anti-SUV39H1 (clone 44.1) antibodies were from Abcam; anti-H3Ac and anti-SIRT1 antibodies were from Upstate; anti-MTA2 antibody (BL1808) was from Bethyl.
Cell, Volume 133
Supplemental Data Epigenetic Control of rDNA Loci in Response to Intracellular Energy Status Akiko Murayama, Kazuji Ohmori, Akiko Fujimura, Hiroshi Minami, Kayoko Yasuzawa-Tanaka, Takao Kuroda, Shohei Oie, Hiroaki Daitoku, Mitsuru Okuwaki, Kyosuke Nagata, Akiyoshi Fukamizu, Keiji Kimura, Toshiyuki Shimizu, and Junn Yanagisawa
Supplemental Experimental Procedures
Antibodies Anti-HA antibodies (clone 12CA5 and 3F10) were purchased from Roche diagnostics; anti-FLAG M2 antibody and anti-FLAG M2-agarose were from Sigma; anti-myc antibody was from Nacalai tesque; anti-UBF(F-9) and anti-GST(B-14) antibodies were from Santa Cruz biotechnology; anti-H3K9me2, anti-H3K4me2, and anti-SUV39H1 (clone 44.1) antibodies were from Abcam; anti-H3Ac and anti-SIRT1 antibodies were from Upstate; anti-MTA2 antibody (BL1808) was from Bethyl laboratories.
Immunoprecipitation To monitor the interaction of NML, SIRT1 and SUV39H1 in vivo, HEK293 cells
1
were transfected
with
expression vectors encoding
the respective proteins
(pcDNA3-FLAG-NML, pcDNA3-HA-SIRT1, and pcDNA3-myc-SUV39H1). At 24 hours after transfection, cells were lysed in lysis buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 2 mM EDTA and 0.1% NP-40) at 4°C for 30 min. The cleared lysate was incubated for 2 hours with 10 µl of anti-FLAG M2-agarose beads. After washing 4 times with the same buffer, FLAG-tagged proteins were eluted with 0.5 mg/mL of FLAG peptide in the lysis buffer at 4°C for 30 min, separated on 10% SDS-polyacrylamide gels and analyzed by immunoblotting with indicated antibodies. For coimmunoprecipitation of the endogenous proteins, HeLa cells were lysed in lysis buffer (20 mM Tris-HCl at pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% NP-40 and 1% Sodium deoxycholate) at 4°C for 30 min.
The cleared lysate was incubated for 4
hours with 4 µg of antibodies against the indicated proteins, added 10 µl of protein G sepharose, and rotated for 1 hour.
After washing 4 times with the same buffer,
immunoprecipitates were separated on 10% SDS-polyacrylamide gels, and analyzed by immunoblotting with the indicated antibodies.
Nucleosome-binding Assay For nucleosome-binding assay, mononucleosomal fractions were prepared from HeLa cell line stably expressing FLAG-NML and parental HeLa cells as a control. The biochemical fractionation procedure and microccocal nuclease treatment of nucleosomes were performed as described (Mendez and Stillman, 2000; Wysocka et al., 2001).
Mononucleosomal fractions were combined and then incubated with
anti-FLAG M2-agarose beads (Sigma). The beads were washed and eluted with 0.5 mg/mL of FLAG peptide (Sigma).
Eluates were analyzed by immunoblotting using
2
anti-H3K4me2 and anti-H3K9me2 antibodies.
Immunofluorescence Cells grown on poly L-Lysine-coated chamber slide were rinsed twice with PBS and fixed in 4% formaldehyde in PBS for 10 min. After rinsing twice PBS, the cells were permeabilized in 0.1% Triton X-100 in PBS, blocked with TBS-T containing 0.5% bovine serum albumin and goat serum for 1 hour at room temperature.
Then the cells
were incubated with anti-NML or anti-UBF antibodies for 1 hour, stained with AlexaFluor-conjugated secondary antibodies (Invitrogen) for 1 hour and mounted with Vectashield (Vector Laboratories). Immunofluorescence was performed using Biozero immunofluorescence microscopy (Keyence, Osaka, Japan).
