Epigenetic Control of rDNA
Loci in Response to
Intracellular Energy Status
Akiko Murayama,1,2,5,6Kazuji Ohmori,1,6Akiko Fujimura,1Hiroshi Minami,4Kayoko Yasuzawa-Tanaka,1
Takao Kuroda,1Shohei Oie,1Hiroaki Daitoku,2Mitsuru Okuwaki,3Kyosuke Nagata,3Akiyoshi Fukamizu,2
Keiji Kimura,1Toshiyuki Shimizu,4and Junn Yanagisawa1,*
1Graduate School of Life and Environmental Sciences
2Center for Tsukuba Advanced Research Alliance
3Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences
University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan
4International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan
5PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan
6These authors contributed equally to this work.
Intracellular energy balance is important for cell sur-
process is ribosome biosynthesis, which adapts to
changes in intracellular energy status. However, the
mechanism that links energy status and ribosome
biosynthesis is largely unknown. Here, we describe
eNoSC, a protein complex that senses energy status
and controls rRNA transcription. eNoSC contains
Nucleomethylin, which binds histone H3 dimethy-
lated Lys9 in the rDNA locus, in a complex with
SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are
required for energy-dependent transcriptional re-
pression, suggesting that a change in the NAD+/
NADH ratio induced by reduction of energy status
could activate SIRT1, leading to deacetylation of his-
tone H3 and dimethylation at Lys9 by SUV39H1, thus
establishing silent chromatin in the rDNA locus. Fur-
thermore, eNoSC promotes restoration of energy
balance by limiting rRNA transcription, thus protect-
sis. These findings provide key insight into the mech-
anisms of energy homeostasis in cells.
Ribosome production is a major biosynthetic and energy-con-
suming activity of eukaryotic cells. Ribosome biosynthesis
adapts rapidly to changes in intracellular energy status. Condi-
tions of energy starvation or glucose deprivation—when cellular
AMP/ATP ratio is increased—lead to the activation of the LKB1-
AMPK (AMP-activated protein kinase) pathway (Hardie, 2004);
this signaling inhibits mammalian TOR (target of rapamycin)/
p70 S6 kinase activity, which is required for rapid and sustained
serum-induced ribosome biosynthesis (Bhaskar and Hay, 2007).
Inhibition of mTOR activity by AMPK suppresses energy expen-
diture and protects cells from energy deprivation-induced apo-
also reduce cellular energy supply. Cells regulate energy de-
mand by sensing the environmental concentration of hydrogen
ions. H+produced under hypoxia promotes interactions be-
tween VHL and rDNA to reduce rRNA synthesis (Mekhail et al.,
rRNA synthesis is tightly regulated in response to metabolic
and environmental changes (Grummt, 2003; Moss et al., 2007).
rRNA genes are present in multiple copies; therefore, rRNA syn-
thesis could be modulated by varying transcription rate per gene
or by varying the number of active genes. Exponentially growing
cells use no more than half of their total complement of rRNA
genes, and it has been shown in both mammalian cells and bud-
ding yeast that the number of active genes decreases when cells
undergo transition from log to stationary phase (Claypool et al.,
2004; Preuss and Pikaard, 2007; Sandmeier et al., 2002). In
yeast, this gene inactivation is dependent on the histone deace-
tylase Rpd3 (Oakes et al., 2006). In mammalian cells, the chro-
matin-remodeling complex NoRC recruits HDAC1 and DNA
methyltransferases to inactive rRNA gene repeats (Santoro
et al., 2002). Furthermore, the activity level of rRNA genes is cor-
related with the type and extent of their chromatin modifications.
Taken together, these data argue that epigenetic mechanisms
control the ratio of active to inactive genes.
In yeast, heterochromatin formation at the ribosomal DNA
(rDNA) locus is also controlled by Sir2p, an NAD+-dependent de-
acetylase that removes acetyl groups from the N-terminal tails of
histone H3 and H4 to regulate nucleosome and chromatin struc-
ture (Buck et al., 2002). Increasing the expression of Sir2p can
bination between rDNA repeat sequences (Guarente, 2000). In
human, the Sir2p homolog SIRT1 deacetylates transcription
factors such as FOXOs, p53, and NF-kB (Yang et al., 2006).
SIRT1 is inducibly transcribed in response to calorie restriction
Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc. 627
Figure 1. NML Binds to Histone H3 Dimethylated at Lys9 at the rDNA Locus
(A) Downregulation of pre-rRNA levels by glucose deprivation. HeLa cells were cultured in medium containing 1000, 300, or 0 mg/l glucose, and pre-rRNA levels
were determined after 24 hr by RT-qPCR, using primers that are specific for the 50external transcribed spacer (ETS) of pre-rRNA and normalizing for cell count.
Values are means ± SD for triplicates.
(B) Glucose deprivation induces heterochromatin formation at the rDNA locus. HeLa cells were cultured in medium containing 1000, 300, or 0 mg/l glucose for 24
hr. ChIP analysis was performed to examine histone modifications at rRNA promoters. Immunoprecipitated DNA was analyzed by qPCR using H0 primers (see
Figure 1H) and normalized to input DNA. Values are means ± SD for triplicates.
628 Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc.
ing a broadrole in mammalian physiology as a mediator of adap-
tation to nutrient deprivation.
Here, we identify a protein complex, eNoSC (energy-depen-
dent nucleolar silencing complex), that contains a previously un-
characterized protein, termed Nucleomethylin (NML), as well as
SIRT1and SUV39H1. Our resultssuggest thatanenergy-depen-
dent change in the NAD+/NADH ratio regulates eNoSC, allowing
the complex to couple changing energy status with level of rRNA
synthesis. In addition, by limiting ribosome biogenesis, eNoSC
promotes the restoration of energy balance and protects cells
from energy deprivation-dependent apoptosis.
