H3K27me3表达缺失是散发性和放射相关性恶性外周神经鞘膜瘤的一种高敏感性标志物

正大多数恶性外周神经鞘膜瘤(MPNST)表现为NF1、CDKN2A及多梳抑制复合体2组成基因——胚胎外胚层发育基因(EED)和Zeste 12抑制基因(SUZ12)的共同缺失。EDD和SUZ12的突变导致组蛋白H3第27号位赖氨酸上三甲基化(H3K27me3)无法进行,导致多梳抑制复合体2抑制性同源盒主调控因子转录活性异常。因此,作者探讨抗H3K27me3单克隆抗体C36B11作为MPNST免疫组化标志     

敲低赖氨酸特异性去甲基化酶1(LSD1)促进人诱导性多能干细胞分化为产胰岛素细胞

目的探索一种人诱导性多能干细胞(hiPSC)体外高效分化为产胰岛素细胞(IPC)的诱导方案。方法 RNAi技术敲低hiPSC赖氨酸特异性去甲基化酶1(LSD1)基因后,利用四步法诱导其体外分化为IPC。流式细胞术分析IPC分化效率,实时定量PCR检测细胞LSD1、POU5同源盒转录因子1(OCT4)、Y染色体性别决定区因子17(SOX17)、叉头盒蛋白A2 (FOXA2)、胰腺和十二指肠同源盒蛋白1 (PDX1)、配对盒转录因子4(PAX4)、PAX6、肝细胞核因子6 (HNF6)、肝细胞核因子1同源盒蛋白A(TCF1)、NK6同源盒蛋白1(NKX6. 1)、葡萄糖转运子2(GLUT2)、葡萄糖激酶(GK)、胰岛素及MAF b ZIP转录因子A(MAFA)的mRNA水平,免疫荧光技术检测细胞PDX1、胰岛素的表达和定位;双硫腙(DTZ)染色和透射电镜分别观察细胞内胰岛素的分泌和分泌颗粒分布情况; ELISA检测诱导后细胞胰岛素和C肽分泌量。结果 LSD1敲低组的IPC分化效率较对照组有明显提高,胰岛β细胞发育相关基因SOX17、PDX1、PAX4、胰岛素的mRNA表达显著上调。LSD1敲低组的IPC共表达成熟β细胞特异性蛋白PDX1和胰岛素。另外,这些IPC能感应葡萄糖刺激并以胰岛素分泌小泡形式释放胰岛素,胰岛素或C肽释放量约为天然胰岛细胞的1/6(而对照组仅为1/8)。结论敲低LSD1基因可以促进hiPSC体外高效分化为IPC。     

人脂肪间充质干细胞向限定性内胚层细胞分化体系的优化

目的探讨activin A、WNT通路激活剂、BMP4以及bFGF信号对人脂肪间充质干细胞(hADSCs)向限定性内胚层(DE)分化的影响。方法基于胚胎干细胞(ESCs)或多潜能干细胞(iPS)向DE分化的诱导体系,建立并优化hADSCs向DE细胞分化的诱导体系。在hADSCs向DE诱导体系中,分别比较不同浓度的activin A、Chir99021替代Wnt3a、添加/去除bFGF以及BMP4等对分化效率的影响。用RT-qPCR检测诱导前后限定性内胚层标志基因FOXA2和SOX17的表达;Western blot检测FOXA2和SOX17蛋白水平;用免疫荧光检测FOXA2和SOX17的表达,鉴定DE分化。结果与ESCs或iPS向DE分化的诱导体系相比,低浓度的activin A,GSK3抑制剂Chir99021替代Wnt3a,去除BMP信号和bFGF信号的诱导方案可以明显促进hADSCs向DE分化的影响(P0.01)。结论 hADSCs向DE分化与hESC/hiPS相比,需要激活不同信号通路,因此最适诱导体系不同。     

组蛋白赖氨酸去甲基化与基因调控

随着组蛋白赖氨酸去甲基化酶的发现,证实组蛋白赖氨酸甲基化是一个可以逆转的组蛋白表遗传修饰。赖氨酸特异性组蛋白去甲基化酶1(lysinespecificdemethylase1,LSD1)是一个FAD依赖性胺氧化酶,它能够特异性脱去单甲基化和二甲基化H3K4和H3K9位点上的甲基基团。JmjC蛋白JHDM1、JHDM2、JMJD23个亚家族都具有组蛋白赖氨酸去甲基化酶活性。目前证实组蛋白甲基化与去甲基化失平衡与肿瘤发生相关。组蛋白赖氨酸去甲基化酶有可能成为一个新的抗肿瘤治疗靶标。     

