Material Bacterial strains

DH5α

F^-Φ80lacZΔM15 Δ(lacZYAargF^-)U169 deoR recA1 endA1 hsdR17 (r_k ^-, m_k) phoA supE44 λ^- thi-1 gyrA96 relA1 tonA (Chemically competent: Gibco BRL)

TOP10

F^- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Str^R) endA1 nupG

(Chemically competent: Invitrogen)

BL21(DE3) Star

F^- ompT hsdS_B (r_B ^- m_B ^-) gal dcm mel31 (DE3)
(Chemically competent: Invitrogen)

Yeast strains

Strain

Genotype

Source

AEY1

MATαade2-101 his3-11, 15 trp1-1 leu2-3, 112 ura3-1

J. Rine

AEY2

MAT a ade2-101 his3-11, 15 trp1-1 leu2-3, 112 ura3-1

J. Rine

AEY2661

AEY1 cse4Δ::kanMX ADE2-1 lys2 + pRS313-3xHA-CSE4

AEY2666

AEY1cse4Δ::kanMX sas2Δ::TRP1 ADE2 lys2 + pRS313-3xHA-CSE4

AEY2781

AEY2661 but pRS426-3xHA-CSE4

AEY2782

AEY2666 but pRS426-3xHA-CSE4

AEY1162

MATαcse4-103 his3 Δ 200 ura3-52 leu2, 112 trp1 Δ 1 pPY13 (CEN/ARS/TRP1/cse4::LEU2)

L. Glowczewski

AEY1781

AEY1162 sas2 Δ ::HIS3

AEY2373

AEY1162 cac1 Δ ::kanMX

AEY2374

AEY1162 cac1 Δ ::kanMX sas2 Δ ::HIS3

AEY1194

MATαcse4 Δ ::LEU2 his3 Δ 200 ura3-52 leu2, 112 trp1 Δ 1 + pPY13 (CEN/ARS/TRP1/CSE4)

L. Glowczewski

AEY1558

MAT a leu2 trp1 ura3-52 prc1-407 pep4-3 prb1-112

E.W. Jones

AEY1559

AEY1558 sas2 Δ ::TRP1

AEY1808

AEY1558 cac1∆::kanMX

AEY2461

AEY1558 sas4∆::kanMX

AEY2463

AEY1558CAC3-9myc::TRP1

AEY2493

AEY1558 ASF1-4HA::TRP1

L40c

MAT a his 3 Δ 200 trp1-901 leu2-3. 112 ade2 lys2-801 am LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ GAL4

E. Wanker

AEY1695

L40c sas2 Δ ::HIS3

Strains between horizontal lines are isogenic

Plasmids

Strain

Genotype

Source

pAE90

URA3, 2μ, GPD-SAS2-PGK

pAE249

pRS315-SAS2-HAT ^-

pAE388

pRS316-SAS2-Zn ^-

pAE431

pRS315-sas2Δ::HIS3

pAE436

pBTM117c- SAS2

pAE439

pACT2-SAS2

pAE451

pBTM117c-SAS4

pAE454

pBTM117c-CAC1

pAE465

pACT2-CSE4 (aa 28-229)

pAE493

pBTM117c-CSE4 (aa 93-229)

pAE535

pACT2-ASF1

pAE613

pRS424-6x-myc-SAS4

pAE614

pRS426-6x-myc-CAC1

pAE625

pRS426-HA-SAS5

pAE686

pACT2-N-term.-CSE4 (aa 11-139)

pAE687

pACT2-C-term.-CSE4 (aa 137-229)

pAE688

pBTM117c-N-term.-CSE4 (aa 11-139)

pAE689

pBTM117c-C-term.-CSE4 (aa 137-229)

pAE690

pBTM117c-SAS5

pAE794

pBTM117c-CSE4 (full length)

pAE817

pBTM117c-SAS2-HAT ^-

pAE818

pBTM117c-SAS2-Zn ^-

pAE820

pRS424-6x myc-CAC2

pAE872

pACT2-SAS4

pAE901

pRS426-6x myc-CSE4

pAE956

pBTM117c-CTF19

pAE974

pRS423-3x HA-CSE4

pAE975

pRS424-6x myc-CSE4

pAE976

pRS425-3x HA-CSE4

pAE977

pRS426-3x HA-CSE4

pAE994

pET15b-CSE4

pET15b

lacI, MCS, HIS-tag

Novagen

pUN60

CEN/ARS, URA3, SUP11

ATCC

pCR^®-Blunt II-TOPO

MCS, TOPO-cloning site

Invitrogen

pACT2

LEU2, MCS, GAL4-AD

Elledge, 1988

pBTM117c

TRP1, MCS, LexA

E. Wanker

Media

LB

10 g/l Caseinpepton, 5 g/l Yeast extract, 5 g/l NaCl, pH 7,2 (Lennox, 1955)