A Nucleolar Protein, NML, Binds to H3K9me2
at the rDNA Locus
Glucose deprivation activates LKB1-AMPK signaling and leads
to the inhibition of ribosome biogenesis (Shaw et al., 2004). Glu-
cose deprivation also reduces rRNA synthesis in HeLa cells
(Figure 1A). Because HeLa cells do not express LKB1 (Tiainen
et al., 1999), this result raises the possibility that, in addition to
the LKB1-AMPK pathway, there are other pathways that control
cellular energy balance.
The LKB1-AMPK pathway reduces the transcription rate of
rRNA genes. In contrast, recent reports mention the importance
of epigenetic regulation of rDNA (McStay, 2006). Thus, we next
investigated whether glucose removal affects the chromatin
structure on rDNA locus. We analyzed the levels of methylation
at Lys9 and acetylation of histone H3 at the rDNA locus by using
quantitative chromatin immunoprecipitation (ChIP) with primers
that amplify the rRNA genes (H0; Figure 1H). Glucose reduction
induced the deacetylation and dimethylation at Lys9 of histone
H3 in rRNA genes (Figure 1B).
To elucidate the mechanisms which link cellular energy status
and epigenetic status of rDNA, we purified proteins that bind to
histone H3 dimethylated at Lys9 (H3K9me2) from HeLa nuclear
extracts, using differentially modified N-terminal tails of histone
H3 peptides. Using mass spectrometry, we identified a protein
that specifically binds to the H3K9me2 peptide as KIAA0409
(accession number BC001071, GeneID 23378) (Figure 1C).
According to a recent proteomic analysis of nucleolar proteins,
KIAA0409 localizes in the nucleolus (Andersen et al., 2005). Sub-
cellular fractionation using the Muramatsu method (Andersen
et al., 2005; Muramatsu and Busch, 1964) and immunofluore-
sence studies confirmed that KIAA0409 is present in the nucleoli
along with UBF (Figures 1D and 1E). Therefore, we designated
KIAA0409 as NML.
Western blot analysis with anti-NML antibody (Figure S1 avail-
preferentially bound to the H3K9me2 peptide (Figures 1F and
S2). NML specifically coimmunoprecipitates with Lys9-dimethy-
lated but not with Lys4-dimethylated histone H3 (Figure 1G).
ChIP and re-ChIP analysis using primers that amplify the rRNA
coding region (H23) indicated that NML bound throughout the
rDNA locus with H3K9me2 (Figures 1H and 1I). These observa-
tions suggest that NML binds to H3K9me2 in the silent rDNA
clusters in vivo in the context of native chromatin.
NML Functions to Suppress rRNA Gene Transcription
Given that NML preferentially binds to silent rDNA clusters, it is
possible that NML might have a suppressive function on rRNA
transcription. Thus, we next examined pre-rRNA synthesis in
various cell lines infected with adenovirus vector containing ei-
ther NML (ad-NML) or LacZ (ad-LacZ) as a control. Infection of
ad-NML resulted in a decrease of pre-rRNA levels in all cell lines
tested (Figure 2A). Conversely, the level of pre-rRNA synthesis
was increased in cells transfected with small-interfering RNAs
against NML (NML siRNAs) (Figures 2B and 2C). We also per-
formed nuclear run-on assays and confirmed that NML reduced
the level of transcription (Figure 2D).
Next, we generated clones that stably expressed a small hair-
pin RNA against NML (NML shRNA), control LacZ (control), or
FLAG-NML (NML stable) (Figure 2E). The growth rates of these
cell lines were almost the same (data not shown). RT quantitative
PCR (qPCR) analysis confirmed the enhancement of pre-rRNA
synthesis in NML shRNA clones and reduction in the NML stable
clones (Figure 2F). In addition, NML reduces the level of protein
synthesis in an NML-dose-dependent manner (Figure 2G).
Taken together, these observations suggest that NML associ-
ates with H3K9me2 in heterochromatic clusters in rDNA and
suppresses rRNA synthesis and ribosome biogenesis. ChIP
analysis revealed that increasing levels of NML caused a dose-
dependent reduction of the acetylation and elevation of the
Lys9 methylation of histone H3 (Figure 2H). These results indi-
cate that alteration of NML levels changes the ratio between
active and silent states in the rDNA repeats.
proteins were resolved by SDS-PAGE, visualized by silver staining, and analyzed by mass spectrometry.
(D) NML is enriched in the nucleolar fraction. HeLa cells were fractionated into cytosolic (Cyto.), nuclear (Nuc.), and nucleolar fraction (Nucleo.) and endogenous
proteins of each fraction were analyzed by immunoblotting using anti-NML, anti-UBF, and anti-b-actin antibodies. UBF was used as a nucleolar marker.
(E) NML localizes in the nucleolus. Endogenous NML and UBF were visualized by immunofluorescence in HeLa cells using anti-NML and anti-UBF antibodies.
(F) Endogenous NML associates with H3K9me2 peptide. Nuclear extracts from HeLa S3 cells were used in the peptide pull-down assays and analyzed by
immunoblotting with anti-NML and anti-MTA2 antibodies.
(G) Endogenous NML associates with H3K9me2 nucleosomes. Mononucleosomal fraction was purified from HeLa cells and subjected to immunoprecipitation
with anti-NML antibody. The immunoprecipitates were analyzed by immunoblotting with anti-H3K9me2, anti-H3K4me2, and anti-NML antibodies.
