Download Supplementary Methods

SUPPLEMENTARY INFORMATION
METHODS
Media and growth conditions
Yeast strains were grown using standard conditions 1. Media containing 5fluoroorotic acid (5-FOA, Toronto Research Chemicals Inc.) were prepared according to
the recipe described by Boeke et al 2. For colony color assay, cells were spotted on a
synthetic complete medium containing 7 mg l –1 of adenine, grown for 3 days at 30°C, and
stored at 4°C for at least two weeks to allow the red color to develop. Expression of the
different reporter genes used in this study was assessed by a spot assay 3. For all the spot
assays, cells were rinsed twice with sterile water before being spotted on the different
media. For the colony color assay and the anchoring of the subtelomeric reporter, cells were
pre-grown in rich medium (YPD). For the anchoring of the TRP1 gene inserted at the HMR
locus or the TPE assay, cells were pre-grown in synthetic medium lacking uracil. For
analysis of the localization of the tetO-tagged telomere (see below), cells were grown in
synthetic complete medium containing 50 µg l –1 uracil. Cells from an early log-phase
culture were collected by centrifugation rinsed twice with water and resuspended in an
uracil-free medium. The resuspended cells were divided in three parts. 5-FOA at a final
concentration of 1g l -1 and uracil at a final concentration of 50 µg l -1 were added to one
part, uracil at a final concentration of 50 µg l -1 was added to a second part, the last one
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being unchanged. Cells were then grown for 2 more hours and prepared for fluorescent in
situ hybridization (see below).
Strains
Genotypes of the strains used in this study are described in Table 1. Gene deletion
or GFP-tagging were performed by homologous recombination with PCR products in
haploid cells 4. Sequences of the oligonucleotides used for the recombination are given in
Table 2. Oligonucleotides 122+123, 124+125, 152+153, 353+354 and 369+370 were used
to delete MLP1, MLP2, NUP60, SIR4 and YKU70 respectively. The Nup145∆606-1341
mutant (nup145∆Cter) was generated by in-frame insertion of the protein A or the GFP
coding sequence followed by a stop codon through homologous recombination in the
genomic copy at amino acid position 606. The PCR product used was generated using
oligonucleotides 113 and 114 (Table 2). GFP-tagged proteins were generated in the same
way as the nup145∆Cter mutant except that primers were designed to integrate the GFP
coding sequence just upstream of the stop codon of the corresponding ORF. For Mlp1p,
Mlp2p, Nup60p and Sir3p tagging, oligonucleotides 29+92, 28+90, 153+154 and 387+388
were used to PCR amplify the GFP coding sequence and an auxotrophy marker (HIS5 from
Schizosaccharomyces pombe or TRP1 from Kluyveromyces lactis). Correct recombination
was checked by PCR and when possible by fluorescence light microscopy.
The LM11 strain carries both a telomere proximal (left end of chromosome VII)
URA3 reporter and a wild-type ADE2 gene flanked by the HML E and I silencers,
integrated at the LYS2 locus, to monitor silencing at an internal chromosomal site 5. Strain
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GF1 (kindly provided by G. Fourel, ENS, Lyon, France) carries a telomere proximal (left
end of chromosome VII) URA3 reporter. Strains LeFFe196, LeFFe219 and LeFFe198,
derivatives of GF1 and LM11 respectively, carry 112 copies of the tetO operator bracketed
by two copies of the URA3 gene inserted at the left end of chromosome VII. LeFFe198 also
carries a tet repressor (tetR) GFP fusion protein under the control of the URA3 promotor
(see below plasmids section) inserted at the LEU2 locus whereas LeFFe219 has a tetRFLAG-CCVC fusion protein under the control of the GAL1 promotor (see below) inserted
at the LEU2 locus. LeFFe200 is a diploid strain obtained by mating LeFFe198 with W3031a. LeFFe288 is a diploid strain obtained by mating LM11 with a W303-1a strain carrying
the tetR-GFP fusion protein under the control of the URA3 promotor.
The YSB2 and YSB1 strains (kind gift of D. Zappulla and R. Sternglanz, Stony
Brook University, New-York, USA) have a TRP1 reporter gene inserted at the HMR locus
and a crippled HMR-E silencer 6. In strain YSB2, the binding sites for the proteins ORC
and Rap1p have been replaced by three binding sites for the protein Gal4p (UASG). YSB1
has a similar silencer to the one in YSB2 but has no Gal4p binding sites.
