表观遗传学对Oct4基因表达的调节作用

第一部分:表观遗传学(Epigenetic)

一、Histone methylation

1、Embryonic stem cells

1.1 Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes. Epigenetics & Chromatin 2010,3:4.(IF=4.731)

1.1.1 实验内容: When the nuclei of mammalian somatic cells are transplanted to amphibian oocytes in the first meiotic prophase, they are rapidly induced to begin transcribing several pluripotency genes, including Sox2 and Oct4.

This study investigated a range of histone modifications in mouse nuclei transplanted to Xenopus laevis oocyte germinal vesicles (GVs,胚泡) in order to ask whether histone modifications are changed at the nuclear or chromatin levels.

1.1.2 实验结果: ①. The transcriptional reprogramming of pluripotency genes, such as Sox2 and Oct4, takes place in transplanted nuclei from C3H 10T1/2 cells and from newly differentiated mouse embryonic stem cells (DmES). ②. The reprogramming of 10T1/2 nuclei is accompanied by an increased phosphorylation, an increased methylation and a rapidly reduced acetylation of several amino acids in H3 and other histones. ③. Histone phosphorylation is increased and histone acetylation is decreased in several regulatory and gene coding regions. An increase of histone H3 lysine 4 dimethylation (H3K4me2) is seen in the regulatory regions and gene coding region of pluripotency genes in reprogrammed nuclei. ④. histone H3 lysine 4 trimethylation (H3K4me3) is observed more strongly in the regulatory regions of pluripotency genes in transplanted nuclei that are rapidly reprogrammed than in nuclei that are reprogrammed slowly and are not seen in β-globin, a gene that is not reprogrammed. ⑤. When 10T1/2 nuclei are incubated in Xenopus oocyte extracts, histone H3 serine 10 (H3S10) is strongly phosphorylated within a few hours.

1.1.3 结论: H3K4 me2 and me3 are likely to be important for the efficient reprogramming of pluripotency genes in somatic nuclei by amphibian oocytes and that Aurora B kinase is required for H3S10 phosphorylation which is induced in transplanted somatic cell nuclei.

1.2 Comparative epigenetic analysis of Oct4 regulatory region in RA-induced differentiated NT2 cells under adherent and non-adherent culture conditions. Mol Cell Biochem. 2012 . 363(1-2):129-34. (IF=2.329)

1.2.1 实验内容：Oct4has a long upstream regulatory region of about 2,600 bp, consisting of proximal enhancer (PE), distal enhancer (DE), and proximal promoter (PP).

In this study, we induced differentiation of a human embryonic carcinoma cell line, NT2, under two different adherent and non-adherent culture conditions, and compared histone modifications as the epigenetic marks on the regulatory region of Oct4 gene after 3 days of differentiation. Comparing the two culturing systems it was shown that methylation of lysine-9 of H3 histone was more drastic in PE region of adherent cells rather than suspension cells.

1.2.2 实验结果：The after induction of differentiation the repressive epigenetic marks of hypoacetylation and methylation on lysine-9 of histone H3 occurred very effectively on the upstream of Oct4, especially in PP region.

1.2.3 结论：cell culture condition as an environmental factor may influence on dynamics of epigenetic marks and consequently the pattern of gene expression.

1.3 PCL2 modulates gene regulatory networks controlling self-renewal and commitment in embryonic stem cells. Cell Cycle, 2011. 10(1):45-51.(IF=5.359)

1.3.1 实验内容：PCL2, a Polycomb Repressive Complex 2 (PRC2) associated protein, is important in cell fate decisions and may play a role in recruitment of PRC2 to target genes and histone methylation.

To better understand the molecular underpinnings of self-renewal and commitment of ESCs, we drafted(设计) and tested gene regulatory networks (GRN) modules.

1.3.2 实验结果：PCL2 is downregulated in a similar fashion as Oct4, Sox2 and Nanog. Depletion of PCL2 in ESCs leads to a decrease in 3meH3K27 at the proximal promoter regions of pluripotency transcription factors Tbx3, Klf4, Foxd3 and a concomitant increase in gene expression. These proteins subsequently activate expression of Oct4, Nanogand Sox2through a feed-forward gene regulatory circuit, altering the core pluripotency network and driving cell fate decisions towards self-renewal.