Lower panel: Endogenous NML binds across the entire rDNA invivo. ChIP assay using HeLa cells was performed with normal IgG and anti-NML antibody. Values
are means ± SD for triplicates.
(I) Endogenous NML colocalizes with H3K9me2 on rDNA. Re-ChIP experiments using HeLa cells were performed by a first immunoprecipitation with anti-NML
antibody, followed by a second precipitation with anti-NML, anti-H3K9me2, and anti-H3K4me2 antibodies. Values are means ± SD for triplicates.
Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc. 629
Figure 2. NML Represses Pre-rRNA Synthesis
(A) NML expression reduces pre-rRNA level in various types of cells. HCT116, NB1RGB, HEK293, and HeLa cells were transiently infected with ad-NML or
ad-LacZ adenoviruses. After 24 hr, pre-rRNA level was determined by RT-qPCR. Values are means ± SD for triplicates.
(B) siRNAs efficiently knock down endogenous NML. HEK293 cells were transfected with siRNAs against NML. The level of endogenous NML protein was
detected by immunoblotting.
(C) Knockdown of NML increases pre-rRNA level. HCT116, NB1RGB, HEK293, and HeLa cells were transfected with siRNA against NML. Forty-eight hours after
transfection, total RNA was isolated, and pre-rRNA level was analyzed by RT-qPCR. Values are means ± SD for triplicates.
(D) NML expression represses rRNA transcription. Nuclear run-on assays were performed to measure transcription of 47S rRNA in ad-LacZ or ad-NML infected
HeLa cells. The assays were performed in duplicate.
630 Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc.
NML Associates with SIRT1
To investigate the molecular bases for the functions of NML,
control or NML-overexpressing cells were treated with the his-
tone deacetylase inhibitors, trichostatin A (TSA) and nicotin-
amide (NIA). TSA inhibits class I/II HDACs; NIA is known as
a class III HDAC inhibitor and a potent inhibitor of SIRT1. As
shown in Figure 3A, reduction of pre-rRNA levels by NML over-
expression was prevented by NIA, but not by TSA, implicating
SIRT1 in the NML-mediated inhibition of rRNA synthesis. Knock-
down of endogenous SIRT1 by siRNAs increased pre-rRNA
levels and prevented the NML-dependent repression (Figures
3B and 3C). Conversely, expression of SIRT1 decreased pre-
rRNA levels and strengthened the NML-dependent repression
We next tested the interaction between NML and SIRT1 by
coimmunoprecipitation and found that SIRT1 interacts with
NML (Figure 3D). ChIP analysis using H0, H8, or H23 primers
showed that SIRT1 associated with chromatin throughout the
rDNA locus (Figure S3). In addition, re-ChIP analysis revealed
that a protein complex containing NML and SIRT1 associates
with the silent chromatin region of rDNA (Figure 3E). The binding
of SIRT1 to rDNA was abrogated by NML siRNA treatment (Fig-
ures 3F and S4). Interestingly, SIRT1 siRNA also reduced the
binding of NML to the rDNA locus (Figure 3F). These results indi-
cate the coordinate binding of NML and SIRT1 to rDNA.
Expression of a deacetylase activity-deficient SIRT1 mutant,
SIRT1(H355A), resulted in increased pre-rRNA levels and pre-
vented the NML-dependent reduction of pre-rRNA levels (Fig-
ures 3G and S5), indicating that the deacetylase activity of
SIRT1 is necessary for the repression of rRNA synthesis medi-
ated by NML. SIRT1 preferentially deacetylates core histones
H3 Lys9, H3 Lys14, and H4 Lys16 in vitro (Vaquero et al.,
by siRNA increased the acetylation of histone H3 at the rDNA lo-
cus (Figure 3H). Interestingly, we also observed a decrease in
Lys9 methylation of H3 as a result of SIRT1 knockdown
(Figure 3H). This result implies that deacetylation at Lys9 of H3
is prerequisite for its methylation (Shankaranarayana et al.,
2003). Considering these results together with the observation
that NML knockdown also reduced the Lys9 methylation of H3
(Figure 2H), it appears that histone methyltransferases partici-
pate in the NML/SIRT1-mediated rRNA gene silencing.
NML Forms a Ternary Complex with SIRT1 and SUV39H1
It is known that Lys9 of histone H3 is methylated by histone
methyltransferases such as SUV39H1, EZH2, and G9a (Lachner
and Jenuwein, 2002). G9a is involved in the activation of rRNA
gene transcription (Yuan et al., 2007). Therefore, we investigated
the possibility of an interaction between NML and either
SUV39H1 or EZH2 and found that NML preferentially associates
with SUV39H1 (Figure 4A). SUV39H1 siRNA prevented the NML-
the other hand, expression of SUV39H1 decreased pre-rRNA
levels (Figure 4D). The SUV39H1-dependent reduction of
pre-rRNA levels requires the methyltransferase activity of
SUV39H1; overexpression of SUV39H1(R235H), a methyltrans-
ferase activity-deficient mutant, resulted in increased pre-rRNA
levels (Figures 4D and S6). A ChIP assay showed that reduction
of SUV39H1 protein levels decreased the Lys9 methylation and
increased the acetylation of histone H3 at the rDNA locus
(Figure 4E). Because knockdown of SUV39H1 increased the
acetylation of H3, it is possible that SUV39H1 is involved in
the recruitment of NML and SIRT1. Consistent with this idea,
the binding of NML and SIRT1 to rDNA was abrogated by
SUV39H1 siRNA (Figure 4F). These results indicate the coordi-
nate binding of NML, SIRT1, and SUV39H1 to rDNA.
To investigate the possibility that these proteins form a single
protein complex, the protein complex containing both NML and
SUV39H1 was sequentially immunoprecipitated by antibodies.
