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Active and repressed ribosomal RNA (rRNA) genes are characterised by specific epigenetic marks and differentially positioned nucleosomes at their promoters. Repression of the rRNA genes requires a non-coding RNA (pRNA) and the presence of the nucleolar remodeling complex (NoRC). ATP-dependent chromatin remodeling enzymes are essential regulators of DNA-dependent processes, and this regulation occurs via the modulation of DNA accessibility in chromatin. We have studied the targeting of NoRC to the rRNA gene promoter; its mechanism of nucleosome positioning, in which a nucleosome is placed over the transcription initiation site; and the functional role of the pRNA. We demonstrate that NoRC is capable of recognising and binding to the nucleosomal rRNA gene promoter on its own and binds with higher affinity the nucleosomes positioned at non-repressive positions. NoRC recognises the promoter nucleosome within a chromatin array and positions the nucleosomes, as observed in vivo. NoRC uses the release mechanism of positioning, which is characterised by a reduced affinity for the remodeled substrate. The pRNA specifically binds to NoRC and regulates the enzyme by switching off its ATPase activity. Given the known role of pRNA in tethering NoRC to the rDNA, we propose that pRNA is a key factor that links the chromatin modification activity and scaffolding function of NoRC., Author Summary Tumour cells overexpress ribosomal RNA (rRNA), which is required for ribosome assembly and cell growth. rRNA gene repression is mediated by the chromatin remodeling complex (NoRC) and a non-coding RNA that binds to this enzyme. This study addresses the mechanism of nucleosome positioning by NoRC and the functional role of the non-coding RNA, which is termed pRNA because it corresponds to the promoter sequence. NoRC recognises the promoter nucleosome in a chromatin array with high affinity and uses a release mechanism to position the nucleosome over the transcription initiation site. The pRNA binds specifically to NoRC and inhibits its ATPase activity. We suggest that the RNA retains NoRC at the gene promoter after remodeling, linking its chromatin modification and scaffolding activity to inactive rDNA copies.

Regulation of gene expression takes place in the nucleus in a highly structured and condensed nucleoprotein environment, called chromatin (Felsenfeld and Groudine, 2003; Khorasanizadeh, 2004; Vaquero et al., 2003). A broad group of factors regulates the properties of chromatin; e.g. by covalently modifying histones and / or by ATP-dependent chromatin remodeling, thereby allowing or preventing gene expression. The mammalian genome contains hundreds of gene copies encoding precursor ribosomal RNA and the transcription of these genes is highly regulated with respect to cellular metabolism (Grummt, 2003). However, even in actively growing cells, only a subset of the rRNA genes are actively transcribed, exhibiting an accessible chromatin conformation (Conconi et al., 1989). In a chromatin context, the activation of rDNA genes involves the transcription termination factor TTF-I (Längst et al., 1998; Längst et al., 1997a). However, the silenced rDNA gene fraction remains in an inaccessible heterochromatic state throughout the cell cycle (Conconi et al., 1989). Until recently, the onset of silencing and the mechanisms that maintain the inactive state of rRNA genes were less understood. Recent studies, including the work presented in this thesis, provide insights into the molecular mechanism of ribosomal RNA gene silencing (Lawrence et al., 2004; Németh et al., 2004; Santoro and Grummt, 2001; Santoro et al., 2002; Strohner et al., 2004; Zhou et al., 2002). Accumulating evidence indicates that the combined action of chromatin modifying mechanisms such as chromatin remodeling, histone modification and DNA methylation contribute to the process of rRNA gene silencing. Here I present data demonstrating an active role of the chromatin remodeling complex NoRC in rDNA gene silencing and propose dual functions of TTF-I in rDNA regulation in chromatin, namely involvement in both activation and silencing of rDNA transcription. 4.1 NoRC, a novel chromatin remodeling complex In this doctoral study, a novel protein complex, composed of the nucleolar protein Tip5 and the ATPase Snf2h, was purified using convential chromatography and affinity purification methods. A detailed chromatin remodeling analysis revealed that this complex is able to induce mononucleosome movement in an ATP and histone H4 tail dependent fashion. Finally, this Tip5-Snf2h complex was termed NoRC (nucleolar remodeling complex), a novel member of the ISWI family of ATP-dependent chromatin remodeling complexes (Strohner et al., 2001). To dissect its functions, the NoRC complex was reconstituted from its recombinant subunits Tip5 and Snf2h, using the baculo virus driven expression system. Reconstitution confirmed the direct interaction between Tip5 and Snf2h. Furthermore, recombinant and cellular NoRC display similar sizes in gel filtration columns. Recombinant NoRC exhibits chromatin stimulated ATPase activity and mobilizes nucleosomes in an energy-dependent manner. Both activities are histone H4 tail dependent. NoRC and its subunits Tip5 and Snf2h were compared in different DNA / Nucleosome binding assays. NoRC shows preferred binding to structured (bent) DNA, e.g. a region within the mouse rDNA promoter, and interacts with mononucleosomes in electrophoretic mobility shift assays (EMSA). While no stable interaction with core nucleosomes could be detected in EMSA, ATPase assays and DNase I protection assays noticeably pinpointed to NoRC / nucleosome interactions with both nucleosomal and protruding linker DNA. 4.2 NoRC specifically represses rDNA transcription in chromatin The functional consequences of the Tip5 / TTF-I interaction were assessed and the influence on chromatin structure of the rDNA promoter in an in vitro system was determined. Tip5 in NoRC interacts with the N-terminal part of full length TTF-I and unmasks its DNA binding site. This interaction is required both for binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter in chromatin. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome. To elucidate a potential role of NoRC in rDNA transcriptional regulation, we used an in vitro transcription system with an rDNA minigene reconstituted into chromatin. These studies revealed a specific function for NoRC in rDNA transcriptional repression on chromatin templates. In contrast, NoRC had no effect on DNA transcription. Transcription experiments were then performed with chromatin templates reconstituted from recombinant histones lacking individual histone tails. The results indicate that NoRC-mediated rDNA gene repression is dependent on the histone H4 tail, suggesting an involvement of chromatin remodeling. Further transcription experiments revealed that NoRC-mediated repression occurs prior to preinitiation complex formation and does not affect activated rDNA genes. NoRC stably associates with the silenced gene, and these early steps of rDNA repression do not depend on DNA and histone modifications (Strohner et al., 2004). NoRC showed preferred binding to a structured (bent) region within the mouse rDNA promoter. Methylation of a single CpG dinucleotide within this region abrogated rDNA transcription in chromatin (Santoro and Grummt, 2001), but did not influence DNA binding of NoRC. Furthermore, nucleosomal DNA is less methylated than free DNA, but chromatin remodeling enhances methylation. The results suggest an important role for the chromatin remodeling complex NoRC in the establishment of rDNA silencing. NoRC then contributes to maintenance of the silenced state throughout the cell cycle by interacting with DNA and histone modifying enzymes. Transcriptional repression by chromatin remodeling factors seems to be a common mechanism to stably inhibit gene expression.