Yeast strains, recombinant DNA and microbiological techniques
The yeast strains used in this study are listed in Additional file 1: Table S2. Microbiological techniques were carried out essentially as previously described 29. Chromosomal integration of a C-terminally tagged cassette into the TAP or GFP tagged strains was performed as previously described [43,44]. For gene disruption, the indicated gene was deleted using a PCR product amplified from either the KanMX4 plasmid pRS400 or the HIS3 plasmid pFA6a. All deletions and genomically tagged strains were confirmed by PCR analysis and/or western blot analysis.
Growth assays were carried out by growing cells at 30 ºC in YPD to an OD600 of 0.3-0.4. Subsequently, ten-fold serial dilutions of an equal number of cells were made and drops of these dilutions were spotted onto YPD plates. Growth was recorded after 48 h of incubation at 30 ºC.
Preparation of yeast cell extracts, TAP purifications and western blot analysis
To prepare yeast cell extracts, yeast cultures were grown to an OD600 of 0.5-0.8 in YPD medium. Total proteins were extracted using the trichloroacetic acid (TCA) method. All tandem affinity purifications (TAPs) were performed as previously described [45]. Briefly, TAP-fusion proteins and their associated proteins were recovered from cell extracts by affinity selection on IgG Sepharose beads. After bead washing, the Tobacco Etch Virus (TEV) protease was added to release the bound material. The eluate was incubated with calmodulin-coated beads in the presence of calcium. After washing, the bound material was released with EGTA. This enriched fraction was called the calmodulin eluate. To analyze the TAP-purified protein complexes, TCA-precipitation, LysC/trypsin digestion and multidimensional protein identification technology (MudPIT) mass spectrometry analyses were performed as described previously [46]. Following electrophoresis and western blotting, membranes were probed with specific antibodies: α-PGK1 was used as a loading control (Invitrogen), α-HA (Roche), α-TAP (Thermo Fisher), α-H2B total (Active Motif), α-H2Bub1 (Cell Signaling), α-Spt8 (Santa Cruz) and α-GFP (Roche). Quantification of the western blot bands was performed by densitometry using ImageJ software and subsequent normalization using the ratio between the protein to study and loading control protein.
Chromatin immunoprecipitation and chromatin enriched fractions
Chromatin immunoprecipitation (ChIP) was performed as previously described [32]. In brief, early log-phase cultures (100 ml), grown in YP medium containing 2% raffinose, were separated into two aliquots and either glucose or galactose was added to one aliquot to a final concentration of 2%. Thirty min after the addition of each carbon source, cultures were cross-linked with 1% of formaldehyde solution (Sigma) for 20 min at room temperature with intermittent shaking. After quenching with 125 mM glycine, cells were collected by centrifugation and washed four times with 25 ml cold Tris–saline buffer (150 mM NaCl and 20 mM Tris–HCl at pH 7.5). Cells were broken in 300 µl of lysis buffer (50 mM HEPES–KOH at pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Tergitol-type NP-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors (Complete, Roche)) plus glass beads. Cell extracts were sonicated in a Bioruptor sonicator (Diagenode) for 30 min of 30 s on/30 s off cycles to yield chromatin fragments with an average size of 300 bp. An aliquot (10 µL) of extract was reserved as the input and the rest was used for immunoprecipitation with magnetic beads (Dynabeads®) coated with monoclonal anti-mouse IgG antibodies. Immunoprecipitations were conducted for 2 h at 4 ºC, and the immune complexes were then washed twice with 1 mL of lysis buffer, twice with 1 mL of lysis buffer supplemented with 360 mM NaCl, twice with 1 mL wash buffer (10 mM Tris–HCl at pH 8.0, 250 mM LiCl, 0.5% NP-40, 5 mg/mL of nadeoxycholol and 1 mM EDTA) and once with 1X TE. Samples were eluted at 65 ºC for 15 min with 100 µl of elution buffer (50 mM Tris–HCl at pH 8, 10 mM EDTA and 1% SDS). Inputs and immunoprecipitation (IP) samples were incubated overnight at 65 ºC to reverse the cross-link. Samples were then treated with proteinase K (Ambion), at 100 mg/250 ml of chromatin for 2 h. Afterwards, DNA was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1), and was then ethanol precipitated and resuspended in 40 µl of 1X TE. DNA was used as a template in the qPCR reaction using specific primers for GAL1, PMA1 and YEF3 promoters (Additional file 1: Table S3).