Nucleoli Purification HeLa cell nucleoli were isolated in high purity by density gradient fractionation as described (Andersen et al., 2005).
Chromatin Immunoprecipitation and Quantitative-PCR (qPCR) Detection Nuclear proteins were cross-linked to genomic DNA by adding formaldehyde for 10 min directly to the medium to a final concentration of 1%. Cross-linking was stopped by adding glycine to a final concentration of 0.125 M and incubating for 5 min at room temperature on a rocking platform. The medium was removed and the cells were washed twice with ice-cold phosphate-buffered saline (PBS) (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4 and 8.1 mM Na2HPO4.2H2O). The cells were collected by scraping in ice-cold PBS supplemented with a protease inhibitor cocktail (Nacalai
3
tesque). After centrifugation the cell pellets were resuspended in lysis buffer [1% SDS, 10 mM EDTA, protease inhibitors and 50 mM Tris–HCl (pH 8.1)] and the lysates were sonicated to result in DNA fragments of 300 to 1,000 bp in length.
Cellular debris was
removed by centrifugation and the lysates were diluted 1:10 in ChIP dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM NaCl, protease inhibitors and 16.7 mM Tris–HCl (pH 8.1)].
Non-specific background was removed by
incubating the chromatin resuspension with a salmon sperm DNA/protein A agarose slurry (Upstate Biotechnology, Lake Placid, NY, USA) for 30 min at 4°C with agitation. The samples were centrifuged and the recovered chromatin solutions were incubated with 5 µl of indicated antibodies overnight at 4°C with rotation.
The
immuno-complexes were collected with 60 µl of protein A agarose slurry (Upstate Biotechnology) for 2 hours at 4°C with rotation.
The beads were pelleted by
centrifugation for 1 min at 4°C at 100 g and washed sequentially for 5 min by rotation with 1 ml of the following buffers: low salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris–HCl (pH 8.1)], high salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl and 20 mM Tris–HCl (pH 8.1)] and LiCl wash buffer [0.25 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris–HCl (pH 8.1)]. Finally, the beads were washed twice with 1 ml TE buffer [1 mM EDTA and 10 mM Tris–HCl (pH 8.0)]. For re-ChIP, the immuno-complexes were eluted with 50 µl of elution buffer (1% SDS and 100 mM NaHCO3) containing 10 mM DTT at room temperature for 30 min, the supernatant was diluted 1:40 in ChIP dilution buffer and the antibody against the second protein of interest was added, the new immuno-complexes were allowed to form by incubating at 4°C overnight on a rocking platform, the immuno-complexes were
4
collected by incubating with 120 µl protein A agarose slurry at 4°C for 2 hours on a rocking platform and finally washed as indicated above.
In both cases the
immuno-complexes were then eluted by adding 250 µl elution buffer and incubation for 15 min at room temperature with rotation.
After centrifugation, the supernatant was
collected and the cross-linking was reversed by adding NaCl to final concentration of 200 mM and incubating overnight at 65°C. The remaining proteins were digested by adding proteinase K (final concentration 40 µg/ml) and incubation for 1 hour at 45°C. The DNA was recovered by phenol/chloroform/isoamyl alcohol (25/24/1) extractions and precipitated with 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol using glycogen as a carrier. The purified DNA were amplified by real-time PCR using the Thermal Cycler DiceTM TP800 (Takara) and Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The primers for real-time PCR are as follows: 5’-GGTATATCTTTCGCTCCGAG-3’ and
5’-GACGACAGGTCGCCAGAGGA-3’
5’-AGTCGGGTTGCTTGGGAATGC-3’ and for
H8,
for
H0,
5’-CCCTTACGGTACTTGTTGACT-3’
5’-CCTTCCACGAGAGTGAGAAGC-3’
and
5’-TCGACCTCCCGAAATCGTACA-3’ for H23 (O'Sullivan et al., 2002).
siRNA and Plasmid DNA Transfection For transfection of siRNAs, cells at 30 to 50% confluency were transfected with 20 nM of siRNA using LipofectAmine RNAi max (Invitrogen) according to the manufacturer’s protocol. duplexes
NML-1
All siRNAs were purchased from Invitrogen.