Immunoblotting of theprecipitate withanti-SIRT1 antibody dem-
formed a ternary complex with both SIRT1 and SUV39H1. To
confirm this interaction among the endogenous proteins, we
immunoprecipitated the proteincomplex fromHeLa cellextracts
using anti-NML antibody and identified SIRT1 and SUV39H1 in
the precipitates (Figure 4H). In purification experiments using
H3K9me2 peptide, NML, SIRT1, and SUV39H1 were copurified
(Figure 4I). Our observations indicate that an NML/SIRT1/
SUV39H1 protein complex suppresses rRNA gene transcription
by establishing silent chromatin at the rDNA locus.
NML Protects Cells from Cell Death Induced
by Glucose Deprivation
In mammalian cells, CR decreases cellular ATP concentration
and increases the NAD+/NADH ratio (Guarente and Picard,
2005). According to our observations and the fact that the
NAD+/NADH ratio regulates the deacetylase activity of SIRT1,
it is possible that the NML/SIRT1/SUV39H1 complex partici-
pates in CR-induced repression of rRNA gene transcription.
ChIP analysis revealed that the binding of NML and SIRT1 to
therDNA locus increased
(Figure 5A). In addition, neither downregulation of pre-rRNA
levels nor alteration of histone modifications induced by glucose
deprivation were observed in cells treated with siRNA against
NML, SIRT1, or SUV39H1 (Figures 5B and 5C). Thus, the NML/
SIRT1/SUV39H1 complex participates in the downregulation of
rRNA transcription under CR. Consequently, we designated
the NML/SIRT1/SUV39H1 complex as eNoSC.
(E) Protein level of NML in HeLa cells stably expressing FLAG-NML (NML stable), shRNA against NML (NML shRNA), or control cells (Cont.) was determined by
immunoblotting using anti-NML antibody.
(F) NML decreases pre-rRNA level in a dose-dependent manner. Pre-rRNA level in control, NML stable, or NML shRNA HeLa cells was determined by RT-qPCR.
Values are means ± SD for triplicates.
(G)ExpressionofNMLdecreasesrateofproteinsynthesis.The indicatedHeLacells wereincubated with35S-methionine for 4hr;incorporationof35S-methionine
into protein was measured by scintillation counting and normalized to total protein levels. Values are means ± SD for triplicates.
(H) Overexpression of NML induces deacetylation and dimethylation in histone H3 at Lys9. ChIP analysis was performed to determine histone modifications on
the rRNA promoter (H0) using the indicated HeLa cells. Values are means ± SD for triplicates.
Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc. 631
Next, we performed parallel timecourse studies of pre-rRNA
levels and total cellular ATP levels in control and NML shRNA
cells. As shown in Figure 5D, in the absence of glucose, pre-
rRNA levels in NML shRNA cells reduced more slowly than those
in the control cells. In contrast, the total cellular ATP levels in
NML shRNA cells decreased faster than those in control cells.
These observations suggest that eNoSC restores energy levels
under low-glucose conditions.
TUNEL assay and poly(ADP-ribose) polymerase 1 (PARP-1)
cleavage assay revealed that insufficient intracellular energy
induced apoptosis (Figures 5E and 5F). Thus, we next mea-
sured the percentage of dead cells in populations of HEK293
cells transfected with either indicated siRNAs or NML expres-
sion vector after various intervals of glucose deprivation
(Figure 5G). When glucose was withdrawn, cells treated with
siRNA against NML, SIRT1, or SUV39H1 died rapidly as
compared with control siRNA-treated cells (Figure 5G). Knock-
down of the components of eNoSC reduced the tolerance of
low-glucose conditions (Figure 5H). The same results were
obtained when we used HeLa cells (Figure S7). Conversely,
Figure 3. NAD+-Dependent Histone Deacetylase SIRT1 Is Required for NML-Mediated Repression of rRNA Synthesis
(A) NIA inhibits NML-mediated rRNA repression. HEK293 cells transfected with NML or control plasmid were cultured for 6 hr in medium containing 40 nM TSA or
5 mM NIA, and pre-rRNA levels were measured by RT-qPCR. Values are means ± SD for triplicates.
(B) siRNAs efficiently knock down endogenous SIRT1. HEK293 cells were transfected with siRNAs against SIRT1. The levels of endogenous SIRT1 and NML
proteins were examined by immunoblotting.
as indicated. Forty-eight hours after transfection, pre-rRNA levels were determined by RT-qPCR. Values are means ± SD for triplicates.
(D) NML interacts with SIRT1. HEK293 cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated by anti-FLAG antibody and
immunoblotted with anti-HA or anti-FLAG antibody.
anti-NML antibody, followed by a second precipitation with the indicated antibodies. Values are means ± SD for triplicates.
(F) Coordinated binding of endogenous NML and SIRT1 to rDNA. Association of NML or SIRT1 to rDNA was analyzed by ChIP using anti-NML and anti-SIRT1
antibodies in HEK293 cells following treatment with either NML siRNA or SIRT1 siRNA. Values are means ± SD for triplicates.
(G) SIRT1 potentiates reduction of pre-rRNA level via its deacetylase activity. HEK293 cells were transiently transfected with NML, SIRT1, or catalytically inactive
SIRT1(H355A) plasmid. Forty-eight hours after transfection, pre-rRNA levels were determined by RT-qPCR. Values are means ± SD for triplicates.
(H) SIRT1 is required for heterochromatin formation at the rDNA locus. Modification of histones at rRNA promoters was analyzed by ChIP in HEK293 cells
following treatment with control or SIRT1 siRNA. Values are means ± SD for triplicates.
632 Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc.