Chromatin enriched fractions (ChEFs) were obtained from 50 mL of cells with an OD600 of 0.5. Cells were collected by centrifugation, were washed with water and were broken by resuspending in 200 µL of buffer 1 (HEPES 20 mM at pH 8, KCl 60 mM, NaCl 15 mM, MgCl2 10 mM, CaCl2 1 mM, butyric acid 10 mM, triton X-100 0.8%, sucrose 0.25 M, spermidine 2.5 mM and spermine 0.5 mM) plus 200 µL of glass beads and vortexing for 4 min at 4 ºC. All of the following steps were conducted at 4 ºC. Lysate was then centrifuged for 5 min at 500 g and the supernatant was recovered and re-centrifuged once more for 5 min at 500 g. The new supernatant was recovered in a new tube and a 20 µL sample from this extract was used as INPUT (IN). The rest of the extract was centrifuged at 20.000 g for 20 min and the pellet was resuspended in 200 µL of buffer 1 and centrifuged at 20.000 g for 20 min. After discarding the supernatant, the pellet was resuspended in 200 µL of buffer 2 (HEPES pH 7.6 20 mM, NaCl 450 mM, MgCl2 7.5 mM, EDTA 20 mM, glycerol 10%, NP-40 1%, sucrose 0.5 M, urea 2 M, DTT 1 mM and PMFS 0.125 mM) and centrifuged at 20.000 g for 20 min. The supernatant was discarded and the pellet was again resuspended in buffer 2 and centrifuged at 20.000 g for 20 min. The supernatant was discarded and the pellet, which was used as CHROMATIN FRACTION (C), was resuspended in 20 µL of Laemmli buffer 1X to be run on a gel.
Quantitative RT-PCR
Total RNA prepared by hot phenol extraction was treated for 30 min at 30 °C with DNase I RNase-free (Roche) prior to use for cDNA synthesis. Subsequently, cDNA was synthetized in 20 µL reactions containing 50 ng/µL of DNase I treated RNA, 250 ng of random hexamers (Invitrogen), 10 units/µL of SuperScript III Reverse Transcriptase (Invitrogen), 1XFirst Strand Buffer, 10 mM DTT, and 0.5 mM dNTPs, following the manufacturer’s instructions. Quantitative real-time PCR was then performed in a LightCycle 480 Thermal Cycler (Roche) using the SYBR® Premix Ex Taq™ kit (Takara) for fluorescent labelling. For each analysis primer pair, a negative control was included. The primers set used in this study is provided in Additional file 1: Table S3. A primer pair for ALG9 was used as a reference gene. Data and errors bars represent the average and standard deviation of three independent biological samples.
In vitro deubiquitination assay
The FLAG-tagged H2B substrate (including H2B ubiquitinated with HA-ubiquitin and unmodified H2B) was obtained by purifying N-terminally Flag-tagged histone H2B using an M2 agarose slurry (Sigma A2220) from cells that lack genomic HTB1 gene and contain two plasmids, one in which histone H2B is tagged with FLAG tag (pZS145 HTA1-Flag-HTB1-CEN-HIS3) and other in which Ubiquitin protein is tagged with HA epitope (GAPDH-3HAUB14::
URA3). The purified substrate was split into equal aliquots each containing 500 ng of FLAG-tagged H2B. Aliquots were incubated with Ubp8-TAP purified complexes at room temperature for 30 min in deubiquitination (DUB) buffer (100 mM Tris–HCl at pH 8.0, 1 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM PMSF and 1% protease inhibitor). One aliquot was subjected to a mock in vitro deubiquitination reaction lacking Ubp8-TAP. Reactions were stopped by adding one volume of 2X Laemmli sample buffer containing 50 mM DTT. Samples were separated on a 15% SDS–PAGE gel, transferred to a nitrocellulose membrane and subjected to western blot analysis with an anti-HA antibody (Roche) that was used to detect HA-tagged ubiquitin.
Fluorescence microscopic analysis of GFP localization
Yeast cultures (10 mL) that were grown to an OD600 of 0.3-0.6 were pelleted and were then fixed by incubating them in methanol for 10 min on ice while vortexing every 2 min. Fixed cells were subsequently centrifuged and washed once with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4). Cells were resuspended in 30-50 µL of 1X PBS and then 10 µL were placed on glass microscope slides. Samples were observed under a Leica TCS-SP2-AOBS confocal microscope.
Quantification of GFP Fluorescence microscopic data
GFP Fluorescence microscopic data were quantified using IPython notebooks. Find full description in Additional file 2: Figure S8. The main library used for the pipeline implementation was Scikit-image [47]. The GPF and DAPI images were first converted to grayscale images and were represented by a NumPy array [48] to construct a histogram of pixel values from the GFP nuclear intensity values. Several components of the Scikit-image library were combined into an image processing workflow as follows: i) Binarization. Images were converted to their binarized version to discriminate GFP nuclear intensity from noise background. We employed the filter threshold Otsu algorithm, and we further removed the artefacts connected to the image border; ii) Segmentation. To count the cells, a segmentation of the DAPI cell nuclei was performed; iii) Statistical analysis. R package software [49] was used to generate the statistical analysis. To determine significant values between different experimental groups, the mean data were compared using one-way analysis of variance (ANOVA). Tukey's multiple comparisons test was also used. Values of *p<0.001 were considered significant.