NoRC, a novel chromatin remodeling complex involved in ribosomal RNA gene silencing

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

A Vaccinia-based system for directed evolution of GPCRs in mammalian cells

Directed evolution in bacterial or yeast display systems has been successfully used to improve stability and expression of G protein-coupled receptors for structural and biophysical studies. Yet, several receptors cannot be tackled in microbial systems due to their complex molecular composition or unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality and uniform expression, we develop a viral transduction system based on Vaccinia virus. By rational design of synthetic DNA libraries, we first evolve neurotensin receptor 1 for high stability and expression. Second, we demonstrate that receptors with complex molecular architectures and large ligands, such as the parathyroid hormone 1 receptor, can be readily evolved. Importantly, functional receptor properties can now be evolved in the presence of the mammalian signaling environment, resulting in receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface. Our approach thus provides insights into the intricate molecular interplay required for GPCR activation

The chromatin remodeling complex NoRC and TTF-I cooperate in the regulation of the mammalian rRNA genes in vivo

The transcription termination factor (TTF)-I is a multifunctional nucleolar protein that terminates ribosomal gene transcription, mediates replication fork arrest and regulates RNA polymerase I transcription on chromatin. TTF-I plays a dual role in rDNA regulation, being involved in both activation and silencing of rDNA transcription. The N-terminal part of TTF-I contains a negative regulatory domain (NRD) that inhibits DNA binding. Here we show that interactions between the NRD and the C-terminal part of TTF-I mask the DNA-binding domain of TTF-I. However, interaction with TIP5, a subunit of the nucleolar chromatin remodeling complex, NoRC, recovers DNA-binding activity. We have mapped the protein domains that mediate the interaction between TTF-I and TIP5. The association of TIP5 with the NRD facilitates DNA binding of TTF-I and leads to the recruitment of NoRC to the rDNA promoter. Thus, TTF-I and NoRC act in concert to silence rDNA transcription

Chromatin targeting signals nucleosome positioning mechanism and non-coding RNA mediated regulation of the chromatin remodeling complex NoRC

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

The DNA chaperone HMGB1 facilitates ACF/CHRAC-dependent nucleosome sliding

Nucleosome remodelling complexes CHRAC and ACF contribute to chromatin dynamics by converting chemical energy into sliding of histone octamers on DNA. Their shared ATPase subunit ISWI binds DNA at the sites of entry into the nucleosome. A prevalent model assumes that DNA distortions catalysed by ISWI are converted into relocation of DNA relative to a histone octamer. HMGB1, one of the most abundant nuclear non-histone proteins, binds with preference to distorted DNA. We have now found that transient interaction of HMGB1 with nucleosomal linker DNA overlapping ISWI-binding sites enhances the ability of ACF to bind nucleosomal DNA and accelerates the sliding activity of limiting concentrations of remodelling factor. By contrast, an HMGB1 mutant with increased binding affinity was inhibitory. These observations are consistent with a role for HMGB1 as a DNA chaperone facilitating the rate-limiting DNA distortion during nucleosome remodelling

Maximizing Detergent Stability and Functional Expression of a GPCR by Exhaustive Recombination and Evolution

To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics(R) technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution

Analysis of NoRC activity and nucleosome binding.

<p>(<b>A</b>) Purified recombinant NoRC and Snf2H proteins were analysed by SDS-PAGE and Coomassie blue staining. Molecular weight markers are shown on the left and arrows on the right point to the recombinant proteins. (<b>B</b>) Remodeling activity of NoRC was tested on nucleosomes reconstituted on 601 DNA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004157#pgen.1004157-Thstrm1" target="_blank">[20]</a>. Nucleosomes positioned at the border (upper panel), or at the center of the DNA fragment (lower panel) were incubated with increasing amounts of NoRC and ATP as indicated. After the remodeling reaction the nucleosome positions were analysed on native PAA gels. (<b>C</b>) The ATPase activity of NoRC in the presence of DNA and nucleosomes exhibiting different linker lengths. ATP hydrolysis was measured using radioactive ATP as a tracer and the hydrolysed phosphate was separated via thin layer chromatography. Total ATP hydrolysis was quantified and plotted. (<b>D</b>) The binding affinity of NoRC to DNA molecules of different length was quantified and plotted. NoRC was incubated with a mixture of DNA molecules of different length and analysed by EMSA. The graph shows a quantification of the relative binding of the individual DNA fragments by NoRC. (<b>E</b>) Analysis of Tip5 binding to cruciform DNA. Radioactive labelled cruciform DNA (panel 2) and the two 40 bp DNA controls (panel 1 and 3), which cover the same nucleotide sequence, were incubated with increasing amounts of recombinant Tip5. DNA binding was analysed on native PAA gels. The structure of the annealed oligonucleotides is given on top. (<b>F</b>) Binding of NoRC to DNA and nucleosomes with different lengths of linker DNA. Purified nucleosomes, assembled on a 247 bp rDNA fragment, are either positioned at the border or the center of the DNA fragment. The reconstituted nucleosomes contained either no linker DNA (146 bp fragment), ∼25 bp linker DNA (171 bp fragment), ∼50 bp linker DNA (247 bp, middle position) or ∼100 bp linker DNA (end-positioned nucleosome). A scheme of the nucleosomes is shown on the top. Arrowheads indicate the DNA/NoRC or nucleosome/NoRC complexes.</p