NML expression protected cells from apoptosis (Figures 5G
and 5H). These results indicate that eNoSC-mediated rDNA
silencing protects cells from energy deprivation-induced apo-
The Methyltransferase-like Domain of NML Plays
an Important Role in Repression of rRNA Transcription
Sequence analysis demonstrates that the N-terminal domain of
NML shows no obvious domain structure; however, it is required
Figure 4. NML/SIRT1/SUV39H1 Complex Suppresses rRNA Synthesis
(A) NML interacts with SUV39H1. HEK293 cells were transfected with FLAG-NML, myc-SUV39H1, or myc-EZH2 as indicated. 24 hr after transfection, the cell
lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-FLAG or anti-myc antibody.
(B)The siRNAs efficiently knock down endogenous SUV39H1. HEK293 cells were transfected with siRNAs against SUV39H1. The level of endogenous SUV39H1
protein was examined by immunoblotting.
(C) SUV39H1 knockdown restores repression of pre-rRNA synthesis by NML. HEK293 cells were transfected in combination with NML plasmid and SUV39H1
siRNA as indicated. Forty-eight hours after transfection, pre-rRNA levels were determined by RT-qPCR. Values are means ± SD for triplicates.
(D) SUV39H1 represses pre-rRNA synthesis via its histone methyltransferase activity. HEK293 cells were transiently transfected with NML, SUV39H1, or
SUV39H1(R235H) plasmid. Forty-eight hours after transfection, pre-rRNA levels were determined by RT-qPCR. Values are means ± SD for triplicates.
(E)SUV39H1isrequired forheterochromatinformationattherDNAlocus.Modification of histone onrDNAwasanalyzedbyChIPusingtheindicated antibodies in
(F) SUV39H1 is required for binding of NML and SIRT1 to rDNA locus. Control or SUV39H1 siRNA-treated HEK293 cells were analyzed by ChIP assay using anti-
NML or anti-SIRT1 antibody. Values are means ± SD for triplicates.
(G)NML formsaternarycomplex withSIRT1 and SUV39H1. HEK293cells weretransfectedincombinationwithFLAG-NML, myc-SUV39H1,andSIRT1 plasmids
as indicated. FLAG-NML was immunoprecipitated using anti-FLAG antibody. NML/SIRT1/SUV39H1 complex was eluted using FLAG peptides then immunopre-
cipitated with anti-myc antibody. SIRT1 in immunoprecipitates was detected by immunoblotting.
(H) Endogenous NML associates with SIRT1 and SUV39H1. The cell lysates from HeLa cells were prepared and immunoprecipitated with normal rabbit IgG or
anti-NML antibody and immunoblotted using antibodies against SIRT1 and SUV39H1.
(I) SIRT1 and SUV39H1 associate with H3K9me2 peptide. Whole-cell extracts of HeLa cells were pulled down with the indicated peptide. Pull-down assay was
performed in a buffer containing 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% NP-40, 1 mM DTT, and 2 mM EDTA. Bound protein was resolved by 10% SDS-
PAGE and analyzed by immunoblotting with anti-NML, anti-SUV39H1, and anti-SIRT1 antibodies.
Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc. 633
for the specific binding to H3K9me2 peptide (Figure S2). The
C-terminal domain of NML contains consensus sequence motifs
(I, post-I, II, and III) found in SAM-dependent methyltransferases
(SAM-MT) (Schluckebier et al., 1995) (Figure 6A). Thus, we have
solved the crystal structure of the methyltransferase-like domain
of NML(242–456 amino acids [aa]) complexed with S-adenosyl-
homocysteine (SAH) at 2.0 A˚resolution using multiwavelength
anomalous diffraction of seleno-methionyl protein crystals
(Figure 6B and Table S1). NML(242–456 aa) can be divided into
two parts: a large domain and a small domain. The large domain
reveals an a/b structure and adopts a well-characterized SAM-
MT fold with a seven-stranded b sheet (b1–b7) with two helices
(a1, a2) on one side and two helices (a3, a4) on the opposite
side. The small domain, comprising three helices (aA–aC), is
Figure 5. NML/SIRT1/SUV39H1 Complex Acts as an Energy-Dependent Repressor of rRNA Transcription and Protects Cells from Apoptosis
Induced by Low Glucose
(A) Treatment of cells with low glucose increases binding of endogenous NML and SIRT1 to rDNA. HEK293 cells were treated with 1000 mg/l or 0 mg/l glucose
(glucose deprivation) medium and association of NML and SIRT1 to rDNA was analyzed by ChIP assay. Values are means ± SD for triplicates.
(B) The NML complex is required for rRNA repression in response to low glucose. Pre-rRNA levels were analyzed by RT-qPCR in HEK293 cells transfected with
indicated plasmids or siRNAs after 36 hr of treatment with 1000 mg/l or 0 mg/l of glucose. Values are means ± SD for triplicates.
(C) Histone modifications at the rDNA locus changein response to glucose concentration.HEK293 cells were transfected with NML plasmid or indicated siRNAs.
The transfected cells were cultured for 36 hr in medium containing 1000 mg/l or 0 mg/l of glucose. Modifications of histones at the rDNA locus were analyzed by
ChIP. Values are means ± SD for triplicates.
(D) Timecourse studies of pre-rRNA levels and total cellular ATP levels in control or NML shRNA cells after glucose deprivation. Control or NML shRNA cells were
cultured withoutglucose. At the indicated time points, pre-rRNA and ATP levels were analyzed as described in the Experimental Procedures. Values are means ±
SD for triplicates.
(E and F) NML/SIRT1/SUV39H1 complex protects cells from apoptosis induced by glucose deprivation. HeLa cells were transfected with the indicated siRNAs.