组蛋白赖氨酸甲基化在表观遗传调控中的作用

组蛋白赖氨酸的甲基化在真核基因表观遗传调控中起着关键作用。迄今已知,在组蛋白H3中有5个赖氨酸(K4、K9、K27、K36、K79)和组蛋白H4中的1个赖氨酸(K20)可被特异的组蛋白赖氨酸甲基转移酶甲基化。这不同位点的甲基化效应是不同的,H3-K9、H3-K27、H4-K20甲基化具有抑制效应;H3-K4、H3-K36、H3-K79甲基化具有激活效应,而且组蛋白甲基化与其它组蛋白共价修饰之间以及DNA甲基化之间存在对话。     

揭示LSD1在胚胎干细胞分化中起关键作用的机制

据Whyte WA 2012年2月1日(Nature,2012 Feb 1.doi:10.1038/nature10805)报道,一种赖氨酸特异性去甲基酶1(lysine-specific demethylase 1,LSD1)在胚胎干细胞分化为其他细胞类型时发挥着关键性作用。LSD1是一种组蛋白去甲基酶,作用在组蛋白H3特异性赖氨酸残基上,从而改变染色体DNA上基因转录。LSD1蛋白包含一个位于氨基末端的SWIRM结构域、一个特殊的双螺旋(coiled-coil)塔结构和一个含FAD的胺氧化酶结构域。一个有缺陷的操作     

组蛋白去乙酰化抑制剂largazole在抗hUCMSCs衰老中的作用

目的采用组蛋白去乙酰化抑制剂largazole作用于人脐带间充质干细胞(h UCMSCs),探讨largazole在防止h UCMSCs衰老作用和机制。方法分离、纯化、鉴定人脐带间充质干细胞(h UCMSCs),筛选组蛋白去乙酰化抑制剂largazole扩增h UCMSCs合适的作用浓度。利用合适浓度进行h UCMSCs传代,通过real-time PCR法检测largazole组P4、P 8、P12和P16代h UCMSCs干性基因表达水平;通过CHIP检测组蛋白乙酰化抑制剂largazole对h UCMSCs增殖、分化相关基因(OCT4、TERT、OPN)组蛋白H3K9、H3K14的乙酰化程度的影响。结果 h UCMSCs体外扩增随着传代数的增加,增殖能力逐渐下降,干性基因的表达水平也随之降低,并表现出来向成骨方向自主分化。低剂量的largazole可提高h UCMSCs增殖能力,延缓h UCMSCs的衰老。Largazole主要通过修饰OCT4、TERT、OPN基因启动子区域的组蛋白H3K9/K14ac,使h UCMSCs细胞增殖(OCT4)和端粒酶基因(TERT)mRNA表达增强,成骨分化基因(OPN)的表达减弱。结论表明组蛋白去乙酰化抑制剂largazole可以通过修饰h UCMSCs组蛋白H3的乙酰化,在调控MSCs的生物学特性方面起到重要的作用。     

LSD1调控hiPSCs高效定向分化为IPCs的作用及机制研究

<正>目的:通过抑制或沉默人皮肤成纤维细胞来源的诱导性多能干细胞(hi PSCs)内赖氨酸特异性去甲基化酶1(LSD1)基因的表达,建立一种hi PSCs高效、定向分化为胰岛素分泌细胞(IPCs)的方案,并初步探讨其调控分化机制。方法:采用shRNA或LSD1抑制剂,沉默或抑制hi PSCs内LSD1基因表达,筛选出利于hi PSCs向定型内胚层分化的LSD1活性;利用四步法将     

IOX1对TGF-β诱导的LX2细胞增殖、凋亡及细胞外基质相关蛋白表达的影响

目的:探讨组蛋白去甲基化酶抑制剂IOX1(5-羧基-8-羟基喹啉)提高组蛋白H3第9位赖氨酸二甲基化(H3K9me2)水平对转化生长因子β(TGF-β)诱导的人肝星状细胞株LX2增殖、凋亡及细胞外基质合成和代谢的影响。方法:采用实时无标记细胞分析技术动态观察不同浓度IOX1对TGF-β诱导的LX2细胞增殖的影响。采用流式细胞术观察IOX1对TGF-β诱导的LX2细胞凋亡的影响。Western blot检测细胞中H3K9me2水平,以及α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(Col I)、基质金属蛋白酶1(MMP-1)和金属蛋白酶组织抑制物1(TIMP-1)蛋白的表达。结果:与对照组相比,不同浓度的IOX1均能抑制LX2细胞增殖。流式细胞术结果表明,IOX1能够促进LX2细胞凋亡(P<0.05)。Western blot结果发现,IOX1能提高LX2中H3K9me2水平,且呈剂量依赖性(P<0.05);与对照组相比,300μmol/L IOX1能够明显抑制TGF-β诱导的LX2细胞中α-SMA、TIMP-1和Col I蛋白的表达(P<0.05),MMP-1蛋白的表达在不同浓度IOX1组中表现为上升趋势(P<0.05)。结论:IOX1可抑制TGF-β诱导的LX2细胞增殖并促进细胞凋亡,还可通过提高H3K9me2水平调节细胞外基质的合成和代谢,从而发挥抗肝纤维化作用。     

