Transcription regulation and functional studies of germ cell specific genes

(I)RNA结合蛋白在细胞命运决定中的生物学作用 在生物体的发育过程中，自我更新和分化的决定决定了多能细胞的命运。我们目前对细胞多能性的控制和维持的知识主要来源于对特定转录因子(如Oct4、Nanog和Sox2)在转录水平上控制其下游靶基因表达的作用的研究。同时，多能性相关基因RNA转录本的可用性、稳定性和效率代表了细胞命运决定背景下的另一种水平的调控。我们感兴趣的是研究RNA结合蛋白在维持多能性中的作用。在我们的研究中，我们使用胚胎干细胞和胚胎癌细胞作为模型系统，研究在多能细胞中优先表达的RNA结合蛋白(Lin28a和Pumilio/Nanos家族)的生物学功能。此外，我们有兴趣阐明控制这些基因产物的细胞类型特定表达模式的调控机制。 1.Lin28a基因表达的转录调控 Lin28a编码一种RNA结合蛋白，是细胞增殖的重要调节因子。Lin28a在胚胎干细胞、胚胎癌细胞、精原干细胞和部分癌细胞中高表达。除心肌和骨骼肌外，Lin28a在成体组织和各种体细胞中均未见表达。我们发现，Lin28a的细胞类型特异性表达受表观遗传调控。具体地说，不表达Lin28a的细胞分别用组蛋白去乙酰酶抑制剂和DNA甲基转移酶抑制剂处理，然后收集用于基因表达分析。我们发现，只有在接受组蛋白去乙酰化酶抑制剂处理的细胞中，Lin28a的表达才被重新激活。我们进一步证明了Lin28a的表达伴随着转录激活的组蛋白修饰在其启动子区域的丰富，这导致染色质构象的松弛，从而增加了转录因子对Lin28a启动子的可及性。 2.Lin28a蛋白的功能研究 以胚胎癌细胞为模型，我们正在测试Lin28a蛋白是否能增强其翻译产物对细胞增殖具有促进作用的RNA转录本的稳定性。我们利用RNA干扰技术成功地沉默了Lin28a蛋白的表达。与先前在胚胎干细胞中的发现一致，我们还观察到当Lin28a表达减弱时，胚胎癌细胞的细胞增殖率降低。在存在RNA生物合成抑制剂的情况下，将检查选定的RNA转录本的稳定性。 3.Pumilio和Nanos蛋白在小鼠胚胎干细胞增殖中的作用 Pumilio和Nanos是进化上保守的RNA结合蛋白，在果蝇的胚胎发育和生殖系发育中发挥着重要作用。具体地说，这两种蛋白质相互作用，抑制目标RNA转录本的翻译。在哺乳动物中，已鉴定出两种Pumilio蛋白亚型(Pumilio-1和-2)和三种异构体Nanos蛋白(Nanos-1至-3)。与其他类型的细胞相比，我们观察到Pumilio和特定的Nanos蛋白在小鼠胚胎干细胞和胚胎癌细胞中的优先表达模式，表明这些蛋白在多能性中具有功能作用。为了测试Pumilio和Nanos蛋白在维持多能性方面的作用，我们将沉默它们在小鼠胚胎干细胞中的表达，并检查细胞的增殖是否受到影响。我们还将测量多能性相关基因在微米或纳米沉默细胞中的表达水平，以评估它们参与维持多能性的过程。 (2)对全球转录组输出的表观遗传调控 除了蛋白质编码转录本的表达外，很明显，哺乳动物基因组也表达大量非蛋白质编码的RNA转录本。其中，一些长的非蛋白质编码的RNAs(LncRNAs)和许多microRNAs(MiRNAs)分别通过在转录和翻译水平上调节其他基因的表达来调节细胞的生理。在哺乳动物细胞中，表观遗传修饰物可以促进组蛋白乙酰化或DNA去甲基化，从而改变单个蛋白质编码转录本的表达水平。然而，在这些修饰物的影响下，完整转录组(包括编码蛋白质的RNA转录本和非编码蛋白质的RNA转录本)的全球变化从未得到系统的分析。 我们开始研究表观遗传修饰物对哺乳动物细胞转录组输出的影响。我们在几个小鼠细胞系中的初步分析表明，特定的lncRNAs的表达受到组蛋白乙酰化水平的影响。我们正在扩大分析，以检查不同的表观遗传修饰物对完整转录组输出的影响。从这项研究中，我们希望确定非蛋白质编码的RNA转录子的子集，这些转录子共同或不同地受不同的表观遗传修饰物的调控，并研究它们在细胞内稳态中的作用。

(I) Biological role of RNA-binding proteins in cell fate decision The decision between self-renewal and differentiation determines the fate of pluripotent cells in the development of an organism. Our current knowledge of the cellular control and maintenance of pluripotency is derived mainly from the studies of the action of specific transcription factors (for example, Oct4, Nanog and Sox2) that control the expression of their downstream target genes at transcriptional level. Meanwhile, the availability, stability and efficiency of translation of RNA transcripts of pluripotency-related genes represent another level of regulation in the context of cell fate decision. We are interested in studying the role of RNA-binding proteins in the maintenance of pluripotency. In our research, we use embryonic stem cells and embryonal carcinoma cells as the model systems to study the biological functions of RNA-binding proteins (Lin28a and Pumilio/Nanos families) that are preferentially expressed in pluripotent cells. Also, we are interested in elucidating the regulatory mechanism that controls the cell-type specific expression patterns of these gene products. 1. Transcriptional regulation of Lin28a expression Lin28a encodes an RNA-binding protein and is an important regulator of cell proliferation. Lin28a is highly expressed in embryonic stem cells, embryonal carcinoma cells, spermatogonial stem cells and a subset of cancer cells. With the exception of cardiac and skeletal muscle, no Lin28a expression is found in adult tissues and various types of somatic cells. We found that the cell-type specific expression of Lin28a is regulated epigenetically. Specifically, non Lin28a-expressing cells were treated separately with histone deacetylase inhibitor and DNA methyltransferase inhibitor and then harvested for gene expression analysis. We found that Lin28a expression was reactivated only in cells receiving histone deacetylase inhibitor treatment. We further demonstrated that the expression of Lin28a is accompanied by an enrichment of transcription-activating histone modification in its promoter region, which leads to a relaxation of the chromatin conformation and subsequently an increased accessibility of transcription factors to the Lin28a promoter. 