This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Santoro, R.
Right arrow Articles by Grummt, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santoro, R.
Right arrow Articles by Grummt, I.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, April 2005, p. 2539-2546, Vol. 25, No. 7
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.7.2539-2546.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Epigenetic Mechanism of rRNA Gene Silencing: Temporal Order of NoRC-Mediated Histone Modification, Chromatin Remodeling, and DNA Methylation

Raffaella Santoro* and Ingrid Grummt

Division of Molecular Biology of the Cell II, German Cancer Research Center, Heidelberg, Germany

Received 25 November 2004/ Accepted 3 January 2005


arrow
ABSTRACT
 
Epigenetic control mechanisms silence about half of the rRNA genes in eukaryotes. Previous studies have demonstrated that recruitment of NoRC, a SNF2h-containing remodeling complex, silences rRNA gene transcription. NoRC mediates histone H4 deacetylation, histone H3-Lys9 dimethylation, and de novo DNA methylation, thus establishing heterochromatic features at the rRNA gene promoter. Here we show that inhibition of any of these activities alleviates NoRC-dependent silencing, indicating that these processes are intimately linked. We have studied the temporal order of epigenetic events at the rRNA gene promoter during gene silencing and demonstrate that recruitment of NoRC by TTF-I is a prerequisite for the deacetylation of histone H4 and the dimethylation of histone H3-Lys9. Inhibition of histone deacetylation prevents DNA methylation, while inhibition of DNA methylation does not affect histone modification. Importantly, ATP-dependent chromatin remodeling is required for methylation of a specific CpG dinucleotide within the upstream control element of the rRNA gene promoter, and this modification impairs preinitiation complex formation. The results of this study reveal a clear hierarchy of epigenetic events that control de novo DNA methylation and lead to silencing of RNA genes.


arrow
INTRODUCTION
 
The genes that encode rRNA are tandemly repeated at one or few chromosomal loci in eukaryotes. The number of rRNA gene repeats varies greatly among organisms, ranging from less than 100 to more than 10,000 (24). One might imagine that the presence of large numbers of rRNA gene copies reflects a demand for high levels of rRNA synthesis for rapid growth. However, there is no good correlation between the cellular growth rate and the number of rRNA genes, indicating that there is a considerable plasticity in the number of rRNA gene repeats. Significantly, only a fraction of these repeats is used for rRNA synthesis at any given time. In metabolically active human or mouse cells, approximately half of the ~400 rRNA gene copies are transcriptionally active, and the other half are silent. The relative amounts of active and silent genes are similar in both growing and resting cells as well as during both interphase and metaphase, indicating that the chromatin structure is stably propagated through the cell cycle and is maintained independently of transcriptional activity (5). Active and silent genes are distinct from one another with respect to their chromatin configurations; active genes have a euchromatic structure, whereas silent genes exhibit heterochromatic features (5, 27, 35). The promoter of active rRNA genes is free of CpG methylation and associated with histones that are acetylated. The opposite pattern is predominant among silent genes (22, 56). In addition, silent genes are associated with histone H3 that is methylated on lysine 9 (H3-Lys9) and with heterochromatin protein 1 (HP1). Thus, active and silent rRNA genes are demarcated both by their patterns of DNA methylation and by specific modifications of their associated histones, a finding that links the histone code to the cytosine methylation code (19, 32, 37).

Recent studies have established that, in human and mouse cells, the key determinant that maintains individual rRNA gene repeats in a closed chromatin state is NoRC (nucleolar remodeling complex), one of the ISWI/SNF2-containing ATP-dependent chromatin remodeling machines (38). The large subunit of NoRC, TIP5 (TTF-I-interacting protein 5), shares a number of functional domains with the large subunits of other mammalian remodeling complexes, e.g., ACF, WCRF, CHRAC, and WICH (2, 3, 17, 23, 30), suggesting a common mechanistic basis of action. Despite their pronounced structural and functional homologies, the individual SNF2h-containing complexes appear to have different functions in transcription, DNA repair, and DNA replication (41). Like other members of ISWI/SNF2-containing remodeling machines, NoRC can induce nucleosomes to move along DNA in an ATP- and histone H4 tail-dependent fashion, thereby positioning the histone octamer along the rRNA gene repeats (38). NoRC is associated with silent rRNA gene copies, and overexpression of TIP5 represses rRNA polymerase I transcription (35, 43). NoRC has been shown to silence rRNA gene transcription through recruitment of histone deacetylase and DNA methyltransferase activity, thereby establishing and/or maintaining a repressive higher-order chromatin structure.

To determine how the different chromatin-modifying activities are coordinated with each other and the transcription machinery, we have analyzed the temporal order and functional interplay among histone modifications, DNA methylation, chromatin remodeling, and transcription initiation complex formation. Our results reveal a hierarchical order and mutual dependence of events that operate along a common mechanistic pathway to repress transcription. A model is suggested in which NoRC is recruited to DNA by interaction with TTF-I bound to its target site adjacent to the rRNA gene promoter. Once recruited to rRNA, NoRC acts as a scaffold for subsequent enzymatic reactions that establish a local heterochromatin environment and methylate a critical CpG residue within the upstream control element (UCE) of the rRNA gene promoter. As a consequence, binding of the basal transcription factor UBF to rRNA genes is impaired, and the formation of preinitiation complexes is prevented.


arrow
MATERIALS AND METHODS
 
Plasmids. pMr600-BH represents a fusion of a 5'-terminal mouse rRNA gene fragment (from positions –328 to +292) with a 3'-terminal BamHI/HinfI fragment (from +334 to +712 with respect to the 3' end of 28S rRNA) including two terminator elements (T1 and T2) separated by 177-bp pUC9 vector sequences. In pMr600–133-BH, the cytosine at –133 has been converted into guanine. pMr1930 contains murine rRNA gene sequences from positions –1930 to +155. pMr-1930{Delta}T0 is similar to pMr1930, except that the upstream terminator T0 (from –174 to –144) has been deleted. pcDNA-FLAG-TIP5 and pMr1930-CBH have been described previously (35). pcDNA-FLAG-Snf2h and -Snf2hK211R were a gift from Ramin Shlekhattar.