The siRNA
(5’-UAGGAAGUCCCUGAUGUUGGUUCCC-3’),
(5’-ACUGCUUGCGGCUUAAUGUAUGAGG-3’),
5
NML-2 NML-3
(5’-AAAGGUUCGAACAUCCUCAAAGCGG-3’),
SIRT1-1
(5’-AUUAAUAUCUGAGGUACUUCAUGGG-3’),
SIRT1-2
(5’-AACAGAUACUGAUUACCAUCAAGCC-3’),
SUV39H1-1
(5’-UCUUGUGGCAAAGAAAGCGAUGCGG-3’),
SUV39H1-2
(5’-UUCUUGCGAAUCUUCUCCAGGGUGC-3’). StealthTM RNAi negative control Med GC was used as a negative control. Protein and RNA were extracted at 48 hours after transfection of siRNA. Expression levels of NML, SIRT1 and SUV39H1 were analyzed by immunoblot and the effect of siRNAs on rRNA synthesis was analyzed by RT-qPCR. For transfection of plasmids DNA, cells at 70-80% confluency were transfected with 1 µg total plasmid DNA using 3 µl of PerFectin transfection reagent (Genlantis, San Diego, CA).
RNA Purification and RT-qPCR Total RNA was isolated from cultured cells with Sepasol RNA I Super reagent (Nacalai tesque) according to the manufacturer’s instruction. Total RNA (5 µg) was reverse transcribed using random hexamers and SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Real-time quantitative PCR analysis was performed in 20-µL reactions using the Thermal Cycler DiceTM TP800 (Takara) and Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen).
Primers for 5’ external transcribed spacer of the human
pre-rRNA and human β-Actin mRNA were designed to amplify 100–300-bp amplicons. All the primer sets used gave no signal in the control reactions lacking template. Dissociation-curve analysis showed that single products with the expected Tm values
6
were generated by each primer set.
Relative gene expression was determined by ∆∆CT
method. To normalize values obtained in the samples, control β-Actin CT values were subtracted from the pre-rRNA CT values for each sample (∆CT). Then, ∆CT of the unstimulated sample was subtracted from ∆CT of the stimulated sample (∆∆CT). relative levels of pre-rRNA were calculated as 2–∆∆CT. specific
primers,
For PCR amplification, the
5’-ATCGTCCACCGCAAATGCTTCTA-3’
5’-AGCCATGCCAATCTCATCTTGTT-3’
The
for
and β-Actin,
5’-GAACGGTGGTGTGTCGTTC-3’ and 5’-GCGTCTCGTCTCGTCTCACT-3’ for pre-rRNA were used.
Metabolic Labeling HeLa cells (2 × 105 cells) were labeled for 2 hours with 100 µCi of 35S-methionine in methionine-free DMEM medium (Gibco) supplemented with 10% dialyzed serum and the incorporation of 35S-methionine into protein was determined.
Briefly, protein
was extracted with TNE buffer containing 50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40 and the protein concentration was determined with BCA protein assay kit (Pierce, Rockford, IL, USA). The protein was resuspended in 1% SDS and boiled for 10 min at 100°C.
The radio-activity of the suspensions was
analyzed using a Beckmann Coulter liquid scintillation counter and normalized to the protein content.
SAM Binding Assay Binding of SAM to wild-type or mutant NML was studied by photolabeling of the protein with SAM (Gowher et al., 2006). Recombinant proteins (1 µg each) were
7
incubated with 6 µM of [methyl-3H]-SAM (GE Healthcare, TRK236) for 5 min on ice and spotted onto Parafilm, followed by irradiation with UV (12,000 J/m2).
The
samples were boiled for 3 min in SDS-sample loading buffer and electrophoresed on 12.5% SDS-polyacrylamide gel.
The gel was then soaked in fixing solution
comprising methanol/acetic acid/water (25:10:65) for 20 min followed by Amplify fluorogenic reagent (GE Healthcare biosciences) for 15 min according to the manufacturer’s protocol.
The gel was dried onto Whatman 3MM paper and exposed to
X-ray film at -80°C for 4 days.