pRNA inhibits the activity of NoRC.

<p>(<b>A</b>) Nucleosomes assembled on the −190 to +90 rDNA DNA fragment were incubated with NoRC or Snf2H, ATP and increasing concentrations of pRNA_−143/−39 and pRNA_−113/−39 (5 to 200 nM). The remodeling reactions were analysed by EMSA. The arrowhead indicates the nucleosome at the −120/+27 position. (<b>B</b>) NoRC specifically recognizes the hairpin-loop structure of the pRNA. Mononucleosomes assembled on −190 to +90 rDNA promoter region were incubated with NoRC and increasing concentrations of pRNA_−127/−39 (20 to 80 nM) or the mutated pRNA missing the hairpin-loop structure in the presence of ATP. Remodeling reactions were stopped after 45 min and analysed by EMSA. The arrowhead indicates the nucleosome at the −120/+27 position.</p

NoRC binds to the entry/exit sites of the nucleosome.

<p>(<b>A</b>) Overview of the experimental approach. (<b>B</b>) Analytical EMSA of the DNase I footprinting reaction. For further analysis of the DNase I digestion pattern the nucleosome and nucleosome/NoRC complexes were isolated from the gel. The arrow indicates the NoRC/nucleosome complexes. (<b>C</b>) DNase I footprinting of DNA and centrally positioned nucleosomes. A 247 bp rDNA promoter fragment (−231 to +16 respective to the start site) was radioactively labelled either at the 5′ or 3′ end. The free DNA (bar) and the centrally positioned nucleosome (gray ellipse) were treated with DNase I and after 10 sec and 30 sec the reactions were stopped with EDTA. Nucleosomes and DNA were resolved by EMSA and the bands were isolated. Purified DNA was subsequently analysed on 7% sequencing gels. A scheme of the central positioned nucleosome is shown on the right. (<b>D</b>) Recombinant NoRC was incubated with a purified nucleosome positioned at the center of the DNA fragment and partially digested with DNase I (10 and 30 sec). The reaction was stopped by the addition of EDTA and the nucleoprotein complexes were separated by native gel electrophoresis. Nucleosomes and NoRC/nucleosome complexes were isolated, DNA purified and analysed on 7% sequencing gels. The nucleosome position (gray ellipse) and the radioactive end-labeling (³²P) are indicated. Changes in the digestion pattern upon NoRC treatment are marked with a gray bar, significant changes are highlighted with stars.</p

Nucleosomes and pRNA compete for the binding of NoRC.

<p>(<b>A</b>) ATPase assay. NoRC was incubated with the indicated pRNAs and radioactive ATP as a tracer. Hydrolysed phosphate was separated via thin layer chromatography and analysed on a PhosphoImager. The quantification of three independent reactions is plotted. Error bars show the standard deviations. (<b>B</b>) Competitive binding assays using NoRC, pRNA and nucleosomes. Nucleosomes assembled on the rDNA promoter (−190 to +90) (lane 1) were incubated with NoRC (lanes 2 to 12), resulting in quantitative complex formation (lanes 2 and 8). These complexes were incubated with increasing concentrations of pRNA as indicated and analysed by EMSA. The nucleosome occupying the position −120/+27 is indicated. Lanes 13 to 24 shows the experiment, but performed with Snf2H. (<b>C</b>) Model describing the putative roles of NoRC and pRNA in rRNA gene silencing.</p