(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.
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