Protein Expression and Purification Genes encoding NML (242–456) was subcloned into the expression vector pGEX4T-2 (GE Healthcare). The plasmid was introduced into Escherichia coli strain BL21 (DE3), and in E. coli strain B834 (DE3) RIL to produce SeMet-labeled protein. The cells were grown at 37°C to a cell density of 0.6~0.8 at 660 nm and incubated for a further 12 hours after the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18°C. NML(242-456), and SeMet- labeled NML(242-456) were purified under the same experimental conditions. The cells were harvested, resuspended in lysis buffer [50 mM Tris-HCl buffer (pH 7.8) containing 200 mM NaCl, 1 mM DTT] and disrupted by sonication at 4°C. After centrifugation, the supernatant was applied to a GST affinity column of glutathione-Sepharose 4B (GS4B) (GE Healthcare) equilibrated with lysis buffer. The fusion protein was cleaved by thrombin (Funakoshi) on the column for 1 hour at 4°C.
The cleaved protein was eluted with elution buffer [50 mM Tris-HCl
buffer (pH 7.8) containing 200 mM NaCl, 1 mM DTT].
8
The protein was applied to an
HiTrap SP HP column (GE Healthcare), equipped with an ÄKTA Prime system (GE Healthcare) equilibrated at 4°C with 50 mM Tris-HCl buffer (pH 7.8) containing 200 mM NaCl, 1 mM DTT. The adsorbed fraction was eluted with a linear gradient from 200 mM to 500 mM NaCl at 4°C.
Eluted protein was applied to HiLoad Superdex 75
26/60 gel filtration column (GE Healthcare). confirmed by SDS-PAGE.
Homogeneity of the purified protein was
Purified protein in 20 mM Tris-HCl buffer (pH 7.8)
containing 500 mM NaCl, 1 mM DTT and 1 mM SAH was concentrated at 4°C to ~4 mg/ml in a Centricon-5 concentrator (Amicon). The concentration of the purified protein was determined by UV absorption.
Histone Methyltransferase Assay Bovine thymus histone octamer was purchased from SIGMA (H-9250). Oligonucleosomes were purified from HeLa cells by micrococcal nuclease digestion (Fang et al., 2004). Histone methyltransferase assay was performed at 37°C for 4 hours using 3 µg of histone octamer or oligonucleosomes with 500 ng of GST-NML or GST-PRMT1 in HMT Buffer containing 50 mM Tris-HCl (pH 8.5), 250 mM NaCl, 5 mM DTT, 0.2 mM PMSF, 1 mM MgCl2, 0.1 mM CaCl2 and 0.1 mM ZnCl2, in the presence of 50 µCi/ml [3H]-S-adenosylmethionine (GE Healthcare). SDS-PAGE.
Samples were separated by 17.5%
The gel was stained with Coomassie brilliant blue, followed by
fluorography with Amplify (GE Healthcare), then dried and exposed on BioMAX MS films (Kodak).
Nuclear Run-on Assay
9
cDNAs probes corresponding to 18S rRNA and 28S rRNA of the rRNA were amplified by polymerase chain reaction, cloned into pGEM-T easy vector and spotted onto Hybond-N+ membrane (GE Healthcare). Run-on assay was performed on HeLa cells infected with ad-LacZ or ad-NML. Approximately 107 cells were collected on ice and suspended in lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2 and 0.5% NP-40 for 10 min. Nuclei were collected by centrifugation at 1,000 × g for 5 min, and washed twice with the same buffer.
The nuclei were suspended in 50 mM Tris-HCl (pH 8.3), 40%
glycerol, 5 mM MgCl2 and 0.1 mM EDTA. The nuclei were mixed with an equal volume of Reaction buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, 0.5 mM of ATP, CTP, GTP and 100 µCi of α-32P UTP and incubated for 30 min at 30°C. Nuclear RNA was extracted and resuspended in RapidHyb hybridization buffer (GE Healthcare). Hybridization was carried out at 42°C for 24 hour. Membrane was washed twice at room temperature in 2 ×SSC, 0.1% SDS and twice at 65°C in 0.2 × SSC, 0.1% SDS and then exposed on BioMAX MS film.