1.3.3 结论：PCL2 associates with the core components of the PRC2 complex and is critical for modulating GRNs involved in ESC differentiation and self-renewal. PCL2 is a critical regulator of ESC fate decisions and lack of PCL2 contributes to a delay in differentiation and maintenance of a population of cells with ESC-like characteristics.

1.4 Protein arginine methyltransferase 6 regulates embryonic stem cell identity. Stem Cells Dev. 2012. 21(14):2613-22.(IF=4.67)

1.4.1 实验内容：Protein arginine methyltransferase 6 (PRMT6)-mediated di-methylation of histone H3 at arginine 2 (H3R2me2) can antagonize(对…起反作用) tri-methylation of histone H3 at lysine 4(H3K4me3), which marks active genes.

We investigated the roles of PRMT6 and PRMT6-mediated H3R2me2 in early embryonic development and ES cell identity using gain and loss of function studies with mouse ES cells as a model system.

1.4.2 实验结果：Prmt6 and histone H3R2 methylation levels increased when ES cells are induced to differentiate. Differentiation of ES cells upon upregulation of Prmt6 is associated with decreased expression of pluripotency genes and increased expression of differentiation markers.

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. knockdown of Prmt6 also leads to downregulation of pluripotency genes and induction of expression of differentiation markers.

1.4.3 结论：A critical level of Prmt6 and histone H3R2me must be maintained in mouse ES cells to sustain their pluripotency.

1.5 G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis. Nat Cell Biol, 2006. 8(2):188-194.(IF=18.485)

1.5.1 实验内容：靶基因抑制是通过染色质结构和组蛋白修饰来调控的。为了检查Oct4是否受这种规则调控，我们使用一种针对2me-或3me-H3(K9)的特定抗体进行分析。接下来，我们研究了其他基因序列是否也通过异染色质化包括组蛋白H3甲基化来标记沉默。

1.5.2 实验结果：Oct3/4是POU结构域的同源异型框的基因，该基因在配子形成和在早期胚胎细胞中表达，已经证明对维持多能性是非常重要的。Oct4转入differentiating embryonic cells后，经历了一个逐步程序性失活过程。Transcriptional repression is followed by a pronounced increase in histone H3 methylation on Lys 9 that is mediated by the SET-containing protein, G9a.

1.5.3 结论：在分化的起始阶段Oct3/4基因经历了转录失活。Epigenetic changes actually have an important role in the inhibition of Oct-3/4 reexpression, thereby preventing reprogramming.

1.6 Histone code modifications on pluripotential nuclei of reprogrammed somatic cells. Mol Cell Biol. 2004 Jul;24(13):5710-20.（IF=7.822）

1.6.1 实验内容：Following hybridization with embryonic stem (ES) cells, somatic genomes are epigenetically reprogrammed and acquire pluripotency.

This study show the role of acetylation and methylation of histone H3 and H4 amino termini in the regulation of gene activity through the modulation of chromatin conformation.

1.6.2 实验结果：The reprogrammed somatic genome of ES hybrid cells becomes hyperacetylated at H3 and H4, while lysine 4 (K4) of H3 becomes globally hyper-di- and -tri-methylated. In the Oct4 promoter region, histones H3 and H4 are acetylated and H3-K4 is highly tri-methylated on both the ES and reprogrammed somatic genomes.

1.6.3 结论：H3-K4 di- and tri-methylation of reprogrammed somatic genomes is independent of gene activity and represents one of the major events that occurs during somatic genome reprogramming towards a transcriptional activation-permissive state.

