Functional Analysis Of Nucleolin-chromatin Interaction In Vivo

Besides the well-known role of the nucleolus in ribosome biogenesis, nucleoli play important roles in the regulation of many fundamental cellular processes, including cell cycle regulation, apoptosis, telomerase production, RNA processing and therefore it is not surprising that many nucleolar proteins appear to be multifunctional proteins.Nucleolin, one of the most abundant non-ribosomal proteins of the nucleolus, has been the focus of many studies since it was first described35years ago. It seems to be involved in many aspects of DNA metabolism, chromatin regulation and appeared to be a good pharmacological target for drug development in addition to its role in RNA polymerase I transcription and pre-ribosomal processing and assembly in pre-ribosomes.In eukaryotic cells, DNA is packed into nucleosomes to form chromatin in the nucleus. The cells develop a variety of strategies to overcome the nucleosomal barriers. These strategies include DNA methylation, histone post-translational modifications, incorporation of histone variants and ATP dependent chromatin remodeling.The aim of this thesis is to study the interaction of nucleolin with chromatin, and to decipher the mechanism of nucleolin in gene regulation.It was reported that nucleolin possesses a histone chaperone activity, helps the transcription through nucleosomes, and it is required for ribosomal DNA gene (rDNA) transcription in vivo, but the mechanism by which nucleolin modulates RNA polymerase I (Pol I) transcription is unknown. In the thesis it is shown that nucleolin knockdown results in an increase of the heterochromatin mark H3K9me2and a decrease of H4K12Ac and H3K4me3euchromatin histone marks in rDNA genes. Nucleolin is associated with unmethylated rDNA genes and ChIP-seq experiments identified a strong enrichment of nucleolin in the promoter and coding regions of rDNA. Nucleolin is able to interfere with the binding of TTF-1on the promoter-proximal terminator TO thus inhibiting the recruitment of the nucleolar remodeling complex (NoRC) subunit TIP5and HDAC1and the establishment of a repressive heterochromatin state. In addition, in absence of nucleolin or after inhibition of Pol I by actinomycin D, a strong relocalization of the histone variant macroH2A1to the nucleolus and on the rDNA genes was observed. This invasion of macroH2A1in the nucleolus plays a major role in the inhibition of Pol I transcription in absence of nucleolin, as knockdown of macroH2A1eliminates the repressive effect of nucleolin depletion. These results reveal the importance of nucleolin for the maintenance of the euchromatin state of rDNA required for an efficient production of ribosomal RNAs and the role of macroH2Al in rDNA transcription.