Transfections and RNA analysis. NIH 3T3 and HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For reporter assays, 5 x 105 NIH 3T3 cells were cotransfected with 1 µg of reporter plasmid and different amounts of pcDNA-FLAG-TIP5 by the calcium phosphate DNA coprecipitation method. Transcripts from reporter plasmids were monitored by hybridization to a 32P-labeled riboprobe complementary to pUC9 sequences that have been inserted between the 5'- and 3'-terminal rRNA gene fragments of pMr600-BH. Alternatively, transcript levels were analyzed by reverse transcription)-PCR with the SYBR detection system (Roche Applied Science) and primers specific for the rRNA gene reporter and GAPDH (glyceraldehyde-3-phospate dehydrogenase) mRNA. The amounts of transcripts and GAPDH cDNA were quantified by comparing them to a standard curve obtained by logarithmic dilution of the corresponding DNA. Polymerase I (Pol I) transcription was calculated by normalizing the amount of rRNA gene transcripts to GAPDH mRNA.

ChIP and DNA methylation assays. Chromatin immunoprecipitation (ChIP) assays were performed as described previously (35, 43). Briefly, chromatin from cross-linked cells was sheared by sonication and incubated overnight with specific antibodies, and precipitated proteins were captured with protein G- or protein A-Sepharose saturated with salmon sperm DNA. After elution and reversion of cross-links by heating for 6 h at 65°C, 1 and 3% of precipitated DNA were amplified by PCR with 30 cycles (30 s at 95°C, 40 s at 55°C, 40 s at 72°C) in the presence of 1.5 mM MgCl2, 200 µM (each) deoxynucleoside triphosphates, and 10 pmol of primers (promoter forward primers, 5'-GACCAGTTGTTCCTTTGAG-3' [positions –165 to –145] and 5'-GATAGGTACTGACACGCTGTCCTTTCCCTATTA-3' [–7 to +16]; promoter reverse primer, 5'-AGGACAGCGTGTCAGTACCTATC-3' [–7 to +16]; 28S rRNA forward primer, 5'-GCGACCTCAGATCAGACGTGG-3' [+8124 to +8145]; and 28S rRNA reverse primer, 5'-CTTAACGGTTTCACGCCCTC-3' [+8549 to +8529]). PCR products were separated by electrophoresis on 2% agarose gels and stained with ethidium bromide. Real-time PCR (RT-PCR) was performed in triplicate with a LightCycler (Roche) and the SYBR green detection system. The relative enrichment of the rRNA genes was determined by calculating the ratio of rRNA genes present in the immunoprecipitates to those present in the input chromatin and normalizing the data to those for control reactions containing no antibodies.

To monitor CpG methylation at the rRNA gene reporter, DNA was digested with 20 U of HpaII or MspI before PCR amplification using two different forward primers that map upstream and downstream of the CCGG site at –143 (positions –165 to –145 or –7 to +16) and a reverse primer that is complementary to chloramphenicol acetyltransferase (CAT) gene sequences inserted into the minigene pMr1930-CBH. For quantification, RT-PCR was performed with a LightCycler (Roche). The relative resistance to HpaII digestion was calculated by normalizing the amount of DNA amplified to either of the two forward primers and the reverse primer.

Antibodies. Antibodies to acetylated histone H4 and the FLAG epitope were from Upstate and Sigma, respectively. Antibodies against di- and trimethylated histone H3-Lys9 were a gift from Thomas Jenuwein.


arrow
RESULTS
 
rRNA gene silencing requires recruitment of NoRC to the rRNA gene promoter by TTF-I. To study NoRC function in the establishment of heterochromatin, we overexpressed TIP5, the large subunit of NoRC, in HEK293T cells and compared the levels of acetylated histone H4 and di- and trimethylated histone H3-Lys9 for both endogenous rRNA genes and a cotransfected reporter plasmid (Fig. 1A). Consistent with previous results (43), the overexpression of TIP5 markedly decreased the acetylation of histone H4 at the rRNA gene promoter. Moreover, the level of H3-Lys9 dimethylation was significantly increased, whereas the trimethylation of H3-Lys9 was much less affected. Importantly, the same histone modifications were observed both on endogenous rRNA genes (Fig. 1A, left) and on the Pol I reporter plasmid (Fig. 1A, right), underscoring the functional relevance of NoRC-mediated epigenetic changes at the rRNA gene promoter.



