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.