ATP Measurement To measure ATP, the ATP Bioluminescence Assay Kit CLS II (Roche) was used. The assay is based on the light-emitting oxidation of luciferin by luciferase in the presence of extremely low levels of ATP. After collecting HeLa cells by scraping, cells were centrifuged for 10 min at 500 g in the cold.
Cell pellets were resolved in
boiling 100 mM Tris buffer containing 4 mM EDTA. Boiling was continued for another 2 min in order to inactivate NTPases. Cell remnants were removed by a further centrifugation step at 1,000 g. Supernatants were separated and placed on ice.
10
Determination of free ATP concentrations was as outlined in the manufacturer’s protocol.
Light emission was measured at 562 nm using a luminometer (Berthold).
ATP levels were normalized to protein content using the BCA Protein assay kit (PIERCE).
Assays for Apoptosis Detection 2×105 of HeLa cells were transfected with siRNA against NML, SIRT1 or SUV39H1. 36 hours after transfection, culture medium was replaced with DMEM containing 1,000 mg/L or 0 mg/L glucose and another 36 hours later both floating (dead) and attached cells were harvested by trypsinization and assayed for DNA fragmentation with DeadEnd fluorometric TUNEL system (Promega). The percentage of TUNEL-positive cells was calculated as (TUNEL-positive cells/total cells) ×100.
Alternatively, to
detect apoptotic cells, PARP-1 cleavage was analyzed by immunoblotting using anti-PARP-1 polyclonal antibody.
Crystallization and Data Collection NML(242-456) was successfully crystallized at 20°C 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 sulfate.
Crystals
of
SeMet-labeled
NML(242-456) were obtained under the same conditions. X-ray diffraction data were collected at 100K on beamline BL41 at Spring8, Japan and on beamline BL5 at The Photon Factory, Japan.
Before the X-ray experiments,
crystals of NML(242-456) and SeMet-labeled NML(242-456) were each soaked in crystallization buffer containing 20% ethylene glycol as a cryoprotectant. Diffraction
11
data were processed with HKL2000 (Otwinowski and Minor, 1997).
The
crystallographic data and data collection statistics of NML(242-456) and SeMet-labeled NML(242-456) are provided in Supplemental Table S1. The native crystal belongs to the hexagonal space group P61, as suggested by the data processing software and confirmed by inspection of pseudo-precession images, while the SeMet-labeled crystal belongs to P6122.
Structure Determination and Refinement The structure of NML(242-456) was solved by multi-wavelength anomalous diffraction (MAD) using the SeMet-labeled NML(242-456) crystal.
Experimental
phases were calculated up to 2.5 Å resolution with SOLVE (Terwilliger and Berendzen, 1999) and improved by solvent-flattening with RESOLVE (Terwilliger, 2001). An initial model was built by ARP/wARP (Perrakis et al., 1997), followed by COOT (Emsley and Cowtan, 2004), and refined with CNS (Brunger et al., 1998). After several cycles of rebuilding with program COOT and refinement with CNS and REFMAC5 (Murshudov et al., 1997), the model finally converged, resulting in a crystallographic R value of 19.2% and a free R value of 23.1% for all diffraction data up to 2.0 Å resolution.
All main chain dihedral angles analyzed by PROCHECK
(Laskowski et al., 1993) fall within allowed regions of the Ramachandran plot. The final refinement statistics is summarized in Supplemental Table S1. generated using PyMol (Delano Scientific).
The figures were
Coordinates and structure factors for
NML(242-456) have been deposited in the Protein Data Bank with accession number 2ZFU.
12
Surface Plasmon Resonance (SPR) Analysis For the SPR analysis, we used the same biotinylated H3 peptides as in the peptide pull-down assay.
SPR studies were performed using an upgraded BIAcore3000
biosensor (GE Healthcare) and streptavidin-coupled SA sensor chips.
In all
experiments, the buffer solution (10 mM TrisHCl (pH 7.2), 250 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 2 mM MgCl2 and 0.1% Nonidet P-40) was used as running buffer. Biotinylated
peptides
were
immobilized
at
the
indicated
levels
over
streptavidin-coupled surfaces. Injection of GST fusion proteins was performed in the buffer solution at a flow rate of 20 µl/min. Surface regeneration was obtained using injection of 1 M NaCl.