View larger version (60K):
[in this window]
[in a new window]
  		FIG. 1. Recruitment of NoRC to the rRNA genes (rDNA) mediates histone deacetylation and dimethylation of histone H3 at Lys9. (A) Overexpression of TIP5 triggers histone H4 deacetylation and H3-Lys9 dimethylation. (Left) ChIP analysis of HEK293T cells overexpressing FLAG-TIP5. Cross-linked chromatin was immunoprecipitated with antibodies against acetylated histone H4 or dimethylated (dMeK9) or trimethylated (tMeK9) H3-Lys9 and coprecipitated. rRNA genes were analyzed by RT-PCR. Data represent the amplification product from immunoprecipitated DNA normalized to input material in cells overexpressing TIP5 (+) compared to those in mock-transfected cells (–). Values are the averages of results from two independent experiments. (Right) ChIP analysis of NIH 3T3 cells cotransfected with the rRNA gene reporter plasmid pMr600-BH in the absence or presence of pcDNA-FLAG-TIP5. One and three percent of the input chromatin and 1.7 and 5% of the immunoprecipitated DNA were amplified with primers specific to the rRNA gene reporter and the 28S rRNA coding region. –, negative control. (B) Binding of TTF-I to its target site, T0, is required for recruitment of NoRC to rRNA genes. (Top) Schematics for the Pol I reporters, with thick lines indicating rRNA gene sequences and thin lines indicating vector sequences. A black box marks the promoter-proximal terminator T0 (positions –174 to –144). Arrows indicate the positions of primers used for PCR amplification. (Bottom) NIH 3T3 cells were cotransfected with pcDNA-FLAG-TIP5 and the reporter plasmids pMr1930 or pMr1930{Delta}T0. Cross-linked chromatin was precipitated with anti-FLAG antibodies, and coprecipitated DNA was analyzed by PCR. (C) Transcriptional repression by NoRC requires binding of TTF-I to T0. NIH 3T3 cells were cotransfected with pcDNA-FLAG-TIP5 and the reporter plasmids pMr1930 or pMr1930{Delta}T0. Transcripts were analyzed by RT-PCR. Data represent the amounts of reporter transcripts relative to those of GAPDH mRNA. Values are averages of the results of two independent experiments. –, negative control. (D) Recruitment of NoRC to the rRNA genes mediates histone H4 deacetylation and dimethylation of H3-Lys9. NIH 3T3 cells were cotransfected with pcDNA-FLAG-TIP5 and a wild-type or mutant reporter plasmid (pMr1930 or pMr1930{Delta}T0, respectively). rRNA gene occupancy of acetylated histone H4 (AcH4) and dimethylated H3-Lys9 (dMeH3) was monitored by ChIP analysis using primers specific to the rRNA gene reporter and the 28S rRNA coding region. {alpha}, anti.

TIP5 has been shown to interact with TTF-I, suggesting that NoRC is recruited to the rRNA gene by TTF-I bound to the promoter-proximal terminator T0 (29, 39). If NoRC was recruited to the rRNA genes by TTF-I, it should not associate with templates lacking the TTF-I binding site. To test this possibility, NIH 3T3 cells were cotransfected with an expression vector encoding FLAG-tagged TIP5 and a reporter plasmid that either contains or lacks the TTF-I binding site T0 (pMr1930 or pMr1930{Delta}T0, respectively). ChIP experiments revealed that TIP5 is associated with the wild-type rRNA gene reporter (Fig. 1B, top), but no binding to the plasmid lacking the TTF-I binding site was observed (Fig. 1B, bottom). Thus, recruitment of NoRC to the rRNA genes depends on the binding of TTF-I to the promoter-proximal terminator T0.

Given that the association of TIP5 with TTF-I is the first step in the chain of events that lead to the silencing of Pol I transcription, NoRC should not repress transcription on the mutant reporter pMr-1930{Delta}T0. Indeed, overexpression of TIP5 strongly reduced transcription from the wild-type but not the mutant template (Fig. 1C). Moreover, neither histone H4 hypoacetylation nor H3-Lys9 dimethylation was observed at the template lacking the terminator T0 (Fig. 1D). Thus, the silencing of Pol I transcription and the establishment of heterochromatic features requires recruitment of NoRC to the rRNA genes by TTF-I bound to the promoter-proximal target site T0.

NoRC-mediated DNA methylation depends on histone deacetylation. To examine whether NoRC-dependent histone modification and de novo DNA methylation may proceed as parallel pathways or are dependent on each other, we assayed the effect of trichostatin A (TSA) and azacytidine (aza-dC), inhibitors of histone deacetylase and DNA methyltransferase, respectively, on rRNA gene silencing. Consistent with the results described above, overexpression of TIP5 reduced the levels of both reporter transcripts and cellular pre-rRNA in a dose-dependent manner (Fig. 2A). The repressive effect of TIP5 on transcriptional activity was relieved by either TSA or aza-dC. No additive or synergistic effect was observed, indicating that histone deacetylation and DNA methylation operate along a common mechanistic pathway. While TIP5-mediated histone H4 deacetylation was impaired by TSA treatment, H3-Lys9 dimethylation was not affected (Fig. 2B). This finding underscores the requirement for histone H4 deacetylation in TIP5-mediated rRNA gene silencing and suggests that histone H3-Lys9 dimethylation occurs independently of histone H4 deacetylation. In contrast, inhibition of DNA methylation by aza-dC treatment affected neither histone H4 deacetylation nor H3-Lys9 dimethylation, indicating that histone deacetylation and methylation occur prior to DNA methylation.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 2. Deacetylation of histone H4 is required for NoRC-mediated de novo CpG methylation. (A) Inhibition of histone deacetylation and DNA methylation alleviates rRNA gene silencing. (Top) Pre-rRNA levels in HEK293T cells overexpressing FLAG-TIP5. Transcripts were analyzed on Northern blots using a riboprobe that hybridizes to nucleotides 1 to 155 of human pre-rRNA. (Bottom) Results from transfection of NIH 3T3 cells with the reporter plasmid pMr600-BH (1 µg) in the absence and presence of pcDNA-FLAG-TIP5 (6 and 8 µg); the transcripts were visualized on Northern blots. Where indicated, the cells were treated with 33 nM TSA and/or 50 µM 5-aza-dC for 24 h. (B) TSA impairs NoRC-mediated histone H4 deacetylation but not H3-Lys9 methylation. Results from ChIP and RT-PCR assays demonstrating the association of anti-AcH4 and anti-dMeH3 antibodies with murine rRNA genes in the absence and presence of overexpressed FLAG-TIP5 are shown. Data represent the amplification product from immunoprecipitated DNA normalized to input material in cells overexpressing TIP5 (+) compared to those in mock-transfected cells (–). Values are averages of the results of two independent experiments. (C) Inhibition of histone deacetylation impairs NoRC-mediated CpG methylation at the rRNA gene promoter. ChIP analysis of HEK293T cells transfected with pMr1930-CBH in theabsence or presence of pcDNA-FLAG-TIP5 is shown. DNA coprecipitated with anti-FLAG antibodies was amplified with reporter-specific primers (upper box) before being digested with HpaII and MspI and amplified with primers that map upstream or downstream of the HpaII site at –143. A diagram of the reporter plasmid pMr1930-CBH showing the positions of the primers is at the top. The thick line represents rRNA gene sequences, and the black boxes represent the upstream terminator T0 and the downstream terminators T1 and T2. A fragment of the CAT gene that has been inserted between the 5'- and 3'-terminal parts of the rRNA minigene is shown. The two forward primers correspond to murine rRNA gene sequences (–165 to –145 and –7 to +16), and the reverse primer corresponds to nucleotides 176 to 196 of the bacterial chloramphenicol acetyltransferase (CAT) gene. {alpha}, anti; –, absence; +, presence.

