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STRUCTUREFUNCTION ANALYSIS OF TRANSCRIPTIONALLY REPRESSIVE

By Barbara Hill,2014-11-26 12:34
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STRUCTUREFUNCTION ANALYSIS OF TRANSCRIPTIONALLY REPRESSIVE

SUPPLEMENTARY INFORMATION

METHODS

Media and growth conditions

    1Yeast strains were grown using standard conditions . Media containing 5-

    fluoroorotic acid (5-FOA, Toronto Research Chemicals Inc.) were prepared according to

    2the recipe described by Boeke et al . For colony color assay, cells were spotted on a

    1synthetic complete medium containing 7 mg l 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

    3different reporter genes used in this study was assessed by a spot assay . 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

    1synthetic complete medium containing 50 µg l 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

    -1-1concentration of 1g l and uracil at a final concentration of 50 µg l were added to one

    -1part, uracil at a final concentration of 50 µg l 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

    4haploid cells . 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,

    5integrated at the LYS2 locus, to monitor silencing at an internal chromosomal site . 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 tetR-

    FLAG-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 W303-1a. 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

    6and a crippled HMR-E silencer . In strain YSB2, the binding sites for the proteins ORC

    and Rap1p have been replaced by three binding sites for the protein Gal4p (UAS). YSB1 G

    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

    7GFP-Nup145C plasmid 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

    8fragment of pRS426 . 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

    9sequencing. A SalI-SpeI fragment of plasmid pFa6a-kanMX6 (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)

    10fusion as described 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

    8HindIII and SmaI sites of the integrative plasmid pRS305 . 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 CCVCsCCVCas 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

    11,12a Cys-Xaa-Cys motif at the C-terminus are geranylgeranylated , 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 two-

    hybrid yeast library which has been generated into Y187 strain was screened by the mating

    13strategy using the yeast strain CG1945 transformed with pAS2??Nup60 as bait. Thirty-

    three 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

    14diploid cells were obtained as described . Yeast telomeres were probed with a mixture of

    two plasmids containing a conserved core fragment of the subtelomeric X and Y? element,

    14,15respectively . The plasmid p306tetO112 was used to probe the inserted genomic tetO-repeat-fragment. The telomere-probe was labeled with dig-11-dUTP and the tetO-probe

    14was labeled with biotin-14-dCTP as previously described . All preparations were

    14,16subjected to two-color FISH as described . 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

    16digoxigenin detection as described . Prior to microscopic inspection, preparations were

    -1embedded in antifade solution (Vector labs, Burlingame) containing 0.5 µg ml 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

    17a clear and distinctive characterization of Sir3p-GFP foci and nuclei . 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 Genotype Reference BMA64-1a Mat a, leu2-3,112, his3-11,15, trp1?2, can1-100, ade2-1, ura3-52

    18 YVG1 BMA64 mlp1::HIS5

    18 YVG3 BMA64 mlp2::HIS5

    18 YVG12 BMA64 mlp1::HIS5, mlp2::HIS5

    YVG246 BMA64 nup60::TRP1 This study

    18 YVG31 BMA64 MLP1-GFP::HIS5

    18 YVG36 BMA64 MLP2-GFP::HIS5

    YVG265 YVG31 nup60::TRP1 This study YVG265b YVG36 nup60::TRP1 This study YVG267 BMA64 NUP60-GFP::HIS5 This study YVG269 YVG267 nup145?606-1341-9-myc::TRP1 This study BM64 Mat /Mat a, leu2-3,112/leu2-3,112, his3-11,15/ his3-11,15, trp1?2/trp1?2, can1-100/can1-