2. Functional studies of Lin28a protein Using embryonal carcinoma cells as the model, we are testing if Lin28a protein acts to enhance the stability of RNA transcripts whose translated products display a promoting effect on cell proliferation. We have successfully silenced the expression of Lin28a protein by RNA interference technique. Consistent with the previous findings in embryonic stem cells, we also observed a reduction of cell proliferative rate in embryonal carcinoma cells when Lin28a expression is attenuated. The stability of the selected RNA transcripts will be examined in the presence of inhibitor of RNA biosynthesis. 3. Role of Pumilio and Nanos proteins in mouse embryonic stem cell proliferation Pumilio and Nanos are evolutionarily conserved RNA-binding proteins that play an important role in embryogenesis and germline development in Drosophila. Specifically, the two proteins interact with each other to repress the translation of target RNA transcripts. In mammals, two isoforms of Pumilio protein (Pumilio-1 and -2) and three isoforms of Nanos protein (Nanos-1 to -3) have been identified. We observed a preferential expression pattern of Pumilio and specific Nanos proteins in mouse embryonic stem cells and embryonal carcinoma cells when comparing with other cell types, suggesting a functional role of these proteins in pluripotency. To test the role of Pumilio and Nanos proteins in the maintenance of pluripotency, we are going to silence their expression in mouse embryonic stem cells and examine if the proliferation of the cells is affected. We will also measure the expression level of pluripotency-related genes in Pumilio- or Nanos-silenced cells to evaluate their involvement in the maintenance of pluripotency. (II) Epigenetic regulation of global transcriptome output Besides the expression of protein-coding transcripts, it is apparent that the mammalian genome expresses a large amount of non protein-coding RNA transcripts as well. Among them, a number of long non protein-coding RNAs (lncRNAs) and many microRNAs (miRNAs) have been found to regulate cellular physiology by tuning the expression of other genes at transcriptional and translational levels, respectively. In mammalian cells, the alteration of expression level of individual protein-coding transcripts by epigenetic modifiers, which promote either histone acetylation or DNA demethylation, has been documented. However, the global change in full transcriptome (comprised of protein-coding RNA transcripts and their non protein-coding counterparts) under the influence of these modifiers has never been analyzed systematically. We initiated to examine the effect of epigenetic modifiers on transcriptome output in mammalian cells. Our preliminary analysis in several mouse cell lines indicated that the expression of specific lncRNAs is affected by histone acetylation level. We are expanding the analysis to examine the effect of different epigenetic modifiers on full transcriptome output. From this study we expect to identify the subsets of non protein-coding RNA transcripts that are commonly or distinctively regulated by the different epigenetic modifiers and to investigate their roles in the homeostasis of the cells.