TY

16 g/l Trypton, 10 g/l Yeast extract, 5 g/l NaCl

SOC

2 g/l Trypton, 500 mg/l Yeast extract, 10 mM NaCl, 2-5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM Glucose

YM

6,7 g/l Yeast Nitrogen Base w/o amino acids, 2 g/l Glucose

YPD

10 g/l Yeast extract, 20 g/l Peptone, 2 g/l Glucose

FOA

14 g/L Yeast Nitrogen Base w/o amino acids, 4 g/l Glucose, 2 g/l FOA, 40 mg/l Uracil

Sporulation medium

19 g/l KAc, 0,675 mM ZnAc

Buffers and Solutions

TAE

4.84 g/l Tris/HCl, 0.744 g/l EDTA, 1.142 mg/l Acetic acid

PI-Mix (1000x)

1 M PMSF, 2 mg/ml Benzamidin, 1.4 mg/ml Pepstatin, 1 mg/ml Leupeptin, 100 mg/ml Bacitracin, 1 ml DMSO

TBST

50 mM Tris/HCl, ph 7.6, 150 mM NaCl, 0.0005 % Tween-20

PBS

8 g/l NaCl, 0.2 g/l KCl, 1.14 g/l Na₃HPO₄, 0.2 g/l KH₂PO₄

TE

10 mM Tris/HCl, pH 8, 1 mM EDTA

Z-Buffer

60 mM Na₂HPO₄, 40 mM Na₃HPO₄, 10 mM KCl, 1 mM MgSO₄, pH 7

2x L-Buffer

250 mM Tris/HCl, pH 7.5, 20 % Glycerol, 200 mM NaCl, 2 mM EDTA, 20 mM MgOAc, 2 mM DTT, 1xPI-Mix

Bead Buffer

50 mM Tris/Hcl, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM DTT, 1x PI-Mix

Dilution Buffer

60 mM Tris-HCl, pH 7.4, 190 mM NaCl, 6 mM EDTA, 1.25% Triton X-100, 1 mM DTT, 1x PI-Mix

Urea-Wash

50 mM Tris/Hcl, pH 7.4, 2 M Urea, 150 mM NaCl, 5 mM EDTA, 1 % Triton X-100, 0.2 % SDS

IP Buffer

50 mM Tris/HCl, pH 7,4, 150 mM NaCl, 5 mM EDTA , 1 % Triton X-100, 0.2 % SDS

Low Salt

150 mM NaCl

4x Laemmli

12.5 % SDS, 2.5 % Glycerin, 5 % β-Mercaptoethanol, 125 mM Tris/HCl, pH 6.8, Bromphenolblue

5x DNA loading buffer

40% Ficoll 400, 0.1 M EDTA, 1% SDS, Bromphenolblue

Zymolyase solution

1 M Sorbitol, 0.1 M Na-Citrat, 60 mM EDTA pH 8.5, 5 mg/ml Zymolyase (Seikagaku)

5x HAT-Buffer

250 mM Tris/HCl, pH 850, 50% Glycerin, 0.5 mM EDTA, 5 mM DTT

Lysis Buffer

50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 10 mM imidazole, 1% Triton X-100, 1 mM PMSF, 1xPI-Mix

Wash Buffer

50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 20 mM imidazole, 1% Triton X-100

Elution Buffer

50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.25-1 M imidazole

BenchMark^TM Prestained Protein Ladder

Invitrogen

ReadyLoad^TM 1 kb DNA ladder

Invitrogen

ECL Reagents

Amersham

Antibodies

α-HA

1:1000

Covance

α-acetyl-lysine

1:1000

Upstate

α-Sas2

1:40.000

Meijsing + Ehrenhofer-Murray

α-myc

1:5000

Invitrogen

α-His

1:1000

Sigma

α-mouse-HRP

1:1000

Sigma

α-rabbit-HRP

1:3000

Sigma

α-guinea pig-HRP

1:1000

Sigma

Peptides

The Cse4 peptide used in this study was synthesized by Sigma Genosys. It consists of the N-terminal amino acids 112-134 with the following sequence (putative acetylation sites are marked in red):

1 mg/ml peptide was solubilized in dH₂O and stored at -80°C.