Plasmids
To localize Nup145p in nup60∆ strain (YVG246), cells were transformed with the
GFP-Nup145C plasmid 7 kindly provided by E. Fabre (Institut Pasteur, Paris).
A PvuII fragment of pAS2∆∆ containing the ADH1 promotor, the Gal4p DNA
binding domain coding sequence and the ADH1 terminator was used to replace the PvuII
fragment of pRS426 8. The resulting plasmid was named p12201. The YIP1 coding
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sequence was PCR amplified with Pfu DNA polymerase (Stratagene) and oligonucleotides
mYIP1-29+ (5'-AAGGATCCAGATGTCTTTCTACAATACTAGTAACAATG-3') and
mYIP1-749- (5'- ATCTGCAGATATCGCCCCTAAGCCAATTCCC-3'). The PCR product
was digested with BamHI and cloned into the BamHI and SalI (previously blunted) sites of
p12201 to create plasmid p13123. The absence of mutations in p13123 was checked by
sequencing. A SalI-SpeI fragment of plasmid pFa6a-kanMX6 9 (kind gift of E. Fabre,
Institut Pasteur, Paris) was sub-cloned into the SalI and SpeI site of the p306tetO112
plasmid containing 112 tandem repeats of the tetO operator (kindly provided by K.
Nasmyth, University of Vienna, Austria) to create plasmid p13903. The TRP1 gene from
plasmid Yrp7 was excised by digestion with BglII and EcoRI and, following treatment with
the Klenow fragment of Escherichia coli DNA polymerase I, cloned into the blunted SalI
and SpeI sites of p306tetO112 to create plasmid p14019. Plasmids p13903 linearized with
StuI and p14019 linearized with NcoI were integrated into the subtelomeric URA3 gene of
strains LM11 and GF1 respectively. Correct integration was checked by Southern blot
analysis. Visualization of the tet operators inserted near the left end of telomere VII, in
strains LeFFe198 and LeFFe200, was achieved by using the tet repressor-GFP (tetR-GFP)
fusion as described 10 or fluorescent in situ hybridization (FISH) using a probe
corresponding to the tetO operator (see below). The promotor of the GAL1 gene of S.
cerevisiae was PCR amplified using Pfu DNA polymerase (Stratagene) and
oligonucleotides gal320+ (5'-AAGAAGCTTGGAACTTCAGTAATACG-3') and gal725(5'-GAATAAGAAGTAATACAAACCGA-3'), digested with HindIII and cloned into the
HindIII and SmaI sites of the integrative plasmid pRS305 8. The terminator of the PGK1
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gene of S. cerevisiae, PCR amplified with oligonucleotides PGKterm1 (5'AACCGCGGAAATTGAATTGAATTGAA-3') and PGKterm2 (5'AAGAGCTCGCAGAATTTTCGAGTTAT-3') was inserted into the SstI and SstII sites of
the resulting plasmid to create p14201. The oligonucleotide CCVCs (5'CGATTACAAGGACGACGATGACAAGTGTTGCGTTTGTTAA-3') was mixed to
equimolarity with the oligonucleotide CCVCas (5'CTAGTTAACAAACGCAACACTTGTCATCGTCGTCCTTGTAAT-3'). The mixture
was boiled for 5 min and left for 20 min at room temperature to allow annealing of the two
oligonucleotides. This allow the creation of a synthetic double stranded linker coding for
the FLAG peptide followed by a CCVC motif and a stop codon with a protruding 5'-CG-3'
at the 5' end and a protruding 5'-CTAG-3' at the 3' end. The tet repressor coding sequence
with a SV40 NLS was PCR amplified with Pfu DNA polymerase (Stratagene) and
oligonucleotides tetR27+ (5'-AAGGATCCAAAAATTAGGAATTAATGATG-3') and
tetR625Cla- (5'-TGATCGATAGACCCACTTTCACATTTAAG-3') using the tetR-GFP
plasmid provided by K. Nasmyth as a matrix. The resulting PCR product digested with
BamHI and ClaI and the CCVCs-CCVCas hybrid were both ligated with p14201 digested
with BamHI and XbaI to create plasmid p15201. This plasmid linearized with EcoRV was
integrated into the LEU2 locus. The construct was checked by sequencing. When
integrated, p15201 should allow for Gal-dependent expression of a tet repressor protein
containing a FLAG peptide and a Cys-Cys-Val-Cys tetrapeptide at its C terminus. Since
proteins bearing a Cys-Xaa-Cys motif at the C-terminus are geranylgeranylated 11,12, this
motif was added to target the tetR-protein to the nuclear envelope.
