H3K36me3及其表观遗传修饰蛋白质SETD2和KDM4B在结直肠癌中的临床病理意义

目的:组蛋白修饰是表观遗传调控的一种重要形式。其中,组蛋白甲基化是染色质状态的关键决定因素。组蛋白H3第36位赖氨酸三甲基化产物(histone H3 trimethylation at lysine 36,H3K36me3)可以介导多种转录相关事件,有助于DNA损伤修复。组蛋白赖氨酸三甲基转移酶SET结构域2(SET domain containing 2,SETD2)、赖氨酸特异性去甲基化酶4B(lysine-specific demethylase 4B,KDM4B)分别是H3K36me3甲基转移酶及去甲基酶,SETD2失活及KDM4B活化均可下调H3K36me3水平从而促进肿瘤的发生和发展。本研究主要探讨H3K36me3、SETD2、KDM4B在结直肠癌中表达的临床病理意义。方法:收集2018年10月至2020年10月就诊于福建中医药大学附属人民医院的80例结直肠癌患者术后石蜡包埋的组织样本。采用免疫组织化学染色法检测H3K36me3、KDM4B、SETD2及4种错配修复(mismatch repair,MMR)蛋白质(MSH2、MSH6、MLH1、PMS2)在结直肠癌及正常肠黏膜中的表达水平。采用多重荧光PCR-毛细管电泳检测结直肠癌及正常肠黏膜的微卫星状态,分为微卫星高度不稳定性(microsatellite instability-high,MSI-H)组与微卫星稳定(microsatellite stable,MSS)组,检测2组结直肠癌中H3K36me3及SETD2、KDM4B、MMR蛋白质表达情况并分析其临床病理意义。结果:STED2、H3K36me3表达与结直肠癌发生呈负相关(分别为r=-0.745,P<0.001;r=-0.160,P<0.05),KDM4B表达与结直肠癌发生呈正相关(r=0.660,P<0.001)。不同的患者性别、年龄,不同的肿瘤发生部位、大体类型、肿瘤最大径、浸润深度、淋巴结转移的临床病理参数之间SETD2及H3K36me的表达差异均无统计学意义(均P>0.05),除在不同的肿瘤浸润深度KDM4B的表达差异有统计学意义(χ^(2)=0.349,P<0.05)外,在其他临床病理参数上KDM4B的表达差异均无统计学意义(均P>0.05)。结直肠癌样本中检测出14例为MSI-H结直肠癌、66例为MSS结直肠癌;SETD2及H3K36me3的表达均与结直肠癌微卫星状态相关(分别为χ^(2)=3.916,P<0.05及χ^(2)=41.608,P<0.001);KDM4B的表达与结直肠癌微卫星状态无明显相关性(χ^(2)=0.067,P>0.05)。SETD2及H3K36me3表达与MSI-H结直肠癌发生呈负相关(分别为r=-0.221,P<0.05及r=-0.721,P<0.001)。KDM4B表达与MSS结直肠癌发生呈正相关(r=0.200,P<0.05)。免疫组织化学染色结果显示13例(16.25%)为dMMR,67例(83.75%)为pMMR;PCR-毛细管电泳验证结果显示14例为MSI-H结直肠癌,其中13例为dMMR,1例为pMMR;66例为MSS结直肠癌。免疫组织化学染色与多重荧光PCR-毛细管电泳检测结果具有强一致率(Kappa=0.955)。结论:H3K36me3及其表观遗传修饰蛋白质SETD2和KDM4B蛋白质的不同表达程度与结直肠癌发生、发展具有相关性。

Effects of DNA Methylation and Histone Modification on Differentiation-associated Gene Expression in ES,NIH3T3,and NIT-1

The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes（Pdx-1,Pax4,MafA,and Nkx6.1,etc） were investigated.The promoter methylation status of islet differentiation-associated genes（Pdx-1,Pax4,MafA and Nkx6.1）,Oct4 and MLH1 genes of mouse embryonic stem cells,NIH3T3 cells and NIT-1 cells were profiled by methylated DNA immunoprecipitation,real-time quantitative PCR（MeDIP-qPCR） techniques.The histone modification status of these genes promoter region in different cell types was also measured by using chromatin immunoprecipitation real-time quantitative PCR methods.The expression of these genes in these cells was detected by using real-time quantitative PCR.The relationship between the epigenetic modification（DNA methylation,H3 acetylation,H3K4m3 and H3K9m3） of these genes and their expression was analyzed.The results showed that：（1） the transcription-initiation-sites of Pdx-1,MafA and Nkx6.1 were highly methylated in NIH3T3 cells; （2） NIH3T3 cells showed a significantly higher level of DNA methylation modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIT-1 cells（P〈0.05）; （3） NIT-1 cells had a significantly higher level of H3K4m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIH3T3 cells（P〈0.05）,with significantly increased level of gene expression; （4） NIH3T3 cell had a significantly higher level of H3K9m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and with NIT-1 cell（P〈0.05）,with no detectable mRNA expression of these genes.It was concluded that histone modification（H3K4m3 and H3K9m3） and DNA methylation might have an intimate communication between each other in the differentiation process from embryonic stem cells into islet cells.