2、Mesenchymal stem cells

2.1 Chromatin states of core pluripotency-associated genes in pluripotent, multipotent and differentiated cells. Biochem Biophys Res Commun, 2010. 391(1): 762–767. (IF=2.595)

2.1.1实验内容：在胚胎癌细胞、MSC和成纤维细胞中，研究Oct4、Nanog和Sox2基因调控区域DNA的甲基化和组蛋白修饰。

2.1.2实验结果：亚硫酸盐测序，揭示了Oct4启动子区CpG甲基化和表达抑制之间的关系，但在Nanog或Sox2中它们之间并没有相关性，即与Oct4表达存在不同的抑制机制。1）在多能和已分化的干细胞中，Oct4、Nanog和Sox2的CpG甲基化状态差异表明显著地转录抑制差异；2）启动子上发生H3K27me3和编码区发生H3K36 me3相互排斥。3）Oct4，NANOG，SOX2启动子的差异富集抑制组蛋白修饰（1. H3K4me3存在于大多数启动子上，与表达抑制无关；2. H3K9me1 也许在Oct4、NANOG 和SOX2上有抑制功能；3. Oct4内部既没有H3K27me3也没有 H3K9me3，但是却有CpG甲基化，说明它被DNA甲基化抑制。

2.1.3结论：多能性相关基因不同抑制机制的确立可防止在发育或分化过程中反应混杂，这种机制构成一个保护系统。

3、其他细胞

3.1 Q2ChIP, a Quick and Quantitative Chromatin Immunoprecipitation Assay, Unravels Epigenetic Dynamics of Developmentally Regulated Genes in Human Carcinoma Cells. Stem Cells, 2007. 25:1037–1046.(IF=7.531)

3.1.1 实验内容：We used Q2ChIP to monitor changes in histone H3 modifications on the 5’ regulatory regions of the developmentally regulated genes OCT4, NANOG, LMNA, and PAX6 in the context of retinoic-acid-mediated human embryonal carcinoma cell differentiation. qPCR analysis of precipitated DNA unravels biphasic heterochromatin assembly on OCT4 and NANOG, involving H3 lysine (K)9 and K27 methylation followed by H3K9 deacetylation and additional H3K27 trimethylation.

3.1.2 实验结果：Q2ChIP unravels an unexpected biphasic heterochromatinization(异染色质化) of OCT4 and NANOG in response to RA stimulation, whereas PAX6 displays histone modifications characteristic of repressed genes with potential for activation in undifferentiated cells. Oct4 dissociation from the NANOG promoter upon differentiation.

3.1.3 结论：This study reveal histone modification changes on human OCT4 and NANOG regulatory sequences. Ordered chromatin rearrangement on developmentally regulated promoters upon differentiation.

3.2 Promoter-exon relationship of H3 lysine 9, 27, 36 and 79 methylation on pluripotency-associated genes. Biochem Bioph Res Co, (2010) 401: 611–617. (IF=2.595)

3.2.1 实验内容：Little is known on profiles of trimethylated H3 lysine residues on coding regions of these genes in pluripotent and differentiated cells, and on the interdependence between promoter and exon occupancy of modified H3.

This study explore how H3K9, H3K27, H3K36 and H3K79 trimethylation on exons of pluripotency genes correlates with promoter occupancy and expression.

3.2.2 实验结果：①. Promoter H3K27 trimethylation extends into the first exon of repressed OCT4, NANOG and SOX2, while H3K9me3 occupies the first exon of these genes irrespective of expression. ②. Down-regulation of the H3K36 methyltransferase SetD2 by siRNA causes global and gene-specific H3K36 demethylation and global H3K27 hypermethylation; but it does not affect promoter levels of H3K27me3, mRNA levels are however affected.

3.2.3 结论：The genes examined independence of occupancy of H3K27me3 on promoters and H3K36me3 on exons. mRNA levels are affected, raising the hypothesis of a role of SetD2 on transcription elongation and/or termination.