Primer

The primers used in this study were synthesized by Metabion and applied in a dilution of 10 pmol/μl. Sequences derived from the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/) were used to design optimal primers with the assistance from the Oligonucleotide Properties Calculator (http://www.basic.nwu.edu/biotools/oligocalc.html).

Methods Molecular methods Cell cultivation

The cultivation of bacteria with plasmids was carried out in LB-media or on LB-plates at 37°C unless indicated otherwise. For the bacterial expression of 3xHA-Cse4, nutrient rich TY-median was used. For the maintenance of plasmids the appropriate antibiotics were added to the media (ampicillin, kanamycin, chloramphenicol).

Yeast strains were grown on YPD full media or on YM minimal media at 30°C or 23°C (temperature sensitive mutants). Minimal media were supplemented as appropriate.

Transformation of E. coli and S. cerevisiae

Chemically competent bacterial strains were obtained from Gibco BRL (DH5α) and Invitrogen (TOP10, BL21 (DE3) Star). The transformation was carried out as suggested by the manufacturer.

For the preparation of competent yeast cells and the transformation of DNA into yeast we used a method described from (Klebe, et al., 1983). Competent yeast cells were used immediately or were stored after the addition of DMSO to 5.5% at

-80°C.

DNA isolation

The isolation of plasmids from bacteria was performed with the Mini- or Midi-Kit from QIAGEN according to the manufacturers instruction.

Genomic yeast DNA was isolated as described from (Hoffman and Winston, 1987). For PCR (polymerase chain reaction) mediated analysis of gene knock-outs in yeast, a single yeast colony was heated for one minute in a microwave. The PCR mix was added subsequently and the DNA amplified in a thermocycler.

Plasmid constructions

The epitope tagged HA-CSE4 plasmid was constructed by inserting three HA-tags into the XbaI-site of CSE4 analogous to (Stoler, et al., 1995). For the insertion of the N- and C-terminal CSE4 fragments into the pACT2 and pBTM117c vector the amino acids 11-139 and the amino acids 137-229 were PCR- amplified with CSE4-primers containing specific restriction sites and subcloned into pCR®BluntII-TOPO (Invitrogen). For insertion into pACT2, the N-terminus was excised with PstI/SpeI and the C-terminus with SacI/PstI. The overlapping ends were filled in with T4 DNA-polymerase and the fragments were cloned into SmaI-linearized pACT2. To obtain the N-/C-terminal CSE4 in the LexA-vector, the fragments were excised with AccI/NotI and inserted into AccI/NotI-linearized pBTM117c.

S. cerevisiae strain construction

To generate yeast strains with a specific genotype, the appropriate strains were crossed, sporulated and the resulting tetrads were dissected. For this purpose, small amounts of the yeast strains to be crossed were mixed in dH₂O and were allowed to mate for ≥ 8 h. To select for diploid cells, the cells were then transferred onto supplemented YM and grown at 30°C. After two days, diploids were restreaked onto YPD before they were transferred to sporulation plates and incubated at 30°C for 3-4 days. The cell wall of the sporulated strain was digested with zymolyase solution for 10 minutes at room temperature; the reaction was stopped by the addition of 160 μl dH₂O. The spores from the asci were separated under the microscope (Zeiss Axioskop FS) with a micromanipulator (Narishige) and analyzed for their genotype.

For specific gene knock-outs, we took advantage of homologous recombination. In most cases the kanMX-system was used, which lead after successful homologous recombination to geneticin (G418) resistance in S. cerevisiae.

The basic principle of the system is to amplify the kanMX-gene from pF6a-kanMX4 (pAE478) with homologous sequences at the ends from the gene to be knocked out via PCR. The PCR product is then transformed into competent yeast cells, which are plated onto G418 plates (YPD + 200 mg/l G418). After 2-3 days at 23°C/30°C, the grown colonies are analyzed with PCR.

Polymerase chain reaction

Standard conditions:

1. Denaturation

7 min

93°C

2. Denaturation

1 min

93°C

3. Annealing

30 sec

depending on primers, in general with formula 72.4 + (0,41 _* % GC) – 650/length

4. Extention

1 min/ 1kb

72°C

5. Extension

10 min

72°C

6. Cool down

∞

4°C

PCR for subsequent cloning: repeat step 2-4 for 20-25 cycles
Control PCR: repeat step 2-4 for 25-30 cycles

DNA sequencing

Sequencing of DNA templates was performed with the ABI PRISM^TM Dye Terminator Cycle Sequencing Ready Reaction Kit. For this purpose, 0.5-1 μl DNA was mixed with 4 μl Terminator Ready Reaction Mix, 1 μl Primer and dH₂O and amplified in the thermocycler.