ZMYND10 downregulates cyclins B1 and D1 to arrest cell cycle by trimethylating lysine 9 on histone 3

The BLU gene coding for zinc finger,MYND-type containing 10(ZMYND10)protein is mapped on chromosomal region 3p21.It is frequently lost in some kinds of cancers due to hypermethylation on its promoter region and identified as a tumour suppressor gene.The underlying mechanisms for BLU-mediated tumor suppression remain unclear.BLU has been reported to disturb cell cycle progression.The present study aims at examining whether ZMYND10 prevents progression of the cell cycle by targeting to repressive histone marks and downregulating the level of cyclins.Proteins structurally similar with ZMYND10 have been shown to recognize DNA sequence upstream of coding portion of the gene encoding cell cycle regulators.Enzymes,notably demethylases modifying the lysine residues are over-expressed line oncoproteins,and targeted in anti-cancer therapy.BLU was re-expressed in H1299 and HepG2 cells.The level of cyclin D1,cyclin B1 and trimethylate lysine 9 on histone 3(H3K9me3)and the binding of BLU with SIN3A(a component of the co-repressor)were detected.Cell cycle profile was measured.The evolutionary relationship between ZMYND10 and other ZMYND proteins was analysed by phylogenetic tree construction.We found that BLU expression induced G1 arrest in H1299 cells,and induced G1/G2 arrest in HepG2 cells.Cell cycle arrest was correlated with reduced activities and levels of cyclins;cyclin D1 was downregulated in H1299 cells;Both cyclin B1 and D1 were downregulated in HepG2 cells;and that BLU was associated with SIN3A.In both cell lines,the expression of H3K9me3 was induced.BLU was clustered with histone methyltransferase SMYD3 and SMYD1 on the same clade of the deduced phylogenetic tree.The results thus suggested that ZMYND10 encoded by BLU inhibited cyclins activity to prevent cell cycle progression through interaction with repressors and histone repressive marks to block the expression of genes coding for cyclins.

27位氨基酸三甲基化的组蛋白3(H3K27me3)及其修饰酶在小鼠不同组织中分布

目的研究出生后7日龄和2月龄小鼠各组织器官中27位氨基酸三甲基化的组蛋白3(H3K27me3)的水平及其修饰酶Zeste基因增强子同源物2(EZH2)、赖氨酸特异性脱甲基酶6B(Kdm6B/JMJD3)和赖氨酸特异性脱甲基酶6A(Kdm6A/UTX)的表达。方法免疫组织化学染色法检测7日龄和2月龄小鼠脑、涎腺、背部脂肪、胸腺、肺、心脏、胃、肠、肝、睾丸、皮肤组织中H3K27me3、EZH2、JMJD3和UTX表达,实时定量PCR验证,统计分析H3K27me3水平与EZH2、JMJD3和UTX表达之间的关系。结果 H3K27me3在7日龄和2月龄小鼠被检测组织中均持续存在;EZH2在7日龄和2月龄小鼠脑、心肌、肝及皮肤中表达,但仅在2月龄小鼠涎腺、肠、睾丸、肺、脂肪组织、胸腺中持续表达;JMJD3在7日龄小鼠脑、皮肤、涎腺、心肌、肠、睾丸、肺、脂肪组织、胃中表达,但在2月龄小鼠的肺、脂肪组织、胃中不再表达;UTX在7日龄小鼠的脑、皮肤、涎腺、心肌、睾丸、肺及脂肪组织中表达,但仅在2月龄小鼠睾丸中表达;大部分免疫组织化学染色阳性的H3K27甲基调节酶相应的mRNA呈中等或较高水平表达。结论 H3K27me3在小鼠不同时期各组织中均持续存在;EZH2多存在于2月龄小鼠脑、皮肤、涎腺、心肌、肠、睾丸、肺、脂肪组织、肝、胸腺;JMJD3、UTX多存在于7日龄小鼠脑、皮肤、涎腺、心肌、睾丸、肺、脂肪组织;H3K27me3分布与EZH2的表达之间无明显一致关系,与JMJD3或UTX不存在明显反向分布关系,EZH2与JMJD3或UTX的表达分布也不存在反向关系。提示EZH2、JMJD3和UTX在小鼠出生后多种组织中可能起重要作用;在体内H3K27me3水平及其修饰酶可能受多因素控制,以完成复杂的生理功能。