GST fusion proteins were dialyzed against the buffer solution
prior to their use. Data were interpreted using BIAevaluation 4.1 software.
13
Supplemental References
Andersen, J.S., Lam, Y.W., Leung, A.K., Ong, S.E., Lyon, C.E., Lamond, A.I., and Mann, M. (2005). Nucleolar proteome dynamics.
Nature 433, 77-83.
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al.
(1998).
Crystallography & NMR system: A new software suite for macromolecular structure determination.
Acta Crystallogr. D. Biol. Crystallogr. 54, 905-921.
Emsley, P., and Cowtan, K. graphics.
(2004).
Coot: model-building tools for molecular
Acta Crystallogr. D. Biol. Crystallogr. 60, 2126-2132.
Fang, J., Wang, H., and Zhang, Y. (2004). Purification of histone methyltransferases from HeLa cells. Methods Enzymol. 377, 213-226.
Gowher, H., Loutchanwoot, P., Vorobjeva, O., Handa, V., Jurkowska, R.Z., Jurkowski, T.P., and Jeltsch, A.
(2006).
Mutational analysis of the catalytic domain of the
murine Dnmt3a DNA-(cytosine C5)-methyltransferase. J. Mol. Biol. 357, 928-941.
Laskowski, R., MacArthur, M., Moss, D., and Thornton, J. (1993). PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283-291.
14
Mendez, J., and Stillman, B.
(2000).
Chromatin association of human origin
recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis.
Mol. Cell Biol. 20,
8602-8612.
Murshudov, G.N., Vagin, A.A., and Dodson, E.J.
(1997).
Refinement of
macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D. Biol. Crystallogr. 53, 240-255.
O'Sullivan, A.C., Sullivan, G.J., and McStay, B. (2002). UBF binding in vivo is not restricted to regulatory sequences within the vertebrate ribosomal DNA repeat.
Mol.
Cell Biol. 22, 657-668.
Otwinowski, Z., and Minor, W.
(1997).
Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 276, 307-326.
Perrakis, A., Sixma, T.K., Wilson, K.S., and Lamzin, V.S.
(1997).
wARP:
improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models. Acta Crystallogr. D. Biol. Crystallogr. 53, 448-455.
Terwilliger, T.C.
(2001).
Maximum-likelihood density modification using pattern
recognition of structural motifs. Acta Crystallogr. D. Biol. Crystallogr. 57, 1755-1762.
15
Terwilliger, T.C., and Berendzen, J. (1999). Automated MAD and MIR structure solution. Acta Crystallogr. D. Biol. Crystallogr. 55, 849-861.
Wysocka, J., Reilly, P.T., and Herr, W. (2001). Loss of HCF-1-chromatin association precedes temperature-induced growth arrest of tsBN67 cells. 3820-3829.
16
Mol. Cell Biol. 21,
Supplementary Table Table S1.
Data Collection and Refinement Statistics
Native
SeMet-labeled Peak
Edge
high remote
Data collection statistics X-ray source
Spring8 BL41
PF BL5
Wavelength (Å)
1.0000
Space group
P61
P6122
a (Å)
43.81
43.91
b (Å)
43.81
43.91
c (Å)
403.91
405.22
Resolution range (Å)
50.00-2.0
Rmerge (%) †‡
7.2 (28.4)
7.7 (17.1)
Average I/σ (I) †
11.0 (3.4)
19.8(10.9) 18.9(9.0)
16.9 (7.3)
No. observations
91682
155951
155702
147301
No. unique reflections
28996
9174
9203
9188
Data completeness (%) †
98.0 (92.0)
99.3 (94.8) 99.4 (95.6) 93.0 (60.6)
0.97912
0.97931
0.96408
Unit-cell parameters
50.00-2.5
Refinement statistics Resolution (Å)
50.0-2.0
Rcryst ¶
0.192
Rfree ¶
0.231
No. atoms
17
8.1 (18.5)
12.0 (23.7)
Protein
3297
SAH
52
Water molecules
283
r. m. s. deviations Bond lengths (Å)
0.009
Bond angles (°)
1.24
†Values in parentheses are for highest resolution shell.
