Ingrid Grummt

The Epigenetics of rRNA Genes: From Molecular to Chromosome Biology

In eukaryotes, the genes encoding ribosomal RNAs (rDNA) exist in two distinct epigenetic states that can be distinguished by a specific chromatin structure that is maintained throughout the cell cycle and is inherited from one cell to another. The fact that even in proliferating cells with a high demand of protein synthesis a fraction of rDNA is silenced provides a unique possibility to decipher the mechanism underlying epigenetic regulation of rDNA. This chapter summarizes our knowledge of the molecular mechanisms that establish and propagate the epigenetic state of rRNA genes, unraveling a complex interplay of DNA methyltransferases and histone-modifying enzymes that act in concert with chromatin remodeling complexes and RNA-guided mechanisms to define the transcriptional state of rDNA. We also review the critical role of the RNA polymerase I transcription factor UBF in the formation of active nucleolar organizer regions (NORs) and maintenance of the euchromatic state of rRNA genes.

c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription

The c-Myc oncoprotein regulates transcription of genes that are associated with cell growth, proliferation and apoptosis. c-Myc levels are modulated by ubiquitin/proteasome-mediated degradation. Proteasome inhibition leads to c-Myc accumulation within nucleoli, indicating that c-Myc might have a nucleolar function. Here we show that the proteins c-Myc and Max interact in nucleoli and are associated with ribosomal DNA. This association is increased upon activation of quiescent cells and is followed by recruitment of the Myc cofactor TRRAP, enhanced histone acetylation, recruitment of RNA polymerase I (Pol I), and activation of rDNA transcription. Using small interfering RNAs (siRNAs) against c-Myc and an inhibitor of Myc–Max interactions, we demonstrate that c-Myc is required for activating rDNA transcription in response to mitogenic signals. Furthermore, using the ligand-activated MycER (ER, oestrogen receptor) system, we show that c-Myc can activate Pol I transcription in the absence of Pol II transcription. These results suggest that c-Myc coordinates the activity of all three nuclear RNA polymerases, and thereby plays a key role in regulating ribosome biogenesis and cell growth.

The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription

Epigenetic control mechanisms silence about half of the ribosomal RNA (rRNA) genes in metabolically active cells. In exploring the mechanism by which the active or silent state of rRNA genes is inherited, we found that NoRC, a nucleolar remodeling complex containing Snf2h (also called Smarca5, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily a, member 5), represses rDNA transcription. NoRC mediates rDNA silencing by recruiting DNA methyltransferase and histone deacetylase activity to the rDNA promoter, thus establishing structural characteristics of heterochromatin such as DNA methylation, histone hypoacetylation and methylation of the Lys9 residue of histone H3. These results indicate that active and inactive rRNA genes can be demarcated by their associated proteins, and link chromatin remodeling to DNA methylation and specific histone modifications.

Nuclear actin and myosin I are required for RNA polymerase I transcription

The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.

Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes

Noncoding RNAs are important components of regulatory networks controlling the epigenetic state of chromatin. We analyzed the role of pRNA (promoter-associated RNA), a noncoding RNA that is complementary to the rDNA promoter, in mediating de novo CpG methylation of rRNA genes (rDNA). We show that pRNA interacts with the target site of the transcription factor TTF-I, forming a DNA:RNA triplex that is specifically recognized by the DNA methyltransferase DNMT3b. The results reveal a compelling new mechanism of RNA-dependent DNA methylation, suggesting that recruitment of DNMT3b by DNA:RNA triplexes may be a common and generally used pathway in epigenetic regulation.