To examine whether histone H4 deacetylation is a prerequisite for DNA methylation, cells overexpressing TIP5 were treated with TSA, and de novo methylation of a cotransfected rRNA gene reporter plasmid (pMr1930-CBH) was analyzed. Cross-linked chromatin was immunoprecipitated with anti-FLAG antibodies (Fig. 2C, middle), DNA was digested with methylation-sensitive isoschizomers HpaII and MspI, and coprecipitated plasmid DNA was amplified with forward primers that map upstream or downstream of the CCGG site (at position –143) in the rRNA gene promoter and a backward primer that is complementary to part of the marker gene (the CAT gene) inserted between the 5'- and 3'-terminal rRNA gene fragments of the reporter construct (Fig. 2C). Consistent with overexpression of TIP5 triggering de novo DNA methylation, a fraction of the reporter plasmid became resistant to HpaII cleavage (Fig. 2C, bottom, lanes 1 and 2). Significantly, de novo DNA methylation did not occur in the presence of TSA (Fig. 2C), indicating that NoRC-mediated DNA methylation depends on prior histone deacetylation. These data demonstrate the mutual interdependence of histone deacetylation and DNA methylation and imply that deacetylation of nucleosomes at the rRNA gene promoter is required for DNA methylation.

Nucleosome remodeling is required for DNA methylation and transcriptional silencing. NoRC has been shown to use the energy of ATP hydrolysis to alter the topology of nucleosomal DNA (39). To investigate the contribution of NoRC-mediated chromatin remodeling to rRNA gene silencing, we monitored Pol I transcription after coexpressing TIP5 either with SNF2h or with SNF2hK211R, an ATPase-deficient mutant. Coexpression of SNF2h enhanced the transcriptional repression mediated by TIP5 from 60 to 80% (Fig. 3A). This finding is consistent with coimmunoprecipitation data showing that the majority of ectopic TIP5 associates with endogenous SNF2h (unpublished results) and explains why moderate overexpression of TIP5 is sufficient to silence the rRNA genes. Strikingly, if SNF2hK211R was coexpressed with TIP5, transcriptional repression was eliminated. This result demonstrates that the ATP-dependent chromatin-remodeling activity of NoRC plays an indispensable role in rRNA gene silencing.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3. ATP-dependent chromatin remodeling is required for rRNA gene methylation and transcriptional silencing. (A) Transcriptional repression requires the ATPase activity of SNF2h. NIH 3T3 cells were transfected with pMr600-BH. Data represent the amplification product from immunoprecipitated DNA normalized to input material in cells overexpressing TIP5 (+) compared to that in mock-transfected cells (–). The reporter transcripts were analyzed by RT-PCR. Values represent averages of the amounts of reporter gene transcripts relative to the amounts of GAPDH mRNA from two independent experiments. (B) NoRC-dependent histone deacetylation and H3K9 dimethylation do not require the ATPase activity of SNF2h. Shown are results from ChIP analysis monitoring the association of AcH4 and dMeK9 H3 histones with the rRNA genes after overexpression of TIP5 and SNF2h or SNF2hK211R. (C) NoRC-dependent DNA methylation requires the ATPase activity of SNF2h. DNA from NIH 3T3 cells overexpressing TIP5 and SNF2h or SNF2hK211R was digested with HpaII and analyzed by RT-PCR using primers that map upstream or downstream of the CCGG sequence at –143. De novo DNA methylation, i.e., resistance to HpaII digestion, is presented as ratios between the amounts of the rRNA genes amplified with primers that map upstream and downstream of the HpaII site. {alpha}, anti; –, absence; +, presence.

If ATP-dependent nucleosome remodeling is necessary for NoRC-mediated rRNA gene silencing, a step may exist that establishes a link between chromatin remodeling, histone modification, DNA methylation, and initiation complex formation. We therefore sought to examine whether NoRC-dependent chromatin remodeling would facilitate the access of histone-modifying enzymes and DNA methyltransferase to the rRNA genes. For this purpose, we monitored histone H4 acetylation and H3-Lys9 dimethylation at the rRNA gene reporter after the coexpression of FLAG-TIP5 with SNF2h and SNF2hK211R, respectively. As shown in Fig. 3B, both histone deacetylation and H3-Lys9 dimethylation occurred at the rRNA gene promoter, regardless of whether wild-type or mutant SNF2h was coexpressed with TIP5. This result indicates that the remodeling activity of NoRC is not required for histone deacetylation and H3-Lys9 dimethylation.

Next, we tested whether the remodeling activity of NoRC is required for de novo methylation of the rRNA genes. Consistent with the transcription data, the overexpression of TIP5 increased the amount of HpaII-resistant methylated rRNA genes by about threefold in both the absence and presence of SNF2h (Fig. 3C). In contrast, the overexpression of TIP5 with SNF2hK211R did not confer HpaII resistance to the rRNA genes. These results indicate that ATP-dependent chromatin remodeling is required for de novo DNA methylation and that DNA methylation occurs subsequent to chromatin remodeling.