The resolution ranges of their outer
shells are 2.07-2.0 Å for native data and 2.59-2.50 Å for Se derivative data. ‡ Rmerge = Σ| I - < I > | / Σ I; calculated for all data. ¶ Rcryst and Rfree = Σ || Fo | - | Fc || / Σ | Fo | , where the free reflections (5% of the total used) were held aside for Rfree through
18
Figure S1.
Anti-NML Antibody Specifically Recognizes NML Protein
Whole cell lysates from HeLa cells were subjected to immunoprecipitation with control IgG (lane 2) or anti-NML antibody in the absence (lane 3) or presence (lane 4) of 0.1 µg/µl of antigen peptide.
The immunoprecipitates were separated by 10% SDS-PAGE
and immunoblotted with anti-NML antibody. As a loading control, 10 % of whole cell lysate was loaded on the same gel (lane 1).
Figure S2.
N-terminal Domain of NML Interacts with Histone H3 Tail
Dimethylated at Lys9 Association of NML with histone H3 N-terminal tail peptides (1-21a.a.) methylated at Lys9. GST-NML and its deletion derivatives (upper panel) were pulled-down with either H3-, H3K9me1-, H3K9me2- or H3K9me3-peptides-immobilized beads, or control beads (no peptide).
Bound proteins were separated by 10% SDS-PAGE and
immunoblotted with anti-GST antibody (lower panel).
We determined that the
dissociation constant between GST-NML-N and H3K9me2 is 121 nM by surface plasmon resonance analysis.
Figure S3.
Endogenous SIRT1 and NML Associates throughout the rDNA Locus
HeLa cells were subjected to ChIP assay using anti-NML or anti-SIRT1 antibody. qPCR was performed on the immunoprecipitated DNA with H0, H8 and H23 primers and normalized to input DNA. Values are means ± SD for triplicates.
Figure S4.
Downregulation of NML Does Not Affect Levels of SIRT1
HeLa cells were transfected with siRNAs against NML. 48 hours later, the levels of
19
endogenous SIRT1 and NML proteins were examined by Western blotting using anti-SIRT1 and anti-NML antibodies.
Figure S5. SIRT1(H355A) Associates with NML, SUV39H1 and rDNA as well as Wild-type SIRT1 (A) HEK293 cells were transfected with plasmids encoding FLAG-NML and HA-SIRT1(H355A) (left panel), or myc-SUV39H1 and HA-SIRT1(H355A) (right panel). Whole-cell lysates were immunoprecipitated with anti-FLAG (left panel) or anti-myc antibody (right panel). The immunoprecipitates were immunoblotted with anti-HA, anti-myc or anti-FLAG antibody. (B) HEK293 cells were transfected with plasmids encoding HA-SIRT1 or HA-SIRT1(H355A).
ChIP assay
was
performed
using
anti-HA antibody.
Immunoprecipitated DNA was quantified by qPCR using H0, H8, H23 primers and normalized to input DNA. Values are means ± SD for triplicates.
Figure S6. SUV39H1(R235H) Associates with NML, SIRT1 and rDNA as well as Wild-type SUV39H1 (A) HEK293 cells were transfected with plasmids encoding FLAG-NML and myc-SUV39H1(R235H) (left panel), or HA-SIRT1 and myc-SUV39H1(R235H) (right panel). Whole-cell lysates were immunoprecipitated with anti-FLAG (left panel) or anti-myc antibody (right panel). The immunoprecipitates were immunoblotted with anti-HA, anti-myc or anti-FLAG antibody. (B) HEK293 cells were transfected with plasmids encoding myc-SUV39H1 or myc-SUV39H1(R235H).
ChIP assay was performed with anti-myc antibody.
20
Immunoprecipitated DNA was quantified by qPCR using H0, H8, H23 primers and normalized to input DNA. Values are means ± SD for triplicates.
Figure S7.
eNoSC Protects Cells from Cell Death Induced by Glucose
Withdrawal (A) The phase-contrast images of HeLa cells during glucose deprivation. HeLa cells were transfected with indicated siRNAs.