二、Histone acetylation

1、Embryonic stem cells

1.1 Global and gene-specific histone modification profiles of mouse multipotent adult germline stem cells. Molecular human reproduction, 2011. 17(3):166-174. (IF=3.852)

1.1.1实验内容：通过调查哺乳动物中第二个主要的表观遗传修饰来拓展maGSCs和male ESC的相对表观遗传学分析。实验对比研究了maGSCs和ESCs的全套基因和特定基因的组蛋白修饰（H3K4me3, H3K9ac, H3K9me3和H3K27me3）过程。

1.1.2实验结果：免疫荧光染色，流式细胞术以及WB实验结果表明，maGSCs的上述表观遗传修饰的整体水平和核分布型态与成人ESCs非常相似；染色质免疫沉淀（ChIP）和实时定量PCR分析表明，maGSCs和ESCs细胞在多能性标记基因Oct4, Sox2, Nanog以及关键的发展谱系控制基因Hoxa11的上述修饰中都显示出了几乎相同的基因特异性模式。

1.1.3结论：maGSCs在关于染色质状态和进一步的多能性证据方面和ESCs都是十分的相似。

1.2 Epigenetic Control of MouseOct-4Gene Expression in Embryonic Stem Cells and Trophoblast Stem Cells. The Journal of Biological Chemistry, 2004. 279:17063-17069. (IF=4.651).

1.2.1 实验内容：Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells.

To determine whether DNA methylation is involved in the regulation of Oct-4 gene expression, we investigated 1) the effect of a DNA methylation inhibitor on Oct-4 non-expressing cells, 2) the methylation status of Oct-4 enhancer/promoter locus in both ES and TS cells, 3) the effect of in vitro methylation on Oct-4 enhancer/promoter activity, 4) Oct-4 expression in the DNA methyltransferase-1 (Dnmt1)-deficient conceptus, and 5) the chromatin structure of Oct-4 enhancer/promoter region in both ES and TS cells.

1.2.2实验结果：用5-氮杂-2-脱氧胞苷或古抑菌素A培养TS细胞，引起了Oct4基因的活化。在胚胎干细胞中Oct4的增强子/启动子区域低甲基化，但在TS细胞中却是高甲基化的。体外检测发现甲基化抑制Oct4增强子/启动子的活性。染色质免疫共沉淀实验结果显示，与TS细胞相比，胚胎干细胞Oct4的增强子/启动子区域是高乙酰化的。

1.2.3结论：Oct4基因表达是在表观遗传学机制包括DNA甲基化与染色质结构重塑的作用下调节的。

1.3 Transcriptional reprogramming and chromatin remodeling accompanies Oct4 and Nanog silencing in mouse trophoblast lineage. Stem Cells and Dev, 2013: 1564-1575.(IF=4.67)

1.3.1 实验内容：In mouse blastocysts CDX2 plays a key role in silencing Oct4 and Nanog expression in the trophectoderm (TE) lineage. A Cdx2-inducible mouse embryonic stem (ES) cell line was utilized as a model system to address the underlying transcriptional and chromatin-based changes that are associated with CDX2-mediated repression.

1.3.2 实验结果：Induction of Cdx2 expression resulted in a decrease in Oct4/Nanog expression, an increase in TE markers, and differentiation into trophoblast-like stem (TS-like) cells within 48 to 120 hours. a significant decrease in histone H3K9/14 acetylation and loss of p300 and HDAC1 binding at the Oct4 and Nanog regulatory elements was observed by 48 hours. Accompanying these changes there was a significant increase in total histone H3 and a loss of chromatin accessibility at both the Oct4 and Nanog regulatory elements (P<0.05), indicative of chromatin remodeling. Lastly, DNA methylation analysis revealed that methylation did not occur at Oct4 and Nanog until 96 to 120 hours after induction of CDX2.

1.3.3 结论：The results show that silencing of Oct4 and Nanog is facilitated by sequential changes in transcription factor binding, histone acetylation, chromatin remodeling, and DNA methylation at core regulatory elements.

2、Mesenchymal stem cells

2.1 Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation. PLoS One, 2011. 6(6):e20526. (IF=4.092)

2.1.1实验内容：对MSC在体外进行表达、成骨分化、自我更新和多向分化潜能以及细胞增殖方面进行检测。同时，对启动子DNA甲基化和组蛋白H3乙酰化水平进行了测定。