NoRC-mediated transcriptional repression requires DNA methylation of CpG at position –133. Previous work has established that methylation of a single CpG residue at –133 within the UCE of the rRNA gene promoter eliminates binding of the basal transcription factor UBF to nucleosomal DNA, thereby preventing transcription complex formation (34). If methylation of CpG at –133 was the final step in the chain of events that silence the rRNA genes, the overexpression of TIP5 should not repress the transcription of a mutant template in which the cytosine at –133 was replaced by guanosine. Indeed, transcription of the mutant (pMr600–133-BH) was not repressed by increasing amounts of FLAG-TIP5 (Fig. 4A), demonstrating that transcriptional repression by TIP5 requires the methylation of a functionally important CpG residue within the UCE. Moreover, ChIP assays revealed that the overexpression of TIP5 caused the deacetylation and dimethylation of histones at both the wild-type and mutant reporter plasmids (Fig. 4B). This finding implies that histone deacetylation and H3-Lys9 methylation, i.e., modifications that mark heterochromatin, are not sufficient for rRNA gene silencing. In addition, this result reveals that CpG methylation is the final step in the chain of events that silence rRNA gene transcription.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 4. NoRC-mediated rRNA gene silencing requires methylation of CpG at –133. (A) Mutation of CpG at –133 eliminates transcriptional repression by NoRC. A Northern blot of RNA from NIH 3T3 cells that were cotransfected with pMr600-BH or pMr600–133-BH in the absence or presence of pcDNA-FLAG-TIP5 is shown. Top, Schematic of pMr600-BH, which uses the same symbols as the diagram in Fig. 2C. CpG at –133 is located within the UCE. In pMr600–133-BH, the cytosine residue at –133 is replaced by guanosine. (B) Histone H4 deacetylation and histone H3-Lys9 dimethylation are not sufficient for rRNA gene silencing. NIH 3T3 cells were transfected with pMr600-BH or pMr600–133-BH in the absence or presence of pcDNA-FLAG-TIP5. ChIP was performed with antibodies against acetylated histone H4 ({alpha}-AcH4) and dimethylated H3-Lys9 ({alpha}-dMeH3). Data represent the n-fold differences between the amounts of precipitated DNA in cells overexpressing TIP5 and those in mock-transfected cells. (C) Overexpression of TIP5 impairs binding of UBF to the rRNA gene promoter. ChIP analysis of NIH 3T3 cells cotransfected with pMr600-BH or pMr600–133-BH in the absence or presence of pcDNA-FLAG-TIP5 is shown. After immunoprecipitation with anti-UBF antibodies, coprecipitated DNA and input chromatin (1 and 3%) were amplified with primers specific to the reporter plasmid. {alpha}, anti; –, absence; +, presence.

We have previously shown that methylation of CpG at –133 impairs UBF binding to the rRNA genes assembled into chromatin (34). We therefore reasoned that overexpression of TIP5 should not decrease the level of UBF associated with the mutant template that cannot be methylated at CpG at –133. Indeed, overexpression of TIP5 eliminated binding of UBF to the wild-type (pMr600-BH) but not the mutant (pMr600–133-BH) template (Fig. 4C), indicating that NoRC-mediated DNA methylation prevents binding of UBF to the UCE. As a consequence, the assembly of productive transcription initiation complexes is impaired. Thus, NoRC establishes rRNA gene silencing by coordinating a hierarchical order of events that modify nucleosomes, methylate DNA, and prevent the formation of Pol I transcription initiation complexes.


arrow
DISCUSSION
 
A clear theme that emerges from studies on the epigenetic regulation of gene expression is the functional interaction and interdependence of chromatin remodeling, histone modification, and DNA methylation as well as the consequent difficulty in dissecting the cause-and-effect relationships of these reactions. In most cases, it remained unknown how the activities that lead to opening or closing of specific genomic areas are coordinated. A growing body of evidence indicates the existence of synergism between chromatin-remodeling factors and DNA- and/or histone-modifying enzymatic activities in the regulation of transcription at the epigenetic level. For example, promoter activation requires the acetylation of nucleosomes, and site-specific histone acetylation acts as a signal for the regulation of nucleosome remodeling (1, 6, 9, 31). Conversely, the interplay between ISWI/SNF2 complexes and histone deacetylases contributes to the establishment and maintenance of heterochromatic structures and transcriptional repression (8, 16, 35). Of note is the mutual dependence of DNA methylation and histone modification. DNA methyltransferases and DNA binding proteins that specifically recognize methylated cytosine residues have been shown to interact with histone deacetylase corepressors (11, 21, 28, 33) and histone methyltransferases (10, 12), and DNA methylation in Neurospora crassa was profoundly altered by mutations that disrupt histone methylation (40).

In the present study, we have investigated the functional interrelationship and temporal order of NoRC-mediated chromatin remodeling, histone modification, and DNA methylation leading to rRNA gene silencing. We show that overexpression of TIP5 establishes heterochromatic marks and silences transcription both at endogenous rRNA genes and at Pol I reporter plasmids, implying that similar mechanisms operate at endogenous and ectopic rRNA genes. We found that histone deacetylation, ATP-dependent chromatin remodeling, and DNA methylation are strictly dependent on each other and occur in a hierarchical and temporal order. Our results reveal that rRNA gene silencing is initiated by the recruitment of NoRC to the Pol I promoter by interaction with TTF-I bound to the promoter-proximal terminator T0. In a subsequent step, NoRC interacts with the Sin3 corepressor complex, leading to deacetylation of nucleosomes at the rRNA genes (43). Though deacetylation of histone H4 per se is not sufficient to silence the rRNA genes, it may act as a flag or signal for SNF2h-mediated nucleosome remodeling. The action of SNF2h may be required beforehand to open the chromatin, thereby either relieving a steric constraint or exposing CpG at –133 to methylation. Methylation of CpG at –133 in the context of chromatin impairs UBF binding to the Pol I promoter and impairs the assembly of productive transcription initiation complexes.