    100, ade2-1/ade2-1, ura3-52/ura3-52

    YVG227 BM64 GFP-?1-234RAP1::TRP1/GFP-?1-234RAP1::TRP1 This study YVG228 BM64 GFP-?1-234RAP1::TRP1/GFP-?1-234RAP1::TRP1, nup145?606-1341-This study

    protA::HIS5/ nup145?606-1341-protA::HIS5

    YVG258 BMA64 nup60::TRP1/ nup60::TRP1 This study YSB2 6 Mat ?;;HML?;;ade2-1, ura3-1, his3-11,15, leu2-3,112, trp1-1, can1-100, aeB::3UASg hmr::TRP1, gal4::LEU2 LeFFe47 YSB2 mlp1::kanMX6 This study LeFFe48 YSB2 mlp2::kanMX6 This study LeFFe50 YSB2 mlp1::HIS5, mlp2::kanMX6 This study LeFFe51 YSB2 yku70::HIS5 This study LeFFe52 YSB2 nup60::HIS5 This study LeFFe54 YSB2 sir4::HIS5 This study LM11 5 Mat , ade2-1, can1-100, his3-11,15, leu2-3,112, trp1-1, ura31, Tel-VII::URA3, Llys2::HML-E-ADE2-HML-I LeFFe124 LM11 mlp1::kanMX6 This study LeFFe125 LM11 mlp2::TRP1 This study LeFFe128 LM11 mlp1::kanMX6, mlp2::TRP1 This study LeFFe129 LM11 nup60::TRP1 This study LeFFe130 LM11 nup145?606-1341-protA::TRP1 This study LeFFe135 LM11 sir4::HIS5 This study LeFFe217 LM11 yku70::kanMX6 This study LeFFe141 LM11 SIR3-GFP::HIS5, nup60::TRP1 This study LeFFe142 LM11 SIR3-GFP::HIS5, nup145?606-1341-protA::TRP1 This study LeFFe143 LM11 SIR3-GFP::TRP1, sir4::HIS5 This study LeFFe146 LM11 SIR3-GFP::TRP1 This study LeFFe149 LM11 SIR3-GFP::TRP1, mlp1::HIS5 This study LeFFe150 LM11 SIR3-GFP::TRP1, mlp2::HIS5 This study LeFFe151 LM11 SIR3-GFP::TRP1, mlp2::HIS5, mlp1::kanMX6 This study LeFFe218 LM11 SIR3-GFP::TRP1, yku70::HIS5 This study LeFFe225 This study Mat /Mat a, leu2-3,112/leu2-3,112, his3-11,15/his3-11,15, trp1-1/trp1-1, can1-100/can1-100, ade2-1/ade2-1, ura3-1/ura3-1, mlp1::kanMX6/mlp1::HIS5 LeFFe226 This study Mat /Mat a, leu2-3,112/leu2-3,112, his3-11,15/his3-11,15, trp1-1/trp1-1, can1-100/can1-100, ade2-1/ade2-1, ura3-1/ura3-1, mlp2::TRP1/mlp2::HIS5 W303-1a Mat a, leu2-3,112, his3-11,15, trp1-1, can1-100, ade2-1, ura3-1 19 LeFFe198 This study Mat , leu2::TetR-GFP, his3-11,15, trp1-1, can1-100, ade2-1, ura3-1, Tel-VII::URA3-L(TetO*112)-kanMX6-URA3, lys2::HML-E-ADE2-HML-I LeFFe200 This study 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-VII::URA3-(TetO*112)-kanMX6-LURA3/Tel-VII, lys2::HML-E-ADE2-HML-I/LYS2 LLeFFe288 This study 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-VII::URA3/Tel-VII, lys2::HML-E-LLADE2-HML-I/LYS2

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GF1 W303-1a Tel-VII::URA3 20 L

LeFFe196 W303-1a Tel-VII::URA3-(TetO*112)-TRP1-URA3 This study LLeFFe219 LeFFe196 leu2::pGAL1-TetRCCVC-LEU2 This study LeFFe221 LeFFe219 mlp2::kanMX6 This study LeFFe222 LeFFe219 nup60::HIS5 This study LeFFe223 LeFFe219 sir4::HIS5 This study LeFFe227 LeFFe219 mlp1::HIS5 This study LeFFe228 LeFFe219 yku70::HIS5 This study LeFFe230 LeFFe219 mlp2::kanMX6, mlp1::HIS5 This study

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