2.1.2实验结果：MSC通过培养扩增会经历老化和自发地成骨分化过程中，telomerase reverse transcriptase (TERT)基因的表达逐步下调，而Runx2 和ALP基因的表达上调。同时，与干细胞自我更新有关的基因，如Oct4和Sox2基因的表达显著下降。值得注意的，这些基因表达的改变，与在H3的K9和K14部位乙酰化的表观遗传学调节异常有关，而不是与这些基因启动子区域的CpG岛甲基化有关。bFGF能够促进MSC增殖并抑制其自发的向成骨细胞分化，促进H3乙酰化，增加了TERT、Sox2和Oct4基因的表达。

2.1.3结论：组蛋白H3乙酰化可被外源信号调节，在调控MSC衰老和分化中起着重要作用。

3、 其他细胞

3.1 Effect of Dynamic DNA Methylation and Histone Acetylation on cPouV Expression in Differentiation of Chick Embryonic Germ Cells. Stem Cells Dev, 2013, 22(20):2725-2735. (IF=4.67)

3.1.1实验内容：实验首先描述了在鸡胚胎生殖细胞分化过程中cPouⅤ(the homologue of Oct4 in avian)基因启动子区DNA甲基化和组蛋白乙酰化的变化；为进一步了解表观遗传修饰对cPouⅤ基因的影响，实验又对用TSA，AzaH和TSA/Aza处理的EG细胞进行了分析；通过CHIP方法对EG细胞进行表观遗传学修饰的分析。

3.1.2实验结果：通过CHIP检测发现：在细胞分化过程中，cPouⅤ启动子区域的DNA甲基化明显增加，而组蛋白的乙酰化则明显的降低；基因表达分析表明，在EG细胞向EB结构转化的过程中，Dnmt3a, Dnmt3b 和HDAC3转录物的水平明显变高，而Dnmt1, HAT和 cPouⅤ mRNA的相对丰度则明显的降低；甲基化分析显示报告基因被cPouⅤ启动子甲基化而明显抑制。

3.1.3结论：在小鸡EG细胞多潜能性维持和分化过程中，通过DNA甲基化和/或组蛋白修饰来调节cPouⅤ基因的激活或抑制作用具有重要作用。

3.2 Zebularine and Scriptaid Significantly Improve Epigenetic Reprogramming of Yak Fibroblasts and Cloning Efficiency. Cellular Reprogramming, 2013,15(4):293-300. (IF=2.744).

3.2.1 实验内容：Abnormal epigenetic reprogramming of the donor nucleus after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiency.

This study have attempted to improve epigenetic reprogramming of the donor nucleus and cloned embryos with Zebularine and Scriptaid. Yak fibroblasts were treated with 20 μl Zebularine alone or 20 μl Zebularine plus 0.5 μl Scriptaid for 24 h, whereas yak cloned embryos were treated exclusively with 0.5 μl Scriptaid for 12 h. There was no effect on cellular viability and proliferation after drug treatment.

3.2.2实验结果：①. The treatment of fibroblasts with Zebularine or Zebularine plus Scriptaid increased histone acetylation of histone 3 lysine 9 (H3K9), but decreased the level of DNA methylation ofOct-4andSox-2promoter regions.

②. When donor cells were used after Zebularine plus Scriptaid treatment to reconstruct cloned embryos and then treated with Scriptaid, the developmental competence and cryosurvival of embryos were improved significantly. In addition, the relative expression of Oct-4andSox-2were increased significantly.

③. The expression levels of Dnmt-1 and Hdac-1 were significantly decreased when fibroblasts and cloned embryos were treated with Zebularine or Scriptaid

3.2.3结论：Treatment with Zebularine and Scriptaid modifies the epigenetic status of yak fibroblasts, subsequently enhancingin vitro developmental potential and the quality of yak cloned embryos.

三、Histone phosphorylation

1、Embryonic stem cells

1.1 Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes. Epigenetics & Chromatin 2010,3:4. (IF=4.731)

1.1.1 实验内容: When the nuclei of mammalian somatic cells are transplanted to amphibian oocytes in the first meiotic prophase, they are rapidly induced to begin transcribing several pluripotency genes, including Sox2 and Oct4.

This study investigated a range of histone modifications in mouse nuclei transplanted to Xenopus laevis oocyte germinal vesicles (GVs,胚泡) in order to ask whether histone modifications are changed at the nuclear or chromatin levels.