组蛋白乙酰化修饰失衡在苯肾上腺素诱导小鼠心肌肥厚中的调控作用

目的:探讨苯肾上腺素诱导小鼠心肌肥厚的组蛋白乙酰化调控机制,为防治肥厚性心肌病提供新的思路。方法:选取健康C57BL/6小鼠,苯肾上腺素皮下注射建立小鼠心肌肥厚模型,实时荧光定量聚合酶链反应(real-time PCR)和染色质免疫共沉淀技术(chromatin immunoprecipitation,ChIP)分别检测心脏核转录因子GATA结合蛋白4(GATA binding protein 4,GATA4)mRNA表达水平及其启动子区组蛋白H3第27位赖氨酸乙酰化(H3K27ac)水平,Western blot检测小鼠心肌组织组蛋白H3K27ac、心房钠尿肽(atrial natriuretic peptide,ANP)及α-肌球蛋白重链(α-myosin heavy chain,α-MHC)的表达,HE染色及超声心动图观察小鼠心肌肥厚。结果:Western blot及ChIP-qPCR表明小鼠心肌组织组蛋白H3K27ac水平及GATA4启动子区组蛋白H3K27ac水平在苯肾上腺素组显著高于生理盐水对照组(P0.05),GATA4及ANP的表达水平在苯肾上腺素组也显著高于生理盐水对照组(P0.05);而组蛋白乙酰化酶抑制剂漆树酸能够降低苯肾上腺素诱导的组蛋白H3K27的高乙酰化,且GATA4mRNA及ANP蛋白的表达水平在漆树酸处理组也显著降低(P0.05);HE染色及超声心动图显示苯肾上腺素能够降低左室收缩末期内径(left ventricular end-systolic diameter,LVESD)和左室舒张末期内径(left ventricular enddiastolic diameter,LVEDD)并增加左室后壁(left ventricular posterior wall,LVPW)厚度,诱导小鼠心肌肥厚,而漆树酸能够提高LVESD和LVEDD并降低LVPW厚度,从而改善小鼠心肌肥厚。结论:组蛋白乙酰化修饰失衡参与了苯肾上腺素诱导的小鼠心肌肥厚,而组蛋白乙酰化酶抑制剂漆树酸能够下调苯肾上腺素诱导的组蛋白高乙酰化进而改善小鼠心肌肥厚。     

LSD1 knockdown confers protection against osteoclast formation by reducing histone 3 lysine 9 monomethylation and dimethylation in ITGB3 promoter

ITGB3, an osteoclast marker, is involved in osteoclast formation. Nevertheless, its related mechanism remains poorly characterized. Herein, this study examines the mechanisms affecting osteoclast formation with the involvement of ITGB3. Osteoclast formation was induced with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL), followed by measurement of the mRNA and protein expression of ITGB3 and LSD1. After gain- and loss-of-function assays, cell viability and the expression of osteoclast marker genes (NFATc1, ACP5, and CTSK) were assessed, and osteoclast formation was evaluated with TRAP staining. ChIP assays were used to examine histone 3 lysine 9 (H3K9) monomethylation (H3K9me1) and H3K9 dimethylation (H3K9me2) modifications and LSD1 protein enrichment in the ITGB3 promoter. During osteoclast formation, ITGB3 and LSD1 were gradually augmented. Knockdown of LSD1 or ITGB3 curbed cell viability, the expression of osteoclast marker genes, and osteoclast formation. Moreover, overexpression of ITGB3 nullified the suppressive impact of LSD1 knockdown on osteoclast formation. Mechanistically, LSD1 promoted ITGB3 expression by reducing H3K9 levels in the ITGB3 promoter. LSD1 enhanced ITGB3 expression by decreasing H3K9me1 and H3K9me2 levels in ITGB3 promoter to boost osteoclast formation.     

Epigenetic signatures and temporal expression of lineage-specific genes in hESCs during differentiation to hepatocytes in vitro

Embryonic stem cells (ESCs) maintain unique epigenetic states to maintain their pluripotency. Differentiation of ESCs into specialized cell types requires changes in these epigenetic states. However, the dynamics of epigenetic marks found in hESCs during differentiation are poorly understood. Here, we report the variation in the dynamics of epigenetic modifications associated with the expression of lineage-specific genes during differentiation of hESCs to hepatocytes in vitro. The promoter regions of pluripotency marker genes characterized by permissive histone marks such as trimethylation of H3 at lysine 4 (H3K4me3) and acetylation of H3 at lysine 9 (H3K9ac) in hESCs were instead enriched with repressive histone marks such as dimethylation of H3 at lysine 9 (H3K9me2), trimethylation of H3 at lysine 9 (H3K9me3) and trimethylation of H3 at lysine 27 (H3K27me3) during differentiation to hepatocytes. Interestingly, expression of definitive endoderm marker genes containing bivalent and non-bivalent domains may be modulated by a marked reduction in H3K27me3 and a significant enhancement of permissive marks such as H3K4me3 and H3K9ac during hESC differentiation. Expression of hepatocyte marker genes regulated by histone modifications was similar to that of pluripotency marker genes. Our findings provide insight into the epigenetic mechanisms regulating expression of developmental genes. Of particular interest, they may be differentially regulated either in a bivalent or non-bivalent domain manner during hESC differentiation.     