This temporal-order model is based on the following observations. TIP5 has been shown to interact with histone deacetylase (HDAC) and DNA methyltransferases (Dnmt1 and Dnmt3), thereby recruiting these activities to the rRNA genes (35, 43). This finding suggests either that a specific acetylation pattern targets CpG methylation or, alternatively, that Dnmt1 targets deacetylation toward regions that are to be silenced. In either scenario, methylation and deacetylation would act together to potentiate the repressed state. Treatment with TSA or 5-aza-dC eliminated NoRC-mediated silencing, indicating that histone deacetylation and cytosine methylation act on the same pathway to repress transcription. In support of this idea, inhibition of histone deacetylation prevented de novo DNA methylation, while inhibition of DNA methylation did not affect histone acetylation. This implies that histone deacetylation is a prerequisite for DNA methylation, a mechanism that has been described only for Neurospora (36). On the other hand, DNA methylation itself can target histone deacetylation via methyl-binding proteins, which are known to recruit histone deacetylase corepressors to methylated DNA (21, 28), indicating that both mechanisms are not mutually exclusive. Recent results have demonstrated that the methyl-binding protein MBD2 is associated with rRNA genes (15). This demonstration suggests that histone deacetylation plays a role in both the establishment and maintenance of heterochromatic features for a fraction of rRNA genes.

Our finding that TSA treatment does not affect NoRC-mediated dimethylation of histone H3-Lys9 is consistent with the results of other groups showing that histone acetylation and methylation act independently of each other (25, 42). Our results reveal that histone deacetylation and H3-Lys9 dimethylation are not sufficient for transcriptional repression and indicate that these heterochromatic marks per se do not prevent the access of transcription factors to chromatin. Importantly, TIP5-dependent histone deacetylation at the rRNA gene promoter does not require the ATPase activity of NoRC. This suggests either that histone deacetylation and chromatin remodeling are independent of each other or that histone deacetylation precedes nucleosome remodeling. We favor the idea that NoRC-mediated deacetylation of histone H4 occurs prior to remodeling and that deacetylation is required to remodel nucleosomes at the rRNA gene promoter. In support of this idea, in vivo and in vitro studies have demonstrated that the acetylation of histone H4 at lysines 12 and 16 inhibits the chromatin-remodeling activity of ISWI, suggesting that the function of ISWI/SNF2h is regulated by the site-specific acetylation of histones (4, 6). Like NoRC, other SWI/SNF2-like proteins have been linked to DNA methylation and histone modification (for a review, see references 13 and 26). Mutations in ddm1 and Lsh result in the loss or alteration of both DNA methylation and histone methylation (7, 14, 18, 20, 42). However, cause-and-effect relationships for these enzymatic activities have yet to be defined. Our data do not allow us to determine when NoRC-mediated dimethylation of H3-Lys9 occurs. It is possible that NoRC directly recruits a histone methyltransferase to the rRNA genes or, alternatively, that once that the rRNA genes are methylated, methyl-binding proteins can target histone methyltransferase activity (10). The presence of HP1 at silent rRNA genes (35) suggests that HP1, along with methylated histone H3-Lys9, may play a role in heterochromatin spreading and/or maintaining and perpetuating the silent chromatin state. Elucidation of the functional interrelationship between nucleosome remodeling, histone modification, and DNA methylation, as well as of the role of individual remodeling complexes in coordinating these different activities, will reveal the mechanisms that the cell uses to inherit specific chromatin states from one generation to the next.


arrow
ACKNOWLEDGMENTS
 
We thank Thomas Jenuwein for antibodies against di- and trimethylated H3-Lys9 and Ramin Shlekhattar for pcDNA-FLAG-SNF2h and pcDNA-FLAG-SNF2hK211R.

This work was supported by the Deutsche Forschungsgemeinschaft, the Epigenome Network of the European Union, and the Fonds der Chemischen Industrie.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: German Cancer Research Center, Molecular Biology of the Cell II, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: 0049-6221-423468. Fax: 0049-6221-423404. E-mail: r.santoro{at}dkfz-heidelberg.de. Back


