Transcription regulation and functional studies of germ cell specific genes

Lin28a蛋白通过促进控制细胞生长的基因的翻译和抑制促进细胞分化的let-7 microRNAs的成熟而发挥积极的细胞增殖调节作用。因此，Lin28a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表达。我们发现，Lin28a启动子DNA片段在不显示内源性Lin28a表达的小鼠细胞中具有转录活性。启动子CpG高甲基化与基因表达沉默之间缺乏相关性，提示Lin28a转录不受DNA甲基化的抑制。5-氮杂-2-脱氧胞苷处理始终不能激活Lin28a转录。相反，用组蛋白去乙酰化酶抑制剂曲古抑素A(TSA)处理诱导了Lin28a的表达，这也增加了Lin28a启动子DNA在体内对核酸酶消化的敏感性。我们还观察到Lin28a的表达状态与组蛋白H3(H3K9)赖氨酸9残基的修饰之间的关系：在未分化的胚胎癌细胞P19和TSA处理的NIH/3T3成纤维细胞中，Lin28a的表达与乙酰化H3K9(H3K9Ac)的丰富和二甲基化H3K9(H3K9me2)在Lin28a启动子上的占有率减少一致。相反，当Lin28a的表达在细胞分化时或在没有化学诱导的情况下被沉默时，情况正好相反。组蛋白修饰模式也影响了RNA聚合酶II在Lin28a启动子上的占位。我们的发现表明，H3K9Ac和H3K9me2的动态变化通过控制Lin28a启动子的可及性参与了Lin28a转录的激活。 MicroRNAs(MiRNAs)是一种小的非编码RNA，在转录后水平上调节其靶基因的表达。在癌细胞中，miRNAs根据其靶基因的生物学功能，可能具有促进或抑制肿瘤的作用。用DNA甲基化和/或组蛋白去乙酰化抑制剂处理癌细胞可以调节miRNAs的表达水平，这为表观遗传调控miRNA的表达提供了证据。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的研究。我们研究了在BIX01294处理或不处理的情况下，miRNAs在非小细胞肺癌细胞系H1299中的表达模式。BIX01294是一种有效的G9a甲基转移酶化学抑制剂，催化组蛋白H3的赖氨酸9残基的单甲基化和双甲基化。通过结合芯片分析和实时定量聚合酶链式反应分析，我们鉴定了两个在BIX01294处理后显示一致下调的miRNAs。我们的结果表明，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新的见解。

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.