After 48 hr, we introduced medium either with (1000 mg/l) or without glucose. (E) These cells were cultured for an additional 48 hr and subjected to TUNEL assay.
Values are means ± SD for triplicates. (F) PARP-1 cleavage was analyzed by immunoblotting with anti-PARP-1 antibody.
(G) Percentage of dead cells after glucose deprivation. Forty-eight hours after transfection with the indicated siRNAs, HEK293 cells were cultured in medium
containing 1000 mg/l (upper panel) or 0 mg/l of glucose (lower panel). At the indicated times, the cell viability was measured by Trypan blue exclusion assay.
Values are means ± SD for triplicates.
(H) siRNA-transfected HEK293 cells were cultured in medium with 0, 100, 300, or 1000 mg/l glucose for 48 hr. The Trypan blue exclusion assay was performed to
measure the viability of the cells. Values are means ± SD for triplicates.
634 Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc.
stabilized by hydrophobic interactions and packs against the
SAM-MT fold via hydrophobic interactions (Figure 6C). It should
be noted that the C-terminal region after b7 turns back toward
The SAH molecule is well-ordered in the crystal structure at
the carboxyl ends of the parallel strands, and its three moieties
are recognizable (Figure 6D). The amino group of the methionine
moiety interacts with the oxygen atom of Cys363 and the car-
boxylate of Asp314 via a water molecule belonging to motif I.
Both hydroxyls of the ribose moiety are recognized by Asp334
and His281, which belong to the post-I and small domains,
respectively. Finally, the nitrogen atoms of the adenine moiety
(N1,N6, and N7) interact, respectively, with the main-chain nitro-
gen atom of Met347, the carboxylate of Asp346, and (via a water
molecule) the main-chain nitrogen atom of Gly368. The smalldo-
mainforms a lid covering the SAH-binding pocket (Figure 6E). As
a consequence, the pocket is almost buried, and the sulfur atom
in SAH is thus only accessible via a narrow entrance (Figure 6F).
NMLm2, in which the Gly residues in motif I are mutated (G316D
and G318R for NMLm1 and G316Q for NMLm2) (Figure 6A).
Neither mutant possesses the ability to bind SAM (Figure 6G).
The aa substitutions introduced into these mutants do not affect
NML binding to either SIRT1, SUV39H1, or chromatin (Figures
7A–7C). However, neither mutant was able to suppress pre-
rRNA levels when expressed in HEK293 (Figure 7D) or HeLa cells
(data not shown). Consistent with this, expression of the mutants
did not alter the modifications of histones in rRNA genes
(Figure 7E). Furthermore, NML expression protected cells from
energy starvation-induced apoptosis, whereas neither mutant
could do so (Figures 7F and S8), suggesting that SAM binding to
yltransferase-like domain, it is possible that there may be target
molecules of NML that are important for silencing of rDNA locus.
Our observations suggest that CR-induced changes in the
NAD+/NADH ratio regulate eNoSC, enabling the complex to
Figure 6. Structure of the Methyltransferase-like Domain of NML
(A) Schematic representation of NML and its point mutants in the methyltransferase-like domain. Conserved motifs in SAM-MTase are shown in red.
(B) Ribbon representation of methyltransferase-like domain of NML. The small domain in the N terminus is shown in gray. The bound SAH molecule is shown as
a ball-and-stick model.
(C) Topological diagram of NML(242–456). Secondary structure elements are colored using the same scheme as in (B).
(D) A schematic diagram showing SAH-protein interactions. Dashed lines correspond to hydrogen bonds.
(E)Accessible surface area of NML(242–456). Solvent-accessible surface colored according to electrostatic potential in the range ?10 kBT (red) to +10 kBT (blue),
where kBis Boltzmann’s constant and T is the absolute temperature.
(F) The SAH binding pocket of (a) NML and (b) Dot1.
and crosslinked by UV irradiation.3H-labeled SAM bound to protein was analyzed by SDS-PAGE followed by autoradiography (upper panel). Lower panel shows
Coomassie blue staining of the same gel.
Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc. 635
couple changing energy status with levels of rRNA synthesis and
ribosome production. Under standard glucose conditions, the
down of these components increases pre-rRNA levels (Figures
5A and 5B). These differences were statistically significant; sta-
tistical analysis in Figure 5B by t test revealed that the p value
of control (lane 1) versus siRNA transfected cells (lanes 5, 7,
and 9) was < 0.05. This result implies that the complex also
has a constitutive role in rDNA silencing.
In mammals, the effect of sirtuins at the rDNA locus is com-
plex. TAFI68, a basal component of the Pol I transcription appa-
ratus, is a relevant target of SIRT1 (Muth et al., 2001). We also
show that SIRT1 suppresses the pre-rRNA levels in the nucleo-
lus. On the other hand, SIRT7 is a nucleolar protein that acts
as a positive regulator of Pol I transcription (Ford et al., 2006).
Our results that NIA, an inhibitor of SIRT family members, abro-
gates the function of eNoSC raised a possibility that NML could
also associate with SIRT7. However, coimmunoprecipitation as-
say revealed that SIRT7 was not able to bind to NML (Figure S9).
The steady-state distribution of SIRT1 is essentially nucleo-
plasmic, showing only a very faint nucleolar localization (Michi-
shita et al., 2005). In immunostaining using anti-SIRT1 antibody,
Figure 7. The Methyltransferase-like Domain of NML Plays an Essential Role in the Repression of rRNA Transcription
(A and B) The NML mutants interact with both SIRT1 and SUV39H1. HEK293 cells were transfected with FLAG-NML, FLAG-NMLm1, or FLAG-NMLm2 in com-
bination with HA-SIRT1 (A) or myc-SUV39H1 (B) as indicated. After 24 hr, lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with the
(C) NML mutants can associate with the rDNA locus. HEK293 cells were transfected with FLAG-NML or FLAG-NMLm plasmid. Association of NML or NMLm on
the rDNA locus was analyzed by ChIP using anti-FLAG antibody. Values are means ± SD for triplicates.