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Two-hybrid plasmid and screen
A PCR-amplified fragment containing the NUP60 coding sequence was inserted
into the BamHI and PstI sites of plasmid pAS2∆∆ to create the Gal4-Nup60p bait plasmid
designated pAS2∆∆Nup60. The construct was checked by sequencing. The FRYL1 twohybrid yeast library which has been generated into Y187 strain was screened by the mating
strategy 13 using the yeast strain CG1945 transformed with pAS2∆∆Nup60 as bait. Thirtythree million diploids were analyzed. Twenty six diploids His+, LacZ+ were selected and
their inserts were identified by sequence.
Epifluorescence microscopy and fluorescent in situ hybridization
Exponentially growing cells with GFP-tagged protein were examined using a Leica
DMRXA fluorescence microscope and images acquisition was done with an Hamamatsu
C4742-95 cooled CCD camera controlled by the Openlab® software (version 2.2.4,
Improvision). For fluorescent in situ hybridization (FISH), structurally preserved nuclei of
diploid cells were obtained as described 14. Yeast telomeres were probed with a mixture of
two plasmids containing a conserved core fragment of the subtelomeric X and Y´ element,
respectively 14,15. The plasmid p306tetO112 was used to probe the inserted genomic tetOrepeat-fragment. The telomere-probe was labeled with dig-11-dUTP and the tetO-probe
was labeled with biotin-14-dCTP as previously described 14. All preparations were
subjected to two-color FISH as described 14,16. The hybridization solution contained the
differentially labeled pan-telomere probe to delineate all telomeres and the tetO-repeat
probe for visualization of the integrated genomic tetO-repeats. Immunofluorescent
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detection of hybrid molecules was carried out using Avidin-FITC (Sigma) for detection of
biotin and rhodamine-conjugated sheep-anti-dig Fab fragments (Roche Biochemicals) for
digoxigenin detection as described 16. Prior to microscopic inspection, preparations were
embedded in antifade solution (Vector labs, Burlingame) containing 0.5 µg ml -1 DAPI (4’6-diamidino-2-phenylindole) as DNA-specific counterstain. Preparations were evaluated
using a Zeiss Axioskop epifluorescence microscope equipped with a double-band-pass
filter for simultaneous excitation of red and green fluorescence, and single band pass filters
for excitation of red, green and blue (Chroma Technologies, Battleborough). Digital images
were obtained using a cooled gray-scale CCD camera (Hamamatsu, Herrsching) controlled
by the ISIS fluorescence image analysis system (MetaSystems, Altlussheim). Localization
of both the tetO and the telomere FISH signals was analyzed in more than 100 successfully
hybridized nuclei for each experiment.
Quantitative image analysis
To assess the spatial distribution of Sir3p-GFP foci in nuclei, we developed a
quantitation method that automatically counts the number of Sir3p-GFP foci and measures
their distance to the nuclear envelope. It is based on the multiscale product of subband
images resulting from an undecimated wavelet transform decomposition of the original
image, after thresholding of non-significant coefficients. The multiscale correlation of the
filtered wavelet coefficients, which allows to enhance multiscale peaks due to objects while
reducing noise, combines information coming from different levels of resolution and gives
a clear and distinctive characterization of Sir3p-GFP foci and nuclei 17. For each nucleus, a
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distance map is computed that gives for each point within a connected nucleus its distance
to the closest border. This information is subsequently used to determine the distance d of
each Sir3p-GFP focus to its containing nuclear envelope. The values obtained for d were
accumulated from over 150 cells and grouped into distance categories of 1 pixel step,
where category 1 corresponds to the outer location.
