调控转录因子(Oct4、Nanog、Sox2和Klf4)的表达与野生型对照无显著差异。通过拟胚体分化实验,发现Ash2l-1^(–/–)m ESCs诱导的拟胚体在Snai2(外胚层标记基因)和Gata4(内胚层标记基因)的表达上显著低于野生型m ESCs诱导的拟胚体(P<0.01)。通过Western blotting,发现Ash2l-1^(–/–)m ESCs中ASH2L-2的表达显著上调(P<0.01),Ash2l-2^(–/–)m ESCs中ASH2L-1的表达显著上调(P<0.01),而Ash2l-1^(–/–)和Ash2l-2^(–/–)m ESCs中,基因组H3K4me3的表达与野生型对照并无显著差异。这表明Ash2l-1和Ash2l-2之间存在补偿效应。利用JASPAR和KEGG预测分析发现,Ash2l-1和Ash2l-2启动子区分别具有3个和16个潜在的多能性转录因子结合位点,这些转录因子可能介导实现Ash2l-1和Ash2l-2之间的补偿效应。以上结果表明,Ash2l-1和Ash2l-2之间的补偿效应可能参与m ESCs多能性的维持和基因组H3K4me3的调控。

白黎芦醇对舒尼替尼诱导肾损伤的作用及机制研究

目的:探究白黎芦醇对舒尼替尼诱导肾损伤的作用及机制。方法:选择WKY大鼠30只随机分为3组:对照组(n=10),舒尼替尼组(n=10),舒尼替尼+白藜芦醇组(n=10)。舒尼替尼组给予舒尼替尼[15 mg/(kg·d)]灌胃28 d,建立舒尼替尼肾损伤大鼠模型。舒尼替尼+白藜芦醇组于建模前7 d给予大鼠白藜芦醇[100 mg/(kg·d)]灌胃,之后给予白藜芦醇[100 mg/(kg·d)]+舒尼替尼[15 mg/(kg·d)]共同灌胃28 d。对照组给予等量生理盐水。鼠尾测压监测大鼠血压变化。舒尼替尼灌胃28 d后处死大鼠,检测各组大鼠血清肌酐、尿素氮水平;苏木素伊红(HE)染色观察肾脏组织形态改变;免疫印迹法检测大鼠肾脏组蛋白第三亚基四号赖氨酸的三甲基化(H3K4me3)、核因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)、单核细胞趋化蛋白1(MCP-1)的蛋白表达变化。结果:与对照组相比,舒尼替尼组大鼠血清肌酐、血尿素氮水平均明显增加[(0.59±0.04)mg/dl vs.(0.47±0.05)mg/dl;(21.10±0.47)mg/dl vs.(17.34±0.59)mg/dl,P均<0.05];肾小管结构破坏,局部近曲小管上皮细胞肿胀、固缩死亡,肾组织肾小管病理损伤评分(paller评分)升高[(39.12±1.91)分vs.(10.92±0.86)分,P<0.05];肾脏H3K4me3、NF-κB、TNF-α、MCP-1蛋白表达水平均增加(1.04±0.10 vs.0.55±0.04;1.09±0.19 vs.0.49±0.05;0.92±0.02 vs.0.32±0.01;0.85±0.06 vs.0.47±0.04,P均<0.05)。与舒尼替尼组相比,舒尼替尼+白藜芦醇组大鼠血清肌酐、血尿素氮水平均明显下降[(0.50±0.03)mg/dl vs.(0.59±0.04)mg/dl;(18.21±0.51)mg/dl vs.(21.10±0.47)mg/dl,P均<0.05];肾脏病理结构改变减轻、paller评分明显降低[(23.40±1.38)分vs.(39.12±1.91)分,P<0.05];肾脏H3K4me3以及NF-κB、TNF-α、MCP-1等促炎因子蛋白表达减少(0.70±0.04 vs.1.04±0.10;0.81±0.11 vs.1.09±0.19;0.42±0.04 vs.0.92±0.02;0.63±0.03 vs.0.85±0.06,P均<0.05)。结论:白黎芦醇通过抑制肾脏H3K4me3水平降低NF-κB、TNF-α、MCP-1的表达,减轻肾脏炎症缓解舒尼替尼诱导的肾损伤。