1.1.2 实验结果: ①. The transcriptional reprogramming of pluripotency genes, such as Sox2 and Oct4, takes place in transplanted nuclei from C3H10T1/2 cells(小鼠胚胎成纤纤细胞) and from newly differentiated mouse embryonic stem cells(DmES). ②. The reprogramming of 10T1/2 nuclei is accompanied by an increased phosphorylation, an increased methylation and a rapidly reduced acetylation of several amino acids in H3 and other histones. ③. Histone phosphorylation is increased and histone acetylation is decreased in several regulatory and gene coding regions. An increase of histone H3 lysine 4 dimethylation (H3K4me2) is seen in the regulatory regions and gene coding region of pluripotency genes in reprogrammed nuclei. ④. histone H3 lysine 4 trimethylation (H3K4me3) is observed more strongly in the regulatory regions of pluripotency genes in transplanted nuclei that are rapidly reprogrammed than in nuclei that are reprogrammed slowly and are not seen in β-globin, a gene that is not reprogrammed. ⑤. When 10T1/2 nuclei are incubated in Xenopus oocyte extracts, histone H3 serine 10 (H3S10) is strongly phosphorylated within a few hours.

1.1.3 结论: H3K4 me2 and me3 are likely to be important for the efficient reprogramming of pluripotency genes in somatic nuclei by amphibian oocytes and that Aurora B kinase is required for H3S10 phosphorylation which is induced in transplanted somatic cell nuclei.

1.2 Recruitment of Oct4 Protein to UV -Damaged Chromatin in Embryonic Stem Cells. PLoS One. 2011;6(12):e27281. (IF=4.092).

1.2.1 实验内容：Oct4是ESC多能性的一个特异性标志，该实验研究了mESC中经紫外激光照射或暴露在不同DNA损伤试剂下Oct4的功能特性，检查Oct4是否能识别稳定表达GFP-Oct4的mESC中染色质的损伤。

1.2.2 实验结果：经紫外线照射后，Oct4以ATP依赖性的方式在照射损伤区域立即积聚，同时伴随着H3K9的去乙酰化和H2A的磷酸化。而且在一些细胞过程被干扰后，观察到γH2AX的ESC核特异性分布。组蛋白去乙酰化酶抑制剂能够阻碍紫外照射的染色中显著的Oct4 积累。

1.2.3 结论：在 DNA 损伤应答（DDR）过程中伴随着一些多能性特异性事件，其中 Oct4在 DDR 过程中具有重要作用。

2、Mesenchymal stem cells

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3、其他细胞

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第二部分：检测方法

一、 Histone methylation

（一） 方法

1、ChIP-qPCR

(1)、ChIP

大致步骤为：在37℃下，1%甲醛处理细胞10min, 使组蛋白和DNA发生交联。随后0.125mol/L甘氨酸在37℃下处理5min终止交联反应。用10mL冷1×PBS清洗细胞2次后，300μL溶解缓冲液( 10mmoL/L Tris-HCl pH8.0, 100mmoL/LNaCl, 1mmoL/L乙二胺四乙酸pH8.0，0.1%脱氧胆酸钠和蛋白酶抑制剂)重悬细胞，并在冰上孵育30min，超声处理细胞悬液4min。随后在4℃下离心10min，弃上清，获得片状碎屑物。用含有蛋白酶抑制剂的稀释缓冲液溶解片状碎屑物， 溶解液分为三个部分：第1份溶解液与抗组蛋白抗体，第2份和第3份溶解液分别作为内参( input control)和阴性对照( negative control)，在第1份溶解液和阴性对照中加入50μL玻璃珠并在44℃孵育1h，然后将玻璃珠分别用低盐，高盐，氯化锂溶液和TE缓冲液洗涤，随后加热65℃ 5h解交联、RNaseA和ProteinaseK消化， 酚氯仿抽提DNA。接下来进行DNA扩增和扩增后的纯化。

(2) 、qPCR

用Real-time PCR 仪对免疫沉淀的DNA、Input control 和Negative control 进行扩增。测定标准曲线和熔解曲线，确保扩增的准确性和专一性， 用Ct值表示样品中模板的相对含量， 采用2-ΔΔCT法进行分析。计算不同组别的 ChIP DNA 样品的 fold enrichment值用于组间比较。

