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Transcription regulation and functional studies of germ cell specific genes

生殖细胞特异性基因的转录调控和功能研究

基本信息

批准号：
8736897
负责人：
Owen Rennert
金额：
$ 27.55万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8736897
关键词：
Biological Biological Models Biological Process Cancer cell line Cell Differentiation process Cell Proliferation Cells Chemicals Coiled-Coil Domain Coupling DNA DNA Methylation Decitabine Development Digestion Down-Regulation Embryonal Carcinoma Embryonal Carcinoma Cell Epigenetic Process Family Fibroblasts Functional RNA Future Gene Expression Gene Proteins Gene Targeting Genes Genetic Transcription Germ Cells Goals H1299 Histone Deacetylase Inhibitor Histone Deacetylation Histone H3 Homologous Gene Hypermethylation Investigation Knowledge Lysine Maintenance Malignant neoplasm of lung Methylation Methyltransferase MicroRNAs Microarray Analysis Mitotic Modification Mono-S Mus NIH 3T3 Cells Non-Small-Cell Lung Carcinoma Organism Pattern Play Polymerase Chain Reaction Predisposition Proteins RNA RNA Polymerase II RNA-Binding Proteins Regulation Research Role Testis Time Transcript Transcriptional Regulation Translations Trichostatin A Undifferentiated cancer cell cell growth cell type embryonic stem cell histone methyltransferase histone modification in vivo inhibitor/antagonist insight interest male novel nuclease pluripotency promoter protein function self-renewal transcription factor tumor

项目摘要

Lin28a protein functions as a positive regulator of cell proliferation by enhancing the translation of genes controlling cell growth and suppressing the maturation of let-7 microRNAs that promote cell differentiation. Accordingly, Lin28a gene products are highly expressed in pluripotent cells and certain cancer cells but are generally absent in differentiated cell types. We found that the Lin28a promoter DNA fragment was transcriptionally active in mouse cells that do not show endogenous Lin28a expression. The lack of correlation between promoter CpG hypermethylation and silencing of gene expression suggested that Lin28a transcription was not repressed by DNA methylation. Consistently, 5-aza-2-deoxycytidine treatment could not activate Lin28a transcription. In contrast, treatment with the histone deacetylase inhibitor Trichostatin A (TSA) induced Lin28a expression; this also increased the in vivo susceptibility of the Lin28a promoter DNA to nuclease digestion. We also observed an association between the expression status of Lin28a and the modification of the lysine 9 residue of histone H3 (H3K9): expression of Lin28a in undifferentiated embryonal carcinoma P19 cells and TSA-treated NIH/3T3 fibroblasts was consistently associated with the enrichment of acetylated H3K9 (H3K9Ac) and a reduction in dimethylated H3K9 (H3K9Me2) occupancy on the Lin28a promoter. The opposite occurred when Lin28a expression was silenced upon cellular differentiation or in the absence of chemical induction. The histone modification pattern also influenced the occupancy of RNA polymerase II on the Lin28a promoter. Our findings suggest that the dynamic change in H3K9Ac and H3K9Me2 occupancy is involved in the activation of Lin28a transcription by controlling the accessibility of the Lin28a promoter. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of their target genes at the post-transcriptional level. In cancer cells, miRNAs, depending on the biological functions of their target genes, may have a tumor promoting or suppressive effect. Treatment of cancer cells with inhibitors of DNA methylation and/or histone deacetylation modulates the expression level of miRNAs, which provides evidence for epigenetic regulation of miRNA expression. The consequences of inhibition of histone methyltransferase on miRNA expression, however, have not been thoroughly studied. We examined the expression pattern of miRNAs in the non-small cell lung cancer cell line H1299 with or without treatment of BIX01294, a potent chemical inhibitor of G9a methyltransferase that catalyzes the mono- and di-methylation of lysine 9 residue of histone H3. By coupling microarray analysis with quantitative real-time polymerase chain reaction analysis, we identified two miRNAs that showed consistent downregulation after BIX01294 treatment. Our results indicate histone H3 methylation regulates miRNA expression in lung cancer cells; this may provide additional insight for future chemical treatment of lung cancer.

Lin 28 a蛋白通过增强控制细胞生长的基因的翻译和抑制促进细胞分化的let-7 microRNA的成熟而作为细胞增殖的正调节剂发挥作用。因此，Lin 28 a基因产物在多能细胞和某些癌细胞中高度表达，但在分化的细胞类型中通常不存在。我们发现Lin 28 a启动子DNA片段在不显示内源性Lin 28 a表达的小鼠细胞中具有转录活性。启动子CpG超甲基化和基因表达沉默之间缺乏相关性，表明Lin 28 a的转录不受DNA甲基化的抑制。因此，5-氮-2-脱氧胞苷不能激活Lin 28 a的转录。相反，用组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）处理诱导Lin 28 a表达;这也增加了Lin 28 a启动子DNA对核酸酶消化的体内易感性。我们还观察到Lin 28 a的表达状态与组蛋白H3（H3 K9）的赖氨酸9残基的修饰之间的关联：Lin 28 a在未分化胚胎癌P19细胞和TSA处理的NIH/3 T3成纤维细胞中的表达与乙酰化H3 K9（H3 K9 Ac）的富集和二甲基化H3 K9（H3 K9 Me 2）的减少一致相关。在Lin 28 a启动子上的占据。当Lin 28 a表达在细胞分化后或在没有化学诱导的情况下沉默时，发生相反的情况。组蛋白修饰模式也影响RNA聚合酶II在Lin 28 a启动子上的占有率。我们的研究结果表明，在H3 K9 Ac和H3 K9 Me 2占用的动态变化参与激活Lin 28 a转录通过控制的可及性Lin 28 a启动子。 MicroRNA（miRNAs）是一类在转录后水平调控靶基因表达的小分子非编码RNA。在癌细胞中，根据其靶基因的生物学功能，miRNA可能具有肿瘤促进或抑制作用。用DNA甲基化和/或组蛋白去乙酰化的抑制剂治疗癌细胞调节miRNA的表达水平，这为miRNA表达的表观遗传调控提供了证据。然而，抑制组蛋白甲基转移酶对miRNA表达的影响尚未得到彻底研究。我们研究了在有或没有BIX 01294处理的非小细胞肺癌细胞系H1299中miRNA的表达模式，BIX 01294是一种有效的G9 a甲基转移酶化学抑制剂，催化组蛋白H3的赖氨酸9残基的单甲基化和二甲基化。通过将微阵列分析与定量实时聚合酶链反应分析相结合，我们鉴定了两种在BIX 01294处理后表现出一致下调的miRNAs。我们的研究结果表明组蛋白H3甲基化调节肺癌细胞中miRNA的表达;这可能为未来肺癌的化学治疗提供额外的见解。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Methylation of an intronic region regulates miR-199a in testicular tumor malignancy.

DOI：
10.1038/onc.2011.60
发表时间：
2011-08-04
期刊：
ONCOGENE
影响因子：
8
作者：
Cheung, H-H;Davis, A. J.;Lee, T-L;Pang, A. L.;Nagrani, S.;Rennert, O. M.;Chan, W-Y
通讯作者：
Chan, W-Y

Methylation profiling using methylated DNA immunoprecipitation and tiling array hybridization.