PCR conditions for 25 cycles were:

Denaturation:

10 sec

96°C

Annealing:

5 sec

50-60°C

Extension:

4 min

60°C

Cool down

∞

4°C

After amplification, the DNA was precipitated with Na-acetate/EtOH, dried and analyzed in the sequencing department of the MPIMG.

Two-hybrid system

The Two-hybrid system can be used to detect protein-protein interactions in yeast. The method is based on the GAL4 gene from Saccharomyces cerevisiae, whose gene product activates several genes from the galactose synthesis pathway. For their activation, Gal4p binds with its DNA-binding domain (DNA-BD) to sequences upstream of the genes (UAS).

Additionally, Gal4p consists of an activation domain (AD), which binds via proteins to the DNA-binding domain and leads to transcription of the reporter genes (lacZ, HIS3). If the two domains remain separated from each other, the transcription from the reporter genes is blocked.

For our experiments we used the pACT2-vector with the activation domain and pBTM117c with the DNA-BD. pBTM117c contains instead of the Gal4 DNA-BD LexA, a bacterial protein that normally binds to lexA promotors. The advantage of LexA based two-hybrid is that less false positive interactions occur and that weak interactions are effectively amplified due to multiple LexA operators. The proteins to be tested were cloned into the vectors and both plasmids were cotransformed into the two-hybrid strain L40c. Protein-protein interactions were monitored with the β-galactosidase filter assay and the expression of HIS3.

β-galactosidase filter assay

Upon activation of the reporter gene lacZ, the enzyme β-galactosidase is expressed in the cells. In this assay, the enzyme uses the colourless X-Gal (5-Brom-4-chlor-3-indolyl-β-D-galactopyranoside) as a substrate instead of galactose, and cleaves it into the dark blue 5-brom-4-chlor-indigo. For this purpose the yeast cells were transferred onto a nitrocellulose filter and broken in liquid nitrogen. The filter was incubated with 2,5 ml buffer Z and 50 μl X-gal for up to 2 h at 30°C. During this time course, a positive interaction between the proteins should lead to a blue coloration.

HIS3 reporter assay

The reporter gene expression was also tested with the HIS3 reporter assay. The two-hybrid strain with the appropriate plasmids was streaked onto media lacking histidine. L40c itself is auxotroph for histidine, but due to the interaction of the proteins to be tested, HIS3 gene expression is activated, and the strain can grow in the absence of histidine.

FACS – fluorescent activating cell sorting

To perform FACS analysis, yeast cells were cultured in an appropriate volume (~3 ml) until they reached OD₆₀₀=0.05-0.1. After brief centrifugation and washing, cells were fixed in 70% EtOH at 4°C over night. Cells were then incubated for 4 h in 20x TE + 1 μg/ml RNase A, washed twice in PBS and subsequently stained over night in PBS + 100 μg/ml propidium-iodide (Sigma). After the suspension was 10x diluted, cell were separated from each other by sonication (3x 5 sec, 60%) and maintained in the dark. FACS analysis was performed with a flow cytometer (FACSCalibur) at the Deutsche Rheumaforschungszentrum in Berlin.

Biochemical methods Protein extract preparation

Protein extract preparation from bacteria

After reaching the desired optical density (OD), the bacterial culture was centrifuged for 10 minutes at 5000 rpm and the pellet resuspended in column buffer. The cells were broken with ultrasound (4 x 1 minute, 60 %) or with the french press. To obtain the protein extract, the broken cells were centrifuged for 30 minutes at 20.000 rpm and the supernatant was frozen at -80°C.

Protein extract preparation from yeast

Yeast cell cultures were grown to an OD₆₀₀= 0,8-1 and harvested by centrifugation for 20 minutes at 5000 rpm at 4°C. After washing the cells with dH₂O, the cell pellet was resuspended in the appropriate buffer.

Native whole cell extracts from yeast cultures <200 ml were prepared in 2x buffer L by glass bead lysis as described by (Moazed and Johnson, 1996), except that the concentration of NaCl was adjusted to 200 mM. For denatured protein extracts, the cells were lysed in Bead Buffer, boiled for 10 minutes at 95°C and diluted in IP dilution buffer for subsequent immunoprecipitation.