(D) NML mutants are not able to reduce pre-rRNA levels. HEK293 cells were transfected with plasmid encoding NML or its mutants. Pre-rRNA level was analyzed
by RT-qPCR and normalized to b-actin mRNA. Values are means ± SD for triplicates.
(E) NML mutants exhibit little effect on the histone modification on the rDNA locus. HEK293 cells were transfected with NML or NMLm plasmid. Modifications of
histones at the rDNA locus were analyzed by ChIP. Values are means ± SD for triplicates.
(F) Percentage of dead cells after glucose deprivation. Forty-eight hours after transfection with the indicated expression vectors, HEK293 cells were cultured in
medium containing 1000 mg/l (upper panel) or 0 mg/l of glucose (lower panel). At the indicated times, the cells viability was measured by Trypan blue exclusion
assay. Values are means ± SD for triplicates.
(G) Proposed model for energy-dependent eNoSC function in the nucleolus.
636 Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc.
we observed that a small fraction of SIRT1 is localized in the nu-
cleolus (Figure S10). These observations imply that nucleoplas-
mic/nucleolar shuttling is required in order for SIRT1 to act in
the nucleolus. Recent evidences indicate that the nucleolus is
associated withdifferent typesofproteindynamics;somenucle-
olar proteins such as UBF are immobile (Roussel et al., 1993),
whereas other proteins such as Nucleophosmin/B23 have
a steady-state nucleolar distribution but continuously shuttle
between nucleolus and nucleoplasm (Colombo et al., 2002). A
quantitative analysis of the proteome of nucleoli revealed that
some proteins are stably copurified with nucleoli, while many
proteins only accumulate transiently in nucleoli (Andersen
et al., 2005). This observation also indicates that many nucleolar
proteins cycle between the nucleolus and nucleoplasm. SIRT1
could be one of these proteins. Our observations demonstrate
that SIRT1 functions in the nucleolus; however, we acknowledge
that data obtained from overexpression experiments could
include artifacts caused by forced recruitment of nucleoplasmic
SIRT1 to the nucleolus.
SIRT1 hasbeen shownto protectagainst apoptosisbydeace-
tylating nonhistone proteins such as p53 (Luo et al., 2001).
Therefore, it is possible that eNoSC could also inhibit cell death
via deacetylating nonhistone proteins. In our hands, SIRT1-de-
pendent p53 deacetylation was not affected by NML expression
(Figure S11). In addition, NML is predominantly localized in the
nucleolus. These results suggest that a major mechanism by
which eNoSC protects cells from energy deprivation-induced
apoptosis is rDNA silencing. However, there remains the possi-
bility that eNoSC protects cells from apoptosis via other path-
ways as well.
Recent evidence indicates that SIRT1 interacts with SUV39H1
and suggests that they are functionally interrelated (Vaquero
et al., 2007). In coimmunoprecipitation experiments, we also ob-
sion levels of NML do not affect the association between SIRT1
and SUV39H1 (Figure S12A), although the recruitment of these
proteins to the rDNA region does require NML (Figures 3F and
S12B). GST pull-down experiments showed that SUV39H1, but
not SIRT1, binds NML in vitro (Figure S13A), suggesting that
SIRT1 binds to NML through SUV39H1. We also found that
glucose deprivation increases the affinity between SIRT1 and
SUV39H1 (Figure S13B) and consequently strengthens the
interaction between NML and SIRT1 (Figure S13C). Therefore,
glucose deprivation reduces pre-rRNA levels partly due to the
enhancement of affinity between SIRT1 and SUV39H1. Consid-
ering these results together with the report that SIRT1 deacety-
lates SUV39H1 (Vaquero et al., 2007), it is possible that the acet-
ylation status of SUV39H1 affects the interaction between SIRT1
Recent accumulating evidence clearly indicates that epige-
netic mechanisms control rRNA gene transcription (McStay,
by recruiting DNA methyltransferase and histone deacetylase
activity to rRNA promoters, thus establishing the structural char-
acteristics of heterochromatin (Santoro and Grummt, 2005; San-
issue: how does silencing, established at the promoter, spread
across the repeat? eNoSC might provide the mechanism: since
eNoSC can change acetylation of Lys9 residue of histone H3 to
dimethylation, which NML can in turn recognize and bind, the
complex is potentially able to spread across the repeat
(Figure 7G). However, there is a possibility that eNoSC requires
silencing initiators, which would dictate which regions are
2000). Therefore, it is possible that eNoSC may also connect
rRNA processing with intracellular energy status.
The sequenceand structuralfeatures ofNMLstrongly suggest
that NML would be a methyltransferase, but the substrate of
NML has not yet been identified. The surface representation of
NML shows that SAH is accessible only via an opening to a nar-
that of Dot1 (Min et al., 2003), an evolutionarily conserved SAM-
dependent histone methyltransferase that methylates Lys79 of
histone H3 in the core domain (Ng et al., 2002). Dot1 requires
additional basic residues outside of the catalytic domain for its
H3 methylation activity (Sawada et al., 2004). Likewise, NML
also has basic residues outside of its methyltransferase-like
domain. However, we could not detect any methyltransferase
activity when NML was incubated with histones (Figure S14). A
recent report indicates that Dot1-mediated H3K79 methylation
requires the basic patch residues (R17H18R19) in the N-terminal
tail of histone H4 (Fingerman et al., 2007). Thus, NML may also
require the N-terminal tail or modified N-terminal tail for target
methylation. Alternatively, the binding of NML to components
of eNoSC might induce a conformational change and make the
entrance wide enough to bind and methylate nonhistone pro-
teins; another possibility is that the target residue(s) could be
aa other than Lys. Because SAM binding to NML is necessary
for eNoSC-mediated rDNA silencing, it will be of great interest
to identify the target of the methylation activity.