arrow
REFERENCES
 
    1
  1. Agalioti, T., S. Lomvardas, B. Parekh, J. Yie, T. Maniatis, and D. Thanos. 2000. Ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. Cell 103:667-678.[CrossRef][Medline]
    2. 2
  2. Bochar, D. A., J. Savard, W. Wang, D. W. Lafleur, P. Moore, J. Cote, and R. Shiekhattar. 2000. A family of chromatin remodeling factors related to Williams syndrome transcription factor. Proc. Natl. Acad. Sci. USA 97:1038-1043.[Abstract/Free Full Text]
    3. 3
  3. Bozhenok, L., P. A. Wade, and P. Varga-Weisz. 2002. WSTF-ISWI chromatin remodeling complex targets heterochromatic replication foci. EMBO J. 21:2231-2241.[CrossRef][Medline]
    4. 4
  4. Clapier, C. R., G. Längst, D. F. V. Corona, P. B. Becker, and K. P. Nightingale. 2001. Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI. Mol. Cell. Biol. 21:875-883.[Abstract/Free Full Text]
    5. 5
  5. Conconi, A., R. M. Widmer, T. Koller, and J. M. Sogo. 1989. Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell 57:753-761.[CrossRef][Medline]
    6. 6
  6. Corona, D. F., C. R. Clapier, P. B. Becker, and J. W. Tamkun. 2002. Modulation of ISWI function by site-specific histone acetylation. EMBO Rep. 3:242-247.[CrossRef][Medline]
    7. 7
  7. Dennis, K., T. Fan, T. Geiman, Q. Yan, and K. Muegge. 2001. Lsh, a member of the SNF2 family, is required for genome-wide methylation. Genes Dev. 15:2940-2944.[Abstract/Free Full Text]
    8. 8
  8. Deuring, R., L. Fanti, J. A. Armstrong, M. Sarte, O. Papoulas, M. Prestel, G. Daubresse, M. Verardo, S. L. Moseley, M. Berloco, T. Tsukiyama, C. Wu, S. Pimpinelli, and J. W. Tamkun. 2000. The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol. Cell 5:355-365.[CrossRef][Medline]
    9. 9
  9. Dilworth, F. J., C. Fromental-Ramain, K. Yamamoto, and P. Chambon. 2000. ATP-driven chromatin remodeling activity and histone acetyltransferases act sequentially during transactivation by RAR/RXR in vitro. Mol. Cell 6:1049-1058.[CrossRef][Medline]
    10. 10
  10. Fujita, N., S. Watanabe, T. Ichimura, S. Tsuruzoe, Y. Shinkai, M. Tachibana, T. Chiba, and M. Nakao. 2003. Methyl-CpG binding domain 1 (MBD1) interacts with the Suv39h1-HP1 heterochromatic complex for DNA methylation-based transcriptional repression. J. Biol. Chem. 278:24132-24138.[Abstract/Free Full Text]
    11. 11
  11. Fuks, F., W. A. Burgers, A. Brehm, L. Hughes-Davies, and T. Kouzarides. 2001. DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat. Genet. 24:88-91.
    12. 12
  12. Fuks, F., P. J. Hurd, R. Deplus, and T. Kouzarides. 2003. The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res. 31:2305-2312.[Abstract/Free Full Text]
    13. 13
  13. Geiman, T. M., and K. D. Robertson. 2002. Chromatin remodeling, histone modifications, and DNA methylation—how does it all fit together? J. Cell. Biochem. 87:117-125.[CrossRef][Medline]
    14. 14
  14. Gendrel, A. V., Z. Lippman, C. Yordan, V. Colot, and R. A. Martiensse. 2002. Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1. Science 297:1871-1873.[Abstract/Free Full Text]
    15. 15
  15. Ghoshal, K., S. Majumder, J. Datta, T. Motiwala, S. Bai, S. M. Sharma, W. Frankel, and S. T. Jacob. 2004. Role of human ribosomal RNA (rRNA) promoter methylation and of methyl-CpG-binding protein MBD2 in the suppression of rRNA gene expression. J. Biol. Chem. 279:6783-6793.[Abstract/Free Full Text]
    16. 16
  16. Goldmark, J. P., T. G. Fazzio, P. W. Estep, G. M. Church, and T. Tsukiyama. 2000. The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p. Cell 103:423-433.[CrossRef][Medline]
    17. 17
  17. Ito, T., M. E. Levenstein, D. V. Fyodorov, A. K. Kutach, R. Kobayashi, and J. T. Kadonaga. 1999. ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev. 13:1529-1539.[Abstract/Free Full Text]
    18. 18
  18. Jeddeloh, J. A., J. Bender, and E. J. Richard. 1998. The DNA methylation locus DDM1 is required for maintenance of gene silencing in Arabidopsis. Genes Dev. 12:1714-1725.[Abstract/Free Full Text]
    19. 19
  19. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science 293:1074-1080.[Abstract/Free Full Text]
    20. 20
  20. Johnson, M. L., X. Cao, and S. E. Jacobsen. 2002. Interplay between two epigenetic marks: DNA methylation and histone H3 lysine 9 methylation. Curr. Biol. 12:1360-1367.[CrossRef][Medline]
    21. 21
  21. Jones, P. L., G. J. Veenstra, P. A. Wade, D. Vermaak, S. U. Kass, N. Landsberger, J. Strouboulis, and A. P. Wolffe. 1998. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19:187-191.[CrossRef][Medline]
    22. 22
  22. Lawrence, R. J., K. Earley, O. Pontes, M. Silva, Z. J. Chen, N. Neves, W. Viegas, and C. S. Pikaard. 2004. A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Mol. Cell 13:599-609.[CrossRef][Medline]
    23. 23
  23. LeRoy, G., A. Loyola, W. S. Lane, and D. Reinberg. 2000. Purification and characterization of a human factor that assembles and remodels chromatin. J. Biol. Chem. 275:14787-14790.[Abstract/Free Full Text]
    24. 24
  24. Long, E. O., and I. B. Dawid. 1980. Repeated genes in eukaryotes. Annu. Rev. Biochem. 4:727-764.[CrossRef]
    25. 25
  25. Maison, C., D. Bailly, A. H. Peters, J. P. Quivy, D. Roche, A. Taddei, M. Lachner, T. Jenuwein, and G. Almouzni. 2002. Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component. Nat. Genet. 30:329-334.[CrossRef][Medline]
    26. 26
  26. Meehan, R. R., S. Pennings, and I. Stancheva. 2001. Lashings of DNA methylation, forkfuls of chromatin remodeling. Genes Dev. 15:3231-3236.[Free Full Text]
    27. 27
  27. Mutskov, V. J., V. R. Russanova, S. I. Dimitrov, and I. G. Pashev. 1996. Histones associated with non-nucleosomal rat ribosomal genes are acetylated while those bound to nucleosome-organized gene copies are not. J. Biol. Chem. 271:11852-11857.[Abstract/Free Full Text]
    28. 28
  28. Nan, X., H. H. Ng, C. A. Johnson, C. D. Laherty, B. M. Turner, R. N. Eisenman, and A. Bird. 1998. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393:386-389.[CrossRef][Medline]
    29. 29
  29. Nemeth, A., R. Strohner, I. Grummt, and G. Längst. 2004. The chromatin remodeling complex NoRC and TTF-I cooperate in the regulation of the mammalian rRNA genes in vivo. Nucleic Acids Res. 32:4091-4099.[Abstract/Free Full Text]
    30. 30
  30. Poot, R. A., G. Dellaire, B. B. Hulsmann, M. A. Grimaldi, D. F. Corona, P. B. Becker, W. A. Bickmore, and P. D. Varga-Weisz. 2000. HuCHRAC, a human ISWI chromatin remodeling complex contains hACF1 and two novel histone-fold proteins. EMBO J. 19:3377-3387.[CrossRef][Medline]
    31. 31
  31. Reinke, H., P. D. Gregory, and W. Hörz. 2001. A transient histone hyperacetylation signal marks nucleosomes for remodeling at the PHO8 promoter in vivo. Mol. Cell 7:529-538.[CrossRef][Medline]
    32. 32
  32. Richards, E. J., and S. C. Elgin. 2002. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 108:489-500.[CrossRef][Medline]
    33. 33
  33. Rountree, M. R., K. E. Bachman, and S. B. Baylin. 2000. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat. Genet. 25:269-277.[CrossRef][Medline]
    34. 34
  34. Santoro, R., and I. Grummt. 2001. Molecular mechanisms mediating methylation-dependent silencing of ribosomal gene transcription. Mol. Cell 8:719-725.[CrossRef][Medline]
    35. 35
  35. Santoro, R., J. Li, and I. Grummt. 2002. The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription. Nat. Genet. 32:393-396.[CrossRef][Medline]
    36. 36
  36. Selker, E. U. 1998. Trichostatin A causes selective loss of DNA methylation in Neurospora. Proc. Natl. Acad. Sci. USA 95:9430-9435.[Abstract/Free Full Text]
    37. 37
  37. Strahl, B. D., and C. D. Allis. 2000. The language of covalent histone modifications. Nature 403:41-45.[CrossRef][Medline]
    38. 38
  38. Strohner, R., A. Nemeth, P. Jansa, U. Hofmann-Rohrer, R. Santoro, G. Längst, and I. Grummt. 2001. NoRC—a novel member of mammalian ISWI-containing chromatin remodeling machines. EMBO J. 20:4892-4900.[CrossRef][Medline]
    39. 39
  39. Strohner, R., A. Németh, K. P. Nightingale, I. Grummt, P. B. Becker, and G. Längst. 2004. Recruitment of the nucleolar remodeling complex NoRC establishes ribosomal DNA silencing in chromatin. Mol. Cell. Biol. 24:1791-1798.[Abstract/Free Full Text]
    40. 40
  40. Tamaru, H., and E. U. Selker. 2001. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414:277-283.[CrossRef][Medline]
    41. 41
  41. Varga-Weisz, P. 2001. ATP-dependent chromatin remodeling factors: nucleosome shufflers with many missions. Oncogene 20:3076-3085.[CrossRef][Medline]
    42. 42
  42. Yan, Q., J. Huang, T. Fan, H. Zhu, and K. Muegge. 2003. Lsh, a modulator of CpG methylation, is crucial for normal histone methylation. EMBO J. 22:5154-5162.[CrossRef][Medline]
    43. 43
  43. Zhou, Y., R. Santoro, and I. Grummt. 2002. The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase I transcription. EMBO J. 21:4632-4640.[CrossRef][Medline]