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Table 1 : Yeast strains used in this study
Name
BMA64-1a
YVG1
YVG3
YVG12
YVG246
YVG31
YVG36
YVG265
YVG265b
YVG267
YVG269
BM64
YVG227
YVG228
YVG258
YSB2
LeFFe47
LeFFe48
LeFFe50
LeFFe51
LeFFe52
LeFFe54
LM11
LeFFe124
LeFFe125
LeFFe128
LeFFe129
LeFFe130
LeFFe135
LeFFe217
LeFFe141
LeFFe142
LeFFe143
LeFFe146
LeFFe149
LeFFe150
LeFFe151
LeFFe218
LeFFe225
LeFFe226
W303-1a
LeFFe198
LeFFe200
LeFFe288
Genotype
Mat a, leu2-3,112, his3-11,15, trp1∆2, can1-100, ade2-1, ura3-52
BMA64 mlp1::HIS5
BMA64 mlp2::HIS5
BMA64 mlp1::HIS5, mlp2::HIS5
BMA64 nup60::TRP1
BMA64 MLP1-GFP::HIS5
BMA64 MLP2-GFP::HIS5
YVG31 nup60::TRP1
YVG36 nup60::TRP1
BMA64 NUP60-GFP::HIS5
YVG267 nup145∆606-1341-9-myc::TRP1
Mat /Mat a, leu2-3,112/leu2-3,112, his3-11,15/ his3-11,15, trp1∆2/trp1∆2, can1-100/can1100, ade2-1/ade2-1, ura3-52/ura3-52
BM64 GFP-∆1-234RAP1::TRP1/GFP-∆1-234RAP1::TRP1
BM64 GFP-∆1-234RAP1::TRP1/GFP-∆1-234RAP1::TRP1, nup145∆606-1341protA::HIS5/ nup145∆606-1341-protA::HIS5
BMA64 nup60::TRP1/ nup60::TRP1
Mat HMLade2-1, ura3-1, his3-11,15, leu2-3,112, trp1-1, can1-100, aeB::3UASg
hmr::TRP1, gal4::LEU2
YSB2 mlp1::kanMX6
YSB2 mlp2::kanMX6
YSB2 mlp1::HIS5, mlp2::kanMX6
YSB2 yku70::HIS5
YSB2 nup60::HIS5
YSB2 sir4::HIS5
Mat , ade2-1, can1-100, his3-11,15, leu2-3,112, trp1-1, ura3–1, Tel-VIIL::URA3,
lys2::HML-E-ADE2-HML-I
LM11 mlp1::kanMX6
LM11 mlp2::TRP1
LM11 mlp1::kanMX6, mlp2::TRP1
LM11 nup60::TRP1
LM11 nup145∆606-1341-protA::TRP1
LM11 sir4::HIS5
LM11 yku70::kanMX6
LM11 SIR3-GFP::HIS5, nup60::TRP1
LM11 SIR3-GFP::HIS5, nup145∆606-1341-protA::TRP1
LM11 SIR3-GFP::TRP1, sir4::HIS5
LM11 SIR3-GFP::TRP1
LM11 SIR3-GFP::TRP1, mlp1::HIS5
LM11 SIR3-GFP::TRP1, mlp2::HIS5
LM11 SIR3-GFP::TRP1, mlp2::HIS5, mlp1::kanMX6
LM11 SIR3-GFP::TRP1, yku70::HIS5
Mat /Mat a, leu2-3,112/leu2-3,112, his3-11,15/his3-11,15, trp1-1/trp1-1, can1-100/can1100, ade2-1/ade2-1, ura3-1/ura3-1, mlp1::kanMX6/mlp1::HIS5
Mat /Mat a, leu2-3,112/leu2-3,112, his3-11,15/his3-11,15, trp1-1/trp1-1, can1-100/can1100, ade2-1/ade2-1, ura3-1/ura3-1, mlp2::TRP1/mlp2::HIS5
Mat a, leu2-3,112, his3-11,15, trp1-1, can1-100, ade2-1, ura3-1
Mat , leu2::TetR-GFP, his3-11,15, trp1-1, can1-100, ade2-1, ura3-1, Tel-VIIL::URA3(TetO*112)-kanMX6-URA3, lys2::HML-E-ADE2-HML-I
Mat /Mat a, leu2-3,112/ leu2::TetR-GFP-LEU2, his3-11,15/his3-11,15, trp1-1/trp1-1,
can1-100/can1-100, ade2-1/ade2-1, ura3-1/ura3-1, Tel-VIIL::URA3-(TetO*112)-kanMX6URA3/Tel-VIIL, lys2::HML-E-ADE2-HML-I/LYS2
Mat /Mat a, leu2-3,112/ leu2::TetR-GFP-LEU2, his3-11,15/his3-11,15, trp1-1/trp1-1,
can1-100/can1-100, ade2-1/ade2-1, ura3-1/ura3-1, Tel-VIIL::URA3/Tel-VIIL, lys2::HML-EADE2-HML-I/LYS2
Reference
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GF1
LeFFe196
LeFFe219
LeFFe221
LeFFe222
LeFFe223
LeFFe227
LeFFe228
LeFFe230
W303-1a Tel-VIIL::URA3
W303-1a Tel-VIIL::URA3-(TetO*112)-TRP1-URA3
LeFFe196 leu2::pGAL1-TetR–CCVC-LEU2
LeFFe219 mlp2::kanMX6
LeFFe219 nup60::HIS5
LeFFe219 sir4::HIS5
LeFFe219 mlp1::HIS5
LeFFe219 yku70::HIS5
LeFFe219 mlp2::kanMX6, mlp1::HIS5
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Table 2 : Oligonucleotide used to knock out genes or to create a GFP fusion.