NoRC - A novel member of mammalian ISWI-containing chromatin remodeling machines

Transcription by RNA polymerase I on nucleosomal templates requires binding of the transcription termination factor TTF‐I to a cognate site 160 bp upstream of the transcription start site. Binding of TTF‐I is accompanied by changes in the chromatin architecture which suggests that TTF‐I recruits a remodeling activity to the rDNA promoter. We have cloned a cDNA that encodes TIP5 (TTF‐I‐interacting protein 5), a 205 kDa protein that shares a number of important protein domains with WSTF (Williams syndrome transcription factor) and hAcf1/WCRF180, the largest subunits of human chromatin remodeling complexes hCHRAC and WCRF. TIP5 co‐localizes with the basal RNA polymerase I transcription factor UBF in the nucleolus and is associated with SNF2h. The cellular TIP5–SNF2h complex, termed NoRC (nucleolar remodeling complex), induces nucleosome sliding in an ATP‐ and histone H4 tail‐dependent fashion. The results suggest that NoRC is a novel nucleolar chromatin remodeling machine that may serve a role in the regulation of the rDNA locus.

The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase I transcription

Mammalian chromatin remodeling complexes are involved in both activation and repression of transcription. Here, we show that NoRC, a SNF2h‐ containing nucleolar chromatin remodeling complex, represses ribosomal gene transcription. NoRC‐mediated rDNA silencing was alleviated by trichostatin A, indicating that histone deacetylation is causally involved in silencing. Chromatin immunoprecipitation experiments demonstrate that overexpression of TIP5, the large subunit of NoRC, mediates deacetylation of nucleosomes in the vicinity of the rDNA promoter. Protein–protein interaction assays reveal association of TIP5 with the histone deacetylase HDAC1 in vivo and in vitro. Deletion of the C‐terminal PHD finger and bromodomain abolishes the interaction of TIP5 and HDAC1, and abrogates transcriptional repression. The results suggest that NoRC silences the rDNA locus by targeting the SIN3 corepressor complex to the rDNA promoter, thereby establishing a repressed chromatin structure.

Cellular stress and nucleolar function

All organisms sense and respond to conditions that stress their homeostaticmechanisms. Here we review current studies showing that the nucleolus, long regardedas a mere ribosome producing factory, plays a key role in monitoring and respondingto cellular stress. After exposure to extra- or intracellular stress, cells rapidly downregulatethe synthesis of ribosomal RNA. Impairment of nucleolar function in responseto stress is accompanied by perturbation of nucleolar structure, cell cycle arrest andstabilization of p53. The nucleolar target for down-regulation of rDNA transcription isTIF-IA, an essential transcription factor that modulates the activity of RNApolymerase I (Pol I). Upon stress, TIF-IA is phosphorylated by c-Jun N-terminalkinase 2 (JNK2). Phosphorylation prevents TIF-IA from interaction with Pol I,thereby impairing transcription complex formation and rRNA synthesis. Furthermore,stress-induced inactivation of TIF-IA is accompanied by translocation of TIF-IA fromthe nucleolus to the nucleoplasm. These findings, together with other data showingstress-induced release of nucleolar proteins to carry out other regulatory functions,reinforce the growing realization that nucleoli orchestrate the chain of events the celluses to properly respond to stress signals.

Acetylation of TAFI68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF‐I. In exploring the functional significance of the promoter‐proximal terminator, we found that TTF‐I associates with the p300/CBP‐associated factor PCAF, suggesting that TTF‐I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAFI68, the second largest subunit of the TATA box‐binding protein (TBP)‐containing factor TIF‐IB/SL1, and acetylation enhances binding of TAFI68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF‐IB/SL1 being required for rDNA transcription, the NAD+‐dependent histone deacetylase mSir2a deacetyl ates TAFI68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF‐IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

Molecular mechanisms mediating methylation-dependent silencing of ribosomal gene transcription.

Epigenetic control mechanisms silence about half of ribosomal RNA genes (rDNA) in metabolically active cells. In the mouse, 40% of rDNA repeats are methylated and can be activated by 5-azacytidine treatment. In exploring the effect of methylation on rDNA transcription, we found that methylation of a single CpG dinucleotide within the upstream control element of the rDNA promoter (at -133) abrogates rDNA transcription both in transfection experiments and in in vitro assays using chromatin templates. Chromatin immunoprecipitation assays demonstrate that methylation of the cytosine at -133 inhibits binding of the transcription factor UBF to nucleosomal rDNA, thereby preventing initiation complex formation. Thus, methylation may be a mechanism to inactivate rDNA genes and propagate transcriptional silencing through cell division.