Both combinatorial K4me0-K36me3 marks on sister histone H3s of a nucleosome are required for Dnmt3a-Dnmt3L mediated de novo DNA methylation

A nucleosome contains two copies of each histone H2A,H2B,H3 and H4.Histone H3 K4me0 and K36me3are two key chromatin marks for de novo DNA methylation catalyzed by DNA methyltransferases in mammals.However,it remains unclear whether K4me0 and K36me3 marks on both sister histone H3s regulate de novo DNA methylation independently or cooperatively.Here,taking advantage of the bivalent histone H3 system in yeast,we examined the contributions of K4 and K36 on sister histone H3s to genomic DNA methylation catalyzed by ectopically co-expressed murine Dnmt3a and Dnmt3L.The results show that lack of both K4me0 and K36me3 on one sister H3 tail,or lack of K4me0 and K36me3 on respective sister H3s results in a dramatic reduction of 5mC,revealing a synergy of two sister H3s in DNA methylation regulation.Accordingly,the Dnmt3a or Dnmt3L mutation that disrupts the interaction of Dnmt3aADD domain-H3K4me0,Dnmt3LADD domain-H3K4me0,orDnmt3aPWWP domain-H3K36me3 causes a significant reduction of DNA methylation.These results support the model that each heterodimeric Dnmt3a-Dnmt3L reads both K4me0 and K36me3 marks on one tail of sister H3s,and the dimer of heterodimeric Dnmt3a-Dnmt3L recognizes two tails of sister histone H3s to efficiently execute de novo DNA methylation.

Npac Is A Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells(ESCs).However,the related mechanisms remain obscure.Here,we show that Npac,a"reader"of histone H3 lysine 36 trimethylation(H3K36me3),is required to maintain mouse ESC(mESC)pluripotency since knockdown of Npac causes mESC differentiation.Depletion of Npac in mouse embryonic fibroblasts(MEFs)inhibits reprogramming efficiency.Furthermore,our chromatin immunoprecipitation followed by sequencing(ChIP-seq)results of Npac reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs.Interestingly,we find that Npac interacts with positive transcription elongation factor b(p-TEFb),Ser2-phosphorylated RNA PolⅡ(RNA PolⅡSer2P),and Ser5-phosphorylated RNA PolⅡ(RNA PolⅡSer5 P).Furthermore,depletion of Npac disrupts transcriptional elongation of the pluripotency genes Nanog and Rif1.Taken together,we propose that Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA PolⅡSer2P and Ser5P.

Colonic vitamin D receptor expression is inversely associated with disease activity and jumonji domain-containing 3 in active ulcerative colitis

BACKGROUND The expression of jumonji domain-containing 3(Jmjd3)and trimethylated H3 lysine 27(H3K27me3)in active ulcerative colitis(UC)and the correlation between vitamin D receptor(VDR)and the Jmjd3 pathway are unknown.AIM To study the relationship between VDR,Jmjd3 and H3K27me3 in patients with active UC.METHODS One hundred patients with active UC and 56 healthy controls were enrolled in this study.The patients with active UC were divided into groups according to mild(n=29),moderate(n=32)and severe(n=29)disease activity based on the modified Mayo score.Vitamin D levels were measured by radioimmunoassay.Colonic mucosal tissues from UC patients and controls were collected by colonoscopy.The expression of VDR,Jmjd3 and H3K27me3 in the intestinal mucosa was determined by immunohistochemistry staining.RESULTS Patients with active UC had lower levels of serum vitamin D(13.7±2.8 ng/mL,P<0.001)than the controls(16.2±2.5 ng/mL).In the UC cohort,serum vitamin D level was negatively correlated with disease activity(r=-0.323,P=0.001).VDR expression in the mucosa of UC patients was reduced compared to that in normal tissues(P<0.001)and negatively correlated with disease activity(r=-0.868,P<0.001).Similar results for VDR expression were noted in the most serious lesion(defined as UC diseased)and 20 cm proximal to the anus(defined as UC normal)(P<0.05).Simultaneously,Jmjd3 expression significantly increased in UC patients(P<0.001),but no difference was found between the different sites in UC patients.H3K27me3 expression in UC patients was significantly down-regulated when compared with normal tissues(P<0.001),but up-regulated in the mild disease activity group in comparison with the moderate disease activity group of UC patients(P<0.05).Jmjd3 Level was negatively correlated with the level of VDR(r=-0.342,P=0.002)and H3K27me3(r=-0.341,P=0.002),while VDR level was positively correlated with H3K27me3(r=0.473,P<0.001).CONCLUSION Serum vitamin D and VDR were inversely correlated with disease activity in active UC.Jmjd3 expression increased in the colonic mucosa of active UC patients and was negatively associated with VDR and H3K27me3 level.