Name
Sequence (5'-3')
28
90
92
113
GAGAGCGGTACATCTTCTGATCCAGACACCAAAAAGGTTAAAGAGAGTCCAGCAAATGATCAAGCTTCCAACGAGattgaaggtagaggtgaagctcaaaa
acttatt
AATGAGTCAAAAAAGATCAAGACTGAAGATGAGGAAGAAAAAGAAACCGATAAGGTGAATGACGAGAACAGTATAattgaaggtagaggtgaagctcaa
aaacttatt
GACATTAGTGACATTTAAAATATGTAGATGTTTCATATTTATATAATTACATTGTTTAATATTACAgtcgacggtatcgataagctt
TAGGGCAGAATGAAGCTCCTCCACATTGAAAAAGGTTTAGTTTGTATTGATCCCTTGTTTTTACTAgtcgacggtatcgataagctt
AGGGAAATGAACTATATATCCTATAATCCCTTTGGCGGGACTTGGACTTTCAAAGTCAATCATTTTattgaaggtagaggtgaagctcaaaaacttatt
114
TTTGACATGGCCCGTCAAATTCTTTAGAAGTGGCAAACACAGAACGTAGTGAAGACCCAGCTAAAATTAATTGgtcgacggtatcgataagctt
122
TAACATTATATCAGGGTGAATATTACTGACAAAAATAATAACTTAAGTCTTCTTTATAATATGATGggaaacagctatgaccatg
123
124
TAGGGCAGAATGAAGCTCCTCCACATTGAAAAAGGTTTAGTTTGTATTGATCCCTTGTTTTTACTAgtaaaacgacggccagt
AGTGGAAGTTTACCAAAAGAAATTTAAGGCGAAAGAACACTGGGCGGAAGCAAACCGGCAggaaacagctatgaccatg
125
152
GACATTAGTGACATTTAAAATATGTAGATGTTTCATATTTATATAATTACATTGTTTAATATTACAgtaaaacgacggccagt
TGTGTCATTTTAAACATCAAATAACAGACCTTTACATCAAATAAGCACCGCAAGATATCCTAAAATCGACATCCAacccatacgatgttcctgactatg
153
154
AATAATATCATCTTGGAATGGTATTTTACACAAACTTACGTATTGAGTTGGGCTATACGGTAATTATGTCACGGCgtcgacggtatcgataagctt
GTGTCTAAGCAGCTAGGAAATGGCTTGGTTGATGAAAATAAAGTTGAGGCTTTCAAGTCCCTATATACCTTTattgaaggtagaggtgaagctcaaaaac
353
354
CGCTTTTCAGGTTTTATAATTCAGTCAGAGTTCTAACTGGACATCGTTTTGCAGGGGATAAAAAAAAAAAGGAgtaaaacgacggccagt
ACTAATCATTAAAATCGATTAAAGACCGTAAAGTAAATTGTTCATTATAGAAGAAAACGACAAAGAAAAACggaaacagctatgaccatg
369
370
387
ACTGTTCTAGTTTTCAACAGTAAAGCTATGATTTGTTAAGTGACTCTAAGCCTGATTTTAAAACGGGAATATTgtaaaacgacggccagt
CGTGCATAAATATCTTGCTAATAGTTGTACAGTACAACGTTTAGCACGACAAAAGTTCTTAATAATAAATAggaaacagctatgaccatg
GTAGAACTAAAACTTCCTTTAGAAATAAATTACGCCTTTTCGATGGATGAAGAATTCAAAAATATGGACTGCATTattgaaggtagaggtgaagctc
388
ATTAAGGAATACAGAAGAGACTGCATGTGTACATAGGCATATCTATGGCGGAAGTGAAAATGAATGTTGGTGGgtcgacggtatcgataagctt
29
Nota : Sequence in upper case letters correspond to the region homologous to the gene,
sequence in lower case letters correspond to the region homologous to the plasmid used as
a matrix
11
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