Investigation of the Regulation of Gene Expression of the bHLH Transcription Factor Neurogenin2 and the Protein Arginine Methyltransferase PRMT8 in the Neuronal Milieu (Doctoral thesis)

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The main goal of this thesis was to investigate the regulatory mechanisms governing the expression of transcription factors and epigenetic modulators which are involved in neurogenesis focusing on the arginine methyltransferase PRMT8, and the bHLH protein Ngn2. Histone methylation on arginine residues is a new mechanism implicated in the dynamic regulation of chromatin structure and epigenetic memory. It is mediated by specialized enzymes called PRMTs. PRMT8 is particularly interesting because it is expressed exclusively in the nervous system, however its putative role in the process of adult neurogenesis is yet unknown. A fundamental role in neurogenesis as well as in neural stem cell maintenance and cell fate determination is played by the bHLH family of transcription factors. One of the proneural genes of this family, and a crucial regulator of neurogenesis is Ngn2. Both the levels and the activity of Ngn2 are under strict control which is essential for the initiation of the differentiation process. However, the regulation of Ngn2 gene expression is still poorly understood. By analyzing the expression profile of PRMT8 in the adult mouse hippocampus, we showed that PRMT8 is localized solely in the nucleus of the mature NeuN+ neurons in the granular zone of the dentate gyrus,and is never expressed together with the early differentiation marker DCX. To investigate the role of PRMT8 in vivo, we generated retroviral vectors in order to overexpress or downregulate the protein in the neurogenic regions of the adult mouse brain. During these experiments we realized that the cDNA sequence which was annotated in the NCBI database, produced only the membrane-bound and not the nuclear form. By analyzing more extensively the 5’ end of the gene using cRACE or 5’ RLM-RACE, we identified several novel alternative transcripts. One of them differs from the established PRMT8 mRNA only in the first exon, and probably represents the nuclear isoform. We also investigated the regulation of Ngn2 gene expression during neuronal differentiation, using as models rat neural progenitor cells (NPCs) as well as human neuroblastoma cells. In the NPCs, the Ngn2 mRNA levels increase fast within the first three hours of differentiation, and then decrease rapidly to undetectable levels. In the neuroblastoma cells the Ngn2 mRNA increases gradually at the early stages of differentiation and then it remains at constantly low levels for the rest of the program. In low serum, but in the absence of RA which is essential for neuronal differentiation, the mRNA levels of Ngn2 rise dramatically and remain high, indicating that continuous presence of RA is important for the control of itssteady state levels. By studying the changes in the protein profile of Ngn2, we noticed a disparity with the corresponding mRNA levels. Further analysis revealed that Ngn2 expression is also regulated at the level of translation and uncovered the presence of a negative feedback mechanism between Ngn2 and HuD, an RNA binding protein which can enhance or suppress the translation of its mRNA targets. The 5’-UTR of the Ngn2 mRNA seems to play a fundamental role in this mechanism, influencing not only the regulation of translation but also the interplay between Ngn2 and HuD. Overexpression experiments revealed that a small number of cells expressing Ngn2 can increase the expression of endogenous HuD in the total cell population, however, co-expression of the HMG family transcription factor Sox11 restricts the expression of HuD exclusively in the Ngn2+ cells, indicating that Ngn2 and Sox11 can synergize to control the expression of target genes. Analysis of the transcriptome profile (RNAseq) at distinct stages of neuronal differentiation in which the expression pattern of Ngn2 changes in a defined manner, resulted in the identification of several genes that provide a source for future studies in order to understand better the network of key regulators of neurogenesis whose expression is adjusted at multiple levels, such as Ngn2.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Βιολογικών Εφαρμογών και Τεχνολογιών
Subject classification: Αναπτυξιακή νευροβιολογία
Keywords: Ngn2,PRMT8,Νευρική διαφοροποίηση,Μεταφραστική ρύθμιση,Εναλλακτικά μετάγραφα,Neuronal differentiation,Translational regulation,Alternative splice variants
Appears in Collections:Διδακτορικές Διατριβές

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