PICOT promotes T lymphocyte proliferation by down-regulating cyclin D2 expression

The mammalian protein kinase C-interacting cousin of thioredoxin(PICOT;also termed glutaredoxin 3)is a multi-domain monothiol glutaredoxin that is involved in a wide variety of signaling pathways and biological processes.PICOT is required for normal and transformed cell growth and is critical for embryonic development.Recent studies in T lymphocytes demonstrated that PICOT can translocate to the nucleus and interact with embryonic ectoderm development,a polycomb group protein and a core component of the polycomb repressive complex 2,which contributes to the maintenance of transcriptional repression and chromatin remodeling.Furthermore,PICOT was found to interact with chromatin-bound embryonic ectoderm development and alter the extent of histone 3 lysine 27 trimethylation at the promoter region of selected polycomb repressive complex 2 target genes.PICOT knockdown in Jurkat T cells led to increased histone 3 lysine 27 trimethylation at the promoter region of CCND2,a cell cycle-regulating gene which encodes the cyclin D2 protein.As a result,the expression levels of CCND2 mRNA and protein levels were reduced,concomitantly with inhibition of the cell growth rate.Analysis of multiple data sets from the Cancer Genome Atlas revealed that a high expression of PICOT correlated with a low expression of CCND2 in a large number of human cancers.In addition,this parameter correlated with poor patient survival,suggesting that the ratio between PICOT/CCND2 mRNA levels might serve as a predictor of patient survival in selected types of human cancer.

Cellular functions of MLL/SET-family histone H3 lysine 4 methyltransferase components

The MLL/SET family of histone H3 lysine 4 methyltransferases form enzyme complexes with core subunits ASH2L, WDR5, RbBP5, and DPY-30 （often abbreviated WRAD）, and are responsible for global histone H3 iysine 4 methylation, a hallmark of actively transcribed chromatin in mammalian cells. Accordingly, the function of these proteins is required for a wide variety of processes including stem cell differentiation, cell growth and division, body segmentation, and hematopoiesis. While most work on MLL-WRAD has focused on the function this core complex in histone methylation, recent studies indicate that MLL-WRAD proteins interact with a variety of other proteins and IncRNAs and can localize to cellular organelles beyond the nucleus. In this review, we focus on the recently described activities and interacting partners of MLL-WRAD both inside and outside the nucleus.

小鼠不同细胞间组蛋白修饰变化对MafA基因转录表达的影响

目的通过比较不同细胞类型之间MafA基因转录起始区的组蛋白修饰差异,探讨组蛋白修饰对MafA基因转录表达的作用。方法采用染色质免疫共沉淀-实时定量PCR法检测小鼠胰岛素瘤β细胞(NIT-1)、NIH小鼠成纤维细胞(NIH3T3)及小鼠胚胎干细胞(mES)三者中的MafA和MLH1基因转录起始区组蛋白修饰(H3K4m3、H3K9m3和H3乙酰化)的状况。同时采用实时定量RT-PCR检测上述三种细胞各基因mRNA表达水平。分析基因的H3K4m3、H3K9m3和H3乙酰化修饰与基因表达之间的相互关系。结果 (1)以mES细胞为参照,NIT-1细胞MafA基因的转录起始区的H3K4m3修饰水平明显增高(P<0.05),H3K9m3修饰水平明显降低(P<0.05);NIH 3T3细胞MafA基因的转录起始区的H3K9m3修饰水平明显增高(P<0.05),H3K4m3修饰水平明显降低(P<0.05);(2)MafA基因的仅在NIT-1细胞表达,其表达与H3K4m3修饰存在直线相关(相关系数0.995);与H3K9m3修饰存在直线负相关(相关系数-0.751);(3)管家基因MLH1的表达与所检测组蛋白修饰无相关性。结论 H3K9m3与H3K4m3修饰能相互协调,共同调控MafA基因的表达,对胚胎干细胞向β细胞分化具有重要的意义。