Cell Culture and Treatments
NB1RGB normal human neonatal skin fibroblast cells were obtained from
Riken Cell Bank (Tsukuba, Japan) and maintained in aMEM (Sigma, St. Louis,
MO). HeLa human cervical carcinoma cells, HEK293 human kidney epithelial
cells, and HCT116 human colorectal cancer cells were maintained in DMEM
(Sigma). All media were supplemented with 10% fetal bovine serum (FBS)
and penicillin-streptomycin mixed solution (Nacalai tesque, Kyoto, Japan).
Cells were maintained at 37?C in an atmosphere containing 5% CO2and
100% humidity. We used medium containing 1000 mg/l glucose, unless other-
wise indicated in the figure legend.
Treatments with trichostatin A (TSA) (40 nM) or NIA (5 mM) were performed
for 6 hr. For glucose treatment, cells were washed twice with PBS(?) and cul-
tured in medium containing 10% FBS with 0 mg/l, 100 mg/l, 300 mg/l, or 1000
mg/l glucose as described in figure legends.
Rabbit anti-human NML antibody was raised against a synthetic peptide cor-
responding to 136–197 aa of NML. The list of the other antibodies is shown in
the Supplemental Data.
Cell 133, 627–639, May 16, 2008 ª2008 Elsevier Inc. 637
Peptide Pull-Down Assay
were prepared from HeLa S3 cells using Dignam protocol (Dignam et al.,
1983), precleared with avidin beads, and incubated with H3 peptides conju-
gated with avidin beads (Promega) for 3 hr at 4?C. About 5 mg of peptide
and 108cells were used for one assay. The beads were then washed eight
times with buffer containing 20 mM HEPES (pH 7.9), 300 mM KCl, 0.2% Triton
X-100, 1 mM PMSF, and protease inhibitor cocktail (Nacalai tesque). The final
wash was performed with buffer containing 4 mM HEPES (pH 7.9), 10 mM
NaCl, 1 mM PMSF, and protease inhibitor cocktail. Bound proteins were
eluted from the resin twice with 100 mM glycine (pH 2.8). The eluates were
combined, neutralized with 1/10 volume of 1 M Tris (pH 8.0), and analyzed
Mononucleosomalfractionpurifiedfrom HeLaNML stable cells wassubjected
to immunoprecipitation using anti-FLAG M2-agarose beads (Sigma). Eluates
were analyzed by immunoblotting using anti-H3K4me2 and anti-H3K9me2
antibodies. Details are provided in the Supplemental Data.
Chromatin Immunoprecipitation and qPCR Detection
ChIP assayswere performed using ChIP assay kit (Upstate) according toman-
ufacturer’s protocol. Primer sequences and experimental details are provided
in the Supplemental Data.
RNA Purification and RT-qPCR
Total RNA was isolated with Sepasol RNA I Super reagent (Nacalai tesque)
and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen).
Real-time quantitative PCR analysis was performed using the Thermal Cycler
Dice TP800 (Takara) and Platinum SYBR Green qPCR SuperMix-UDG (Invi-
trogen). Primers and experimental details are provided in the Supplemental
HeLa cells were labeled for 2 hr with 100 mCi of35S-methionine in methionine-
free DMEM medium (GIBCO) supplemented with 10% dialyzed serum. The
incorporation of35S-methionine into protein was determined using a Beck-
mann Coulter liquid scintillation counter and normalized to the protein content.
Details are provided in the Supplemental Data.
Intracellular ATP was measured by using ATP Bioluminescence Assay Kit CLS
II (Roche) and a luminometer (Berthold). ATP levels were normalized to protein
content using the BCA Protein assay kit (PIERCE). Details are provided in the
Assays for Apoptosis Detection
Apoptosis was detected by TUNEL and PARP-1 cleavage analysis. Details are
provided in the Supplemental Data.
Crystallization and Data Collection
NML(242–456) and SeMet-labeled NML(242–456) were successfully crystal-
lized by the hanging-drop vapor-diffusion method, using 30% PEG8000 as
a precipitant in 0.1 M MES buffer (pH 6.0) containing 200 mM ammonium sul-
fate. X-ray diffraction data were collected at 100K on beamline BL41 at
Spring8, Japan and on beamline BL5 at The Photon Factory, Japan. Details
are provided in the Supplemental Data.
Structure Determination and Refinement
The structureofNML(242–456) wassolved bymultiwavelength anomalous dif-
fraction (MAD) using the SeMet-labeled NML(242–456) crystal. Details for
structure determination and refinement are provided in the Supplemental
Data. The final refinement statistics are summarized in Table S1.
The KIAA0409 protein sequence was deposited in GenBank with accession
number O43159. Coordinates and structure factors for NML(242–456) have
been deposited in the Protein Data Bank with accession number 2ZFU.
Supplemental Data include Supplemental Experimental Procedures, one
table, and fourteen figures and can be found with this article online at http://
We thank Dr. Masami Muramatsu and Dr. Kosuke Morikawa for valuable dis-
cussion and for technical advice.
Received: October 11, 2007
Revised: February 4, 2008
Accepted: March 24, 2008
Published: May 15, 2008
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