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

• 批准号: 8553938
• 负责人: Owen Rennert
• 金额: $ 31.27万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 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/8553938
• 关键词: Adult; Affect; Attenuated; Biological; Biological Models; Biological Process; Cell Proliferation; Cell physiology; Cells; Chromatin; Code; Coiled-Coil Domain; DNA; DNA Methyltransferase Inhibitor; Development; Drosophila genus; Embryonal Carcinoma Cell; Embryonic Development; Epigenetic Process; Family; Gene Expression; Gene Expression Profile; Gene Proteins; Gene Targeting; Genes; Genetic Transcription; Germ Cells; Global Change; Goals; Harvest; Histone Acetylation; Histone Deacetylase Inhibitor; Homeostasis; Homologous Gene; Individual; Investigation; Knowledge; Maintenance; Mammalian Cell; Mammals; Measures; MicroRNAs; Mitotic; Modeling; Molecular Conformation; Mouse Cell Line; Mus; Myocardium; Organism; Output; Pattern; Play; Promoter Regions; Protein Isoforms; Proteins; RNA; RNA Interference; RNA Stability; RNA biosynthesis; RNA-Binding Proteins; Regulation; Relaxation; Research; Role; Skeletal Muscle; Somatic Cell; Stem cells; Techniques; Testing; Testis; Tissues; Transcript; Transcriptional Regulation; Translating; Translations; cancer cell; cell type; demethylation; embryonic stem cell; histone modification; inhibitor/antagonist; interest; male; mammalian genome; novel; pluripotency; promoter; protein expression; self-renewal; transcription factor

(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.

(I)RNA结合蛋白在细胞命运决定中的生物学作用 自我更新和分化之间的决定决定了多能细胞在生物体发育中的命运。 我们目前对多能性的细胞控制和维持的知识主要来自于对特定转录因子（例如，Oct 4、Nanog和Sox 2）的作用的研究，这些转录因子在转录水平上控制其下游靶基因的表达。 与此同时，多能性相关基因的RNA转录物的可用性、稳定性和翻译效率代表了细胞命运决定背景下的另一个水平的调节。 我们有兴趣研究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来研究在多能细胞中优先表达的RNA结合蛋白（Lin 28 a和Pumilio/Nanos家族）的生物学功能。 此外，我们有兴趣阐明的调控机制，控制这些基因产物的细胞类型特异性表达模式。 1. Lin 28 a表达的转录调控 Lin 28 a编码RNA结合蛋白，是细胞增殖的重要调节因子。 Lin 28 a在胚胎干细胞、胚胎癌细胞、精原干细胞和一部分癌细胞中高度表达。 除心肌和骨骼肌外，在成体组织和各种类型的体细胞中均未发现Lin 28 a表达。 我们发现Lin 28 a的细胞类型特异性表达受表观遗传学调控。 具体地，分别用组蛋白脱乙酰酶抑制剂和DNA甲基转移酶抑制剂处理非Lin 28 a表达细胞，然后收获用于基因表达分析。 我们发现Lin 28 a的表达仅在接受组蛋白去乙酰化酶抑制剂处理的细胞中被重新激活。 我们进一步证明了Lin 28 a的表达伴随着其启动子区域中转录激活组蛋白修饰的富集，这导致染色质构象的松弛，随后增加了转录因子对Lin 28 a启动子的可及性。 2. Lin 28 a蛋白的功能研究 使用胚胎癌细胞作为模型，我们正在测试Lin 28 a蛋白是否可以增强RNA转录物的稳定性，RNA转录物的翻译产物显示出对细胞增殖的促进作用。 我们成功地利用RNA干扰技术沉默了Lin 28 a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Lin 28 a表达减弱时，胚胎癌细胞中的细胞增殖率降低。 将在存在RNA生物合成抑制剂的情况下检查所选RNA转录物的稳定性。 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，在果蝇的胚胎发生和生殖系发育中发挥重要作用。 具体而言，这两种蛋白质相互作用以抑制靶RNA转录物的翻译。 在哺乳动物中，已经鉴定了Pumilio蛋白的两种同种型（Pumilio-1和-2）和Nanos蛋白的三种同种型（Nanos-1至-3）。 与其他细胞类型相比，我们观察到Pumilio和特定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的功能作用。 为了测试Pumilio和Nanos蛋白在维持多能性中的作用，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Pumilio或Nanos沉默细胞中多能性相关基因的表达水平，以评估它们在多能性维持中的参与。 (II)表观遗传调控的整体转录组输出 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的非蛋白质编码RNA转录本。 其中，一些长链非蛋白质编码RNA（lncRNA）和许多微小RNA（miRNAs）分别在转录和翻译水平上通过调节其他基因的表达来调节细胞生理。 在哺乳动物细胞中，表观遗传修饰剂（促进组蛋白乙酰化或DNA去甲基化）改变了单个蛋白编码转录物的表达水平。 然而，在这些修饰剂的影响下，全转录组（包括蛋白质编码RNA转录物和它们的非蛋白质编码对应物）的全局变化从未被系统地分析过。 我们开始研究表观遗传修饰剂对哺乳动物细胞中转录组输出的影响。 我们在几种小鼠细胞系中的初步分析表明，特异性lncRNA的表达受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对全转录组输出的影响。 从这项研究中，我们希望确定的子集的非蛋白质编码RNA转录，通常或独特的调节不同的表观遗传修饰剂，并调查他们的作用，在细胞的稳态。