三甲基转移酶SETD2表达下调促进结肠直肠癌细胞的迁移和增殖

目的:研究组蛋白H3K36三甲基转移酶SETD2对结肠直肠癌(CRC)细胞发生和生长的影响。方法:通过短发夹RNA转染降低CRC细胞系HCT116和SW480中SETD2的mRNA和蛋白质表达水平。检测转染后的CRC细胞系中三甲基化H3K36的蛋白质表达水平和肿瘤细胞增殖及迁移能力的变化,通过裸鼠成瘤实验观察KD组和对照组SETD2对于肿瘤生长的影响。结果:在CRC细胞中,随着SETD2的mRNA及蛋白质表达水平下降,检测到三甲基化H3K36蛋白质表达下降,且CRC细胞的增殖和迁移能力显著提升(P<0.05)。在裸鼠成瘤实验中通过免疫组织化学染色法,检测到KD组的小鼠肿瘤组织内三甲基化H3K36的蛋白质表达水平明显低于对照组。KD组裸鼠肿瘤的发生时间明显早于对照组,肿瘤的直径也明显大于对照组(P<0.01)。结论:下调SETD2的mRNA和蛋白质表达导致肿瘤组织三甲基化H3K36蛋白质表达下降,迁移和增殖能力增强,研究提示SETD2在CRC发生中的重要作用。

ERα promotes transcription of tumor suppressor gene ApoA-I by establishing H3K27ac-enriched chromatin microenvironment in breast cancer cells

Apolipoprotein A-I(Apo A-I),the main protein component of high-density lipoprotein(HDL),plays a pivotal role in reverse cholesterol transport(RCT).Previous studies indicated a reduction of serum Apo A-I levels in various types of cancer,suggesting Apo A-I as a potential cancer biomarker.Herein,ectopically overexpressed Apo A-I in MDA-MB-231 breast cancer cells was observed to have antitumor effects,inhibiting cell proliferation and migration.Subsequent studies on the mechanism of expression regulation revealed that estradiol(E2)/estrogen receptorα(ERα)signaling activates Apo A-I gene transcription in breast cancer cells.Mechanistically,our Ch IP-seq data showed that ERαdirectly binds to the estrogen response element(ERE)site within the Apo A-I gene and establishes an acetylation of histone 3 lysine 27(H3 K27 ac)-enriched chromatin microenvironment.Conversely,Fulvestrant(ICI 182780)treatment blocked ERαbinding to ERE within the Apo A-I gene and downregulated the H3 K27 ac level on the Apo A-I gene.Treatment with p300 inhibitor also significantly decreased the Apo A-I messenger RNA(m RNA)level in MCF7 cells.Furthermore,the analysis of data from The Cancer Genome Atlas(TCGA)revealed a positive correlation between ERαand Apo A-I expression in breast cancer tissues.Taken together,our study not only revealed the antitumor potential of Apo A-I at the cellular level,but also found that ERαpromotes the transcription of Apo A-I gene through direct genomic effects,and p300 may act as a co-activator of ERαin this process.

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。

H3K4m3和H3K9m3修饰对胰岛组织特异性基因表达的影响

目的通过比较不同细胞类型之间胰腺十二指肠同源盒1（Pdx-1）、配对盒基因4（Pax4）、MafA（mast cell function associated antigen）和Nkx6．1等胰岛组织特异性基因其转录起始区的H3K4m3和H3K9m3修饰的差异，探讨H3K4nd和H3K9m3修饰对胰岛组织特异性基因表达的作用。方法采用染色质免疫共沉淀．实时定量聚合酶链反应（PCR）法检测小鼠胚胎干细胞（mES，1×10^7）、小鼠成纤维细胞株NIH3T3细胞（1×10^7）和小鼠B细胞株NIT-1细胞（1×10^7）三者中的胰岛组织特异性基因、Oct4基因和MLH1基因转录起始区H3K4rrd和H3K9m3修饰的状况。同时采用实时定量逆转录（RT）-PCR检测上述3种细胞各基因mRNA表达水平。分析H3K4m3和H3K9m3修饰改变与基因表达之间的关系。结果NIT-1细胞中Pdx-1、Pax4、MafA、Nkx6．1等胰岛组织特异性基因转录起始区的H3K4m的修饰水平分别为：（4．84±0．05）％、（9．91±1．33）％、（10．64±0．87）％、（0．23±0．03）％，与mES细胞比较明显增高（P〈0．05），基因表达；NIH3T3细胞中Pdx-1、Pax4、MafA、Nkx6．1等胰岛组织特异性基因转录起始区的H3Kgm3的修饰水平分别为：（0．64±0．21）％、（7．04±1．29）％、（0．39±0．10）％、（2．35±0．81）％，与mES细胞比较明显增高（P〈0．05），基因不表达。结论mK4n13与H3K9m3修饰能相互协调，共同调控胰岛组织特异性基因的表达。