Molecular and Cellular Biology, April 2005, p. 2539-2546, Vol. 25, No. 7
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.7.2539-2546.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sanij, E., Poortinga, G., Sharkey, K., Hung, S., Holloway, T. P., Quin, J., Robb, E., Wong, L. H., Thomas, W. G., Stefanovsky, V., Moss, T., Rothblum, L., Hannan, K. M., McArthur, G. A., Pearson, R. B., Hannan, R. D. (2008). UBF levels determine the number of active ribosomal RNA genes in mammals. JCB 183: 1259-1274 [Abstract] [Full Text]  
  • Murano, K., Okuwaki, M., Hisaoka, M., Nagata, K. (2008). Transcription Regulation of the rRNA Gene by a Multifunctional Nucleolar Protein, B23/Nucleophosmin, through Its Histone Chaperone Activity. Mol. Cell. Biol. 28: 3114-3126 [Abstract] [Full Text]  
  • Brown, S. E., Szyf, M. (2007). Epigenetic Programming of the rRNA Promoter by MBD3. Mol. Cell. Biol. 27: 4938-4952 [Abstract] [Full Text]  
  • Yan, P. S., Venkataramu, C., Ibrahim, A., Liu, J. C., Shen, R. Z., Diaz, N. M., Centeno, B., Weber, F., Leu, Y.-W., Shapiro, C. L., Eng, C., Yeatman, T. J., Huang, T. H.-M. (2006). Mapping Geographic Zones of Cancer Risk with Epigenetic Biomarkers in Normal Breast Tissue.. Clin. Cancer Res. 12: 6626-6636 [Abstract] [Full Text]  
  • Majumder, S., Ghoshal, K., Datta, J., Smith, D. S., Bai, S., Jacob, S. T. (2006). Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription. J. Biol. Chem. 281: 22062-22072 [Abstract] [Full Text]  
  • Viiri, K. M., Korkeamaki, H., Kukkonen, M. K., Nieminen, L. K., Lindfors, K., Peterson, P., Maki, M., Kainulainen, H., Lohi, O. (2006). SAP30L interacts with members of the Sin3A corepressor complex and targets Sin3A to the nucleolus. Nucleic Acids Res 34: 3288-3298 [Abstract] [Full Text]  
  • Cavellan, E., Asp, P., Percipalle, P., Farrants, A.-K. O. (2006). The WSTF-SNF2h Chromatin Remodeling Complex Interacts with Several Nuclear Proteins in Transcription. J. Biol. Chem. 281: 16264-16271 [Abstract] [Full Text]  
  • McStay, B. (2006). Nucleolar dominance: a model for rRNA gene silencing.. Genes Dev. 20: 1207-1214 [Full Text]  
  • Tongaonkar, P., French, S. L., Oakes, M. L., Vu, L., Schneider, D. A., Beyer, A. L., Nomura, M. (2005). Histones are required for transcription of yeast rRNA genes by RNA polymerase I. Proc. Natl. Acad. Sci. USA 102: 10129-10134 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Santoro, R.
Right arrow Articles by Grummt, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santoro, R.
Right arrow Articles by Grummt, I.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»