Étude du contexte chromatinien de la transcription des gènes codant les ARN ribosomiques chez la souris

Authors: Mars, Jean-Clement
Advisor: Moss, Tom
Abstract: At the heart of the ribosome, itself the center of synthesis of all proteins, are four RNA molecules, called ribosomal RNAs. These RNAs are synthesized in the nucleolus of eukaryotic cells from the tandemly repeated genes, the ribosomal RNA genes. A very strict regulation of the transcription of ribosomal RNA needs to be made to allow a controlled and above all a controllable cell cycle. Indeed, the imbalance of ribosomal RNA synthesis has been observed in many diseases such as cancer. Consequently, the study of transcriptional regulation of ribosomal RNAs is of crucial importance in the understanding and subsequent treatment of these diseases. Due to the number of repeats of ribosomal RNA genes, evaluated at around 175 per haploid genome for mammals, the in vivo study of their regulation by mutagenesis techniques is difficult. The approach I developed during this doctoral thesis is that of chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) applied to cell lines conditional for the basal transcription factors. The ribosomal RNA genes have the particularity of being transcribed thanks to an entirely dedicated transcriptional machinery. In mice, these general transcription factors act in concert to perform their role effectively. In the model organism used here, namely Mus musculus, RNA polymerase I transcribes the genes with the help, during initiation, of the factors UBF, Rrn3 / TIF-1A and SL-1 / TIF-1B. The TTF-1 termination factor makes it possible to terminate transcription of precursor ribosomal RNA, and also plays roles in gene regulation. In addition to high-throughput sequencing, we have developed a deconvolution approach to improve the interpretation of ChIP-Seq data. This approach has been validated by the improvement in particular of immunoprecipitation profiles obtained for RNA polymerase I that confirm the electron microscopy images of Miller spread type. This improvement of the data could also highlight a dual role of UBF depending on its complementarity with SL-1 and its affinity for GC-rich sequences of DNA. Subsequently we have been able to highlight the upstream localization of the regulatory regions and of a nucleosome barrier allowing to create and maintain a zone without nucleosomes along the rRNA genes. This barrier has two peculiarities, the first is that it contains the epigenetic marks usually associated with the activation of transcription such as H3K4me3, H2A.Z or the acetylation of H2A.Z. The second particularity is that it is independent of the presence of UBF, which is itself independent of transcription. Challenging certain assumptions about the regulation of ribosomal transcription, our work did not detect the presence of epigenetic markers of transcriptional activation throughout the rRNA gene body. Finally, parallel studies in embryonic stem cells (ESC) and embryonic fibroblasts (MEFs) made it possible to identify 3 categories of ribosomal genes in the same cell. First, a heterochromatic DNA methylated and nucleosomal form, a nucleosomal but non-DNA methylated form, and finally a transcribed form. A quantitative comparison of rRNA synthesis in ESCs and MEFs has shown that the number of active genes is not a significant factor in the regulation of rRNA synthesis. In embryonic cells, all genes are transcribed at the same time with low efficiency. In differentiated cells, a small portion of the genes are transcribed but very efficiently, this efficiency being related to the number of polymerase being transcribed. The different interaction profiles of Rrn3 and PolI near the transcription initiation site suggest that this difference is due to the regulation of initiation.
Document Type: Thèse de doctorat
Issue Date: 2019
Open Access Date: 11 July 2019
Permalink: http://hdl.handle.net/20.500.11794/35443
Grantor: Université Laval
Collection:Thèses et mémoires

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