Larger cell cultures were resuspended in 2x buffer L or bead buffer, respectively, and the cells were broken using a french press. After 1 h centrifugation at 40.000 rpm, protein extracts were analyzed or frozen at -80°C for further use.

SDS-PAGE and immunoblotting

Proteins were analyzed according to their molecular weight with 8/10/12% SDS-PAGE gels. The transfer from the gel to a nitrocellulose membrane was performed either at 0,8 A/cm² for 1 h with a Semi-Dry Blot from BIO-RAD, or at 110 V for 45 minutes with a wet blot from BIO-RAD. The efficiency of the transfer was visualized with Poinceau S dye. Subsequently, the membrane was blocked for 1 h with 5% fat free milk/TBST and incubated with the primary antibody in 5% fat free milk/TBST at 4°C overnight. After washing the membrane four times with TBST, the secondary HRP-conjugated antibody was added for 30 minutes. The membrane was washed with TBST for six times and proteins were then detected with ECL-solution from Amersham.

Detection methods for proteins

To detect proteins directly in SDS-PAGE gels, the gel was stained for 1 h with Coomassie Brilliant Blue R250. Subsequently, the gel was destained with 25% methanol/10% acetic acid, such that the staining of the proteins was maintained.

If small protein concentrations (~0,1 ng/mm²) were to be detected, we used the Silver Stain Kit from Bio-Rad. The staining of the proteins in an SDS-PAGE gel was performed according to the manufacturer’s instructions. Protein concentrations in solutions were determined with the Bradford Assay (Bio-Rad Protein Assay Kit).

Concentration of protein solutions

To concentrate proteins in a smaller volume, Centricon-columns (amicon) with an exclusion matrix of 10 kD were used. After one hour centrifugation at 5000 g, protein solutions were concentrated from 2 ml to approximately 50-200 μl. If necessary, an additional buffer change was performed with three washing steps in 2 ml buffer.

Solo- and Co-immunoprecipitation

Immunoprecipitation experiments were performed to isolate proteins and protein complexes. If the immunoprecipitation was carried out under denaturing conditions, the protein extract was boiled before use for 10 minutes at 95°C. The appropriate antibody was added to the whole cell protein extract and incubated for one hour with shaking at 4°C. Subsequently, protein G sepharose beads (Pharmacia) were added and the lysate-antibody-protein G mix was incubated over night.

Immunoprecipitates from native extracts were washed four times with 1x buffer L and resuspended in SDS sample buffer; precipitates from denatured extracts were washed with Urea wash buffer, IP buffer and detergent free wash buffer before resuspension in SDS sample buffer. After boiling for 10 min at 70°C and centrifugation, the immunoprecipitates were analyzed by immunoblotting.

Bacterial expression of Cse4

For bacterial expression of large amounts of Cse4, N-terminal His-tagged Cse4 was generated by inserting CSE4 into the XhoI/BamHI-site of pET15b and transforming the resulting plasmid (pAE994) into BL21(DE3) Star cells. The expression of His-Cse4 in 2 l LB-culture was induced by adding 1 mM IPTG to the medium at OD₆₀₀=0.8 and subsequent growth for additional 2h at 37°C. The cells were harvested and proteins extracted in Lysis Buffer by sonication (3x 20 sec, 40-50%). After centrifugation, the supernatant was added to 2 ml 50% Ni-NTA matrix and incubated with rotation for 1 h at 4°C. Proteins that did not bind the to matrix were washed off with 15 ml Wash Buffer.

The bound His-Cse4 was eluted by adding 8x 500 μl Elution Buffer with 250 mM imidazole, 1x with 350 mM imidazole and 1x with 1 M imidazole to the matrix. The samples were dialyzed against water, concentrated and further analyzed for His-Cse4 by immunoblotting.

Acetylation assay

To investigate the acetylation of proteins, we took advantage of an in vitro acetylation assay. In a total volume of 25 μl we mixed 2 μg recombinant histone H4 and/or 2 μg His-Cse4 together with the enzyme (200 μg rSAS-I or 500 μg recombinant PCAF), 5xHAT-Buffer and 0,5 μl [¹⁴C] acetyl-CoA (50-62 mCi/mmol Amersham). After one hour incubation at 30°C, the mix was run on a 15 % SDS-PAGE gel. The gel was dried in a gel dryer (BioRad) for 1 hour at 80°C, and the acetylated proteins were detected after over night exposure with a phosphoimager.