Protein arginine methyltransferase 6 regulates embryonic stem cell identity

Histone arginine methylation has emerged as an important histone modification involved in gene regulation. Protein arginine methyltransferase (PRMT) 4 and 5 have been shown to play essential roles in early embryonic development and in embryonic stem (ES) cells. Recently, it has been reported that PRMT6-mediated di-methylation of histone H3 at arginine 2 (H3R2me2) can antagonize tri-methylation of histone H3 at lysine 4 (H3K4me3), which marks active genes. However, whether PRMT6 and PRMT6-mediated H3R2me2 play crucial roles in early embryonic development and ES cell identity remain unclear. Here, we have investigated their roles using gain and loss of function studies with mouse ES cells as a model system. We report that Prmt6 and histone H3R2 methylation levels increased when ES cells are induced to differentiate. Consistently, we find that differentiation of ES cells upon upregulation of Prmt6 is associated with decreased expression of pluripotency genes and increased expression of differentiation markers. We also observe that elevation of Prmt6 increases the methylation level of histone H3R2 and decreases H3K4me, Chd1, and Wdr5 levels at the promoter regions of Oct4 and Nanog. Surprisingly, knockdown of Prmt6 also leads to downregulation of pluripotency genes and induction of expression of differentiation markers suggesting that Prmt6 is important for ES cell pluripotency and self-renewal. Our results indicate that a critical level of Prmt6 and histone H3R2me must be maintained in mouse ES cells to sustain their pluripotency.     

Activin and BMP4 Synergistically Promote Formation of Definitive Endoderm in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic β cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage. STEM CELLS 2012; 30:631-642.     

The epigenetic promotion of osteogenic differentiation of human adipose-derived stem cells by the genetic and chemical blockade of histone demethylase LSD1

Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. It has become increasingly clear that chromatin regulators play an important role in cell fate determination. However, how osteogenic differentiation of hASCs is controlled by epigenetic mechanisms is not fully understood. Here we use genetic tools and chemical inhibitors to modify the epigenetic program of hASCs and identify lysine-specific demethylase 1 (LSD1), a histone demethylase that specifically catalyzes demethylation of di- and mono- methyl histone H3 lysine 4 (H3K4me2/1), as a key regulator in osteogenic differentiation of hASCs. Specifically, we demonstrated that genetic depletion of LSD1 with lentiviral strategy for gene knockdown promoted osteogenic differentiation of hASCs by cell studies and xenograft assays. At the molecular level, we found that LSD1 regulates osteogenesis-associated genes expression through its histone demethylase activity. Significantly, we demonstrated LSD1 demethylase inhibitors could efficiently block its catalytic activity and epigenetically boost osteogenic differentiation of hASCs. Altogether, our study defined the functional and biological roles of LSD1 and extensively explored the effects of its enzymatic activity in osteogenic differentiation of hASCs. A better understanding of how LSD1 influences on osteogenesis associated epigenetic events will provide new insights into the modulation of hASCs based cell therapy and improve the development of bone tissue engineering with epigenetic intervention.     

Stem cell transcription factor SOX2 in synovial sarcoma and other soft tissue tumors

Background

SOX2 has gained considerable interest as a pluripotency inducing gene. Co-transfection of SOX2 together with NANOG, KLF4 and c-MYC into adult fibroblasts was able to generate pluripotent stem cells. SOX2 has been reported to be expressed in synovial sarcoma, a tumor being characterized by the SS18-SSX gene fusion forming part of the SWI/SNF chromatin remodeling complex that affects histone methylation. The role of SOX2 in this tumor type as well as other soft tissue tumor entities however is still poorly characterized. We analyzed SOX2 protein expression in soft tissue tumors. Alongside we tested Histone H3 expression (H3K27me3) in SOX2 positive cases to investigate this epigenetic mark and its correlation with the SOX2 status and clinicopathological parameters.

Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development

Background : Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage‐specific genes. The lineage‐determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation‐specific genes is established remains unclear. Results: Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co‐activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification before myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co‐immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP. Conclusions: MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo. Developmental Dynamics 244:43–55, 2015. © 2014 Wiley Periodicals, Inc.