48 hours later, medium was exchanged for
medium containing 1,000 mg/L, 100 mg/L or 0 mg/L glucose.
Cells were
photographed at 48 hours after medium change, using a phase-contrast microscopy without washing. Representative images are shown. (B) Percentage of dead cells after glucose deprivation. 48 hours after transfection with the indicated siRNAs, HeLa cells were cultured in medium with 1,000 mg/L, 300 mg/L, 100 mg/L or 0 mg/L glucose for additional 48 hours. Percentage of dead cells was measured by Trypan blue exclusion assay. Values are means ± SD for triplicates. Figure S8. NML Mutants Do Not Suppress Glucose Deprivation-induced Cell Death HEK293 cells were transfected with plasmids encoding wild-type or mutant NMLs. After 24 hours, we introduced medium containing 1,000 mg/L or 0 mg/L glucose. Cells were photographed at 24 hours after medium change, using a phase-contrast microscopy without washing.
Figure S9.
Representative images are shown.
NML Interacts with neither SIRT6 nor SIRT7
HA-SIRT6 (upper panel) or HA-SIRT7 (lower panel) was co-transfected into HeLa cells in combination with myc-NML plasmid. 24 hours after transfection, whole cell lysates
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were immunoprecipitated with anti-myc agarose beads. The immunoprecipitates were separated by 10% SDS-PAGE and immunoblotted with anti-HA or anti-myc antibody.
Figure S10.
A part of SIRT1 localizes in the nucleolus
Endogenous SIRT1 and UBF were visualized by immunofluorescence in HEK293 cell using anti-SIRT1 and anti-UBF antibodies. The nucleus was stained with DAPI.
Figure S11. NML Does Not Affect Deacetylase Activity of SIRT1 on p53 HEK293 cells were transfected with plasmids encoding p53, SIRT1, CREB-binding Protein (CBP), or NML. After 24 hours, acetylation level of p53 was analyzed by immunoblotting using anti-Ac-Lys382(p53) antibody.
Figure S12.
NML is Required for Association of SUV39H1 with rDNA
(A) Association between SIRT1 and SUV39H1 is not affected by expression of NML. HEK293 cells were transfected with plasmids encoding HA-SIRT1, myc-SUV39H1 in combination with FLAG-NML or NML siRNA. 48 hours after transfection, cells were lysed and immunoprecipitated with anti-myc antibody.
Immunoprecipitates were
resolved by 10% SDS-PAGE and immunoblotted with anti-HA and anti-myc antibodies. (B) NML is required for association of SUV39H1 with rDNA.
HEK293 cells
transfected with a plasmid encoding myc-SUV39H1 in combination with control siRNA (columns 1-3) or NML siRNA (columns 4-6). 48 hours after transfection, the cells were subjected to ChIP analysis using anti-myc and anti-NML antibodies. Immunoprecipitated DNA was quantified with qPCR using H0 primers. Values are means ± SD for triplicates.
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Figure S13. Glucose Deprivation Enhances Association between SIRT1 and SUV39H1 (A) NML directly binds to SUV39H1 but not to SIRT1.
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S-labeled, in vitro translated
SUV39H1 and SIRT1 were pulled down with GST or GST-NML beads.
Bound
proteins were separated with 10% SDS-PAGE and analyzed by autoradiography. (B-D) Glucose deprivation enhances association of SIRT1 with both NML and SUV39H1.
HEK293 cells were transfected with plasmids encoding HA-SIRT1,
HA-SIRT1(H355A), FLAG-NML and myc-SUV39H1 as indicated.
24 hours after
transfection, cells were cultured in the presence (1,000 mg/L) or absence (0 mg/L) of glucose for an additional 16 hours. Associations between these two proteins were analyzed by immuprecipitation followed by immunoblotting with indicated antibodies.
Figure S14.
Recombinant NML Dose Not Methylate Histones In Vitro
Histone methyltransferase activities of the indicated recombinant proteins, which were expressed and purified from E. coli, were assayed using 3 µg of core histones or oligonucleosomes as substrates.
Representative autoradiograms (upper) and CBB
stainings (lower) are shown.
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