（二） 相关文献

1、 Emodin Modulates Epigenetic Modifications and Suppresses Bladder Carcinoma Cell Growth. Mol Carcinog. 2013 Sep 20. (IF=4.269)

2、 The genomic landscapes of histone H3-Lys9 modifications of gene promoter regions and expression profiles in human bone marrow mesenchymal stem cells. J Genet Genomics, 2008 . 35(10):585-93. (IF=2.076)

3、 Genome-wide characterization of menin-dependent H3K4me3 reveals a specific role for menin in the regulation of genes implicated in MEN1-like tumors. PLoS One. 2012;7(5):e37952. (IF=3.73)

4、 Histone H3 lysine 4 monomethylation and H3 lysine 9 monomethylation: distribution and their association in regulating gene expression under hyperglycaemic/hyperinsulinemic conditions in 3T3 cells. Biochimie. 2012 Dec;94(12):2656-64.(IF=3.142)

5、 Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells, 2007. 25:1037–1046.(IF=7.531).

6、 Epigenetic mechanisms regulate stem cell expressed genes pou5f1 and gfra1 in a male germ cell line. PLoS ONE, 2010 5(9): e12727. (IF=4.411)

7、 Effects of dna methylation and histone modification on differentiation-associated gene expression in es, NIH-3T3, and NIT-1. J Huazhong Univ Sci Technol, 2011. 31(1):10-16.

8、 Pluripotency-related, valproic acid (VPA)-induced genome-wide histone H3 lysine 9 (H3K9) acetylation patterns in embryonic stem cells. J Biol Chem. 2011;286(41):35977-35988.(IF=4.651)

二、 Histone acetylation

（一） 方法

1、ChIP-qPCR

与Histone methylation方法大致相同，其中针对不同的组蛋白修饰所用的抗体不同。

（二）相关文献

1、 Spinocerebellar ataxia type 8 larger triplet expansion alters histone modification and induces RNA foci. BMC Mol Biol. 2009, 10;10:9. (IF=2.796)

2、 Histone deacetylase inhibitors induce epithelial-to-mesenchymal transition in prostate cancer cells. PLoS One. 2012;7(9):e45045. (IF=3.73)

3、Pluripotency-related, valproic acid (VPA)-induced genome-wide histone H3 lysine 9 (H3K9) acetylation patterns in embryonic stem cells. J Biol Chem. 2011;286(41):35977-35988.(IF=4.651)

4、Genome-wide reduction in H3K9 acetylation during human embryonic stem cell differentiation. J Cell Physiol. 2009; 219(3):677-687.(IF=4.586)

5、 Integration of cytological features with molecular and epigenetic properties of rice chromosome 4. Mol Plant. 2008 ;1(5):816-29. (IF=6.126)

6、Epigenetic Control of MouseOct-4Gene Expression in Embryonic Stem Cells and Trophoblast Stem Cells. J Biol Chem. 2004, 279(17):17063-9.(IF=6.355)

三、 Histone phosphorylation

（一） 方法

1、 ChIP-qPCR

与Histone acetylation方法大致相同，其中针对不同的组蛋白修饰所用的抗体不同。

（二）相关文献

1、Loss of FBP1 by Snail-mediated repression provides metabolic advantages in basal-like breast cancer. Cancer Cell. 2013, 18;23(3):316-31.(IF=24.755)

2、 Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes. Epigenetics & Chromatin 2010,3:4.(IF=4.731)

3、 Synchronous recruitment of epigenetic modifiers to endotoxin synergistically activated Tnf-α gene in acute kidney injury. PLoS One. 2013,30;8(7):e70322.(IF=3.73)

4、 CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells. Nat Cell Biol. 2011, 13(1):87-94. (IF=19.488)

5、丁酸钠对K562细胞γ-珠蛋白基因启动子组蛋白磷酸化的影响. 儿科临床杂志, 2010, 25(12): 926-930.

10.21-OCT4综述