Acetylation of Histone H3 on Lysine 4 Regulates Pericentromeric Silencing in Fission Yeast

Xhemalce_8

Manuscript GENESDEV/2009/135202

Supporting Online Materials

Materials and Methods:

Colony color silencing assay Five fold serial dilutions of logarithmically growing cells (OD600~0.5) containing the ade6+ gene inserted at centromere 1 (otr1R::Sph1) and the ade6-210 mutation at the endogenous ade6 locus, were spotted onto adenine poor YE plates and incubated for 4 days at 30°C and 2 days at 4°C before scanning.

DNA damage assay Five fold serial dilutions of logarithmically growing cells (OD600~0.5) were spotted onto YES plates containing the indicated amounts of genotoxic agents. For UV and IR (g rays) sensitivity, cells were spotted onto YES medium and then treated as indicated. Plates were incubated at 30°C for 4 days before scanning.

RNA manipulation Total RNA extraction was performed on the equivalent of 4 OD of logarithmically growing cells (OD600~0.5) as in Lyne et al. 2003. 5µg of RNA was DNase treated with the Turbo DNA-freeä kit (Ambion). 500ng of the resulting RNA was used for first-strand complementary DNA synthesis with the Superscript IIIä Reverse Transcriptase (Invitrogen) with 50 ng of either random hexamers (Invitrogen) or specific primers as stated. Subsequently, 1/10th of the reaction was used for PCR. siRNA purification and Northern Blot were performed as previously described (Buhler et al., 2006)

DNA purification and Southern Blot DNA purification and telomere length analysis by Southern Blotting was performed as previously described (Xhemalce et al. 2007).

Whole Cell Extracts The pellet from 4 OD of logarithmically growing cells (OD600~0.5) was suspended in 200 µl of Laemmli buffer, beat with a volume of 500µl of 0.5 mm glass beads (Biospec) two times 20 seconds at speed 6 in a Fastprepâ instrument. The supernatant was recovered and heated for 5 min at 95°C prior to Western blotting.

Western Blot Proteins separated in a 15 % SDS-PAGE gel were transferred onto a 0.45 µM nitrocellulose membrane (Whatman) in Carbonate Buffer (pH 9.5) with 20% Methanol for 1h at 400 mA. The membranes were blocked for 30-60 min at room temperature in TBS-T+BSA buffer (Tris-buffered saline, 0.1% Tween 20, 5% BSA) and incubated overnight at 4°C with TBS-T+BSA buffer containing the indicated antibodies. The membranes were washed 3 times 10 minutes with TBS-T, incubated 1h with TBS-T containing the appropriate secondary antibodies, washed and revealed with ECL (Amersham).

Mst1 expression and purification Mst1 ORF was cloned into pFAST-BAC HC in frame with a 6xHis tag and a TEV cleavage site. The corresponding bacculovirus was generated following the Bac-to-Bac® Baculovirus Expression System (Invitrogen) instructions. Exponentially growing SF9 cells (~2x106 cells/ml) were infected with P3 viral stock at 1/500 and 109 cells were harvested 48h after the infection. Native purification of His6-Mst1 was performed with HisPure® Cobalt beads (Pierce) according to the manufacturer’s instructions. The proteins bound to the beads were eluted with Elution buffer containing 50, 75 or 150mM Imidazole. 100µl of the third elution (150mM Imidazole) were diluted ten times with TEV buffer (50 mM Tris-HCl pH8, 0.5 mM EDTA, 1 mM dTT, 0.01% Tween-20) and digested for 4h at 4°C with 2.25 µg of His6-TEV. The digestion product was incubated with 100µl of HisPure® Cobalt beads (Pierce) for 2h at 4°C to eliminate the His6-TEV from the supernatant. The supernatant was collected, concentrated to 50µl with Sartorius 10,000 MW filters and 2.5 µl was used in the in vitro HAT assay.

in vitro HAT assay The assay was performed in a final volume of 25 µl of HAT buffer (50 mM Tris-HCl pH8, 0.5 mM EDTA, 1 mM dTT, 10% glycerol and 20 µM Acetyl CoA) containing 0.625 µg of recombinant hH3.1 and purified enzymes at 30°C for 1h. For the radioactive assays, 2µl per reaction of [3H]-AcetylCoA at 150GBq/mmol (Amersham, TRK688) was used.

Fluorescence microscopy Logarithmically growing cells (OD600~0.5) were fixed with 3.6% paraformaldehyde at room temperature for 45 min. Cells processed as described (Alfa et al. 1993) were sequentially stained with the anti H3K4ac antibody, an Alexa Fluorâ 594 conjugated anti-rabbit IgG secondary antibody (Molecular Probes) and DAPI. Images were acquired with an Olympus FV1000 Upright confocal microscope and processed using Adobe Photoshop® CS software.

Chromatin Immunoprecipitation 40 OD of logarithmically growing (OD600~0.5) or synchronized cells were fixed with 1% formaldehyde (Sigma) at room temperature for 1h and quenched with 20mM Glycine for 5 min. Cells were washed 3 times with 25 ml of PBS, 3 times with 1 ml of PEMS (100 mM PIPES, 1 mM EGTA, 1 mM MgSO4, 1.2 M Sorbitol) and spheroplasted in 1 ml of PEMS containing 250 µg of Novozym-234 and 100 µg of Zymolyase-20T at 37°C. Spheroplasts were washed once in 1 ml of cold Washing Buffer (140mM NaCl, 1.2mM EDTA, 16.8 mM Tris-HCl, pH 8.1 supplemented with Complete EDTA-free Protease Inhibitor cocktail from Roche) and suspended in 1.8 ml of cold Lysis Buffer (0.1% SDS, 1% TritonX-100, 140 mM NaCl, 1.2mM EDTA, 16.8 mM Tris-HCl, pH 8.1 supplemented with Protease Inhibitors as above). The suspension was sonicated in 15 ml conical tubes 3 times 10 minutes at High, 30 sec ON/OFF cycles in a cooled Bioruptor® (Diagenode) and cleared by centrifugation for 15 min at 13,000 rpm. The chromatin was quantified and diluted with Lysis Buffer to contain the equivalent of 20 ng/µl of DNA. 500 µl of chromatin corresponding to 10 µg of DNA was incubated with the appropriate antibodies O/N at 4°C, then with 25 µl of pre-washed Protein G Dynabeads® (Invitrogen) for 2-3h at room temperature. The beads were washed for 5 min at RT with 1 ml of TSE-150 (0.1%SDS, 1% Triton X-100, 150mM NaCl, 2mM EDTA, 20mM Tris-HCl, pH 8), TSE-500 (0.1%SDS, 1% Triton X-100, 500mM NaCl, 2mM EDTA, 20mM Tris-HCl, pH 8), LiCl buffer (0.25M LiCl, 1% NP-40, 1%DOC, 1mM EDTA, 10mM Tris, pH 8) and TE (10 mM Tris-HCl, pH8, 0.1 mM EDTA). The immuno-complexes were eluted from the beads using a total of 125µl of elution buffer (100 mM sodium bicarbonate, 1% SDS) for 30 min at 30°C and de-crosslinked overnight at 65°C. The ChIP samples were purified with the Qiaquick® PCR purification kit (Qiagen) and the DNA was eluted from the columns with 50 µl of water.

Quantitative PCR Real-time PCR analysis was performed on an ABI PRISM 7000 sequence detection system with the use of SYBR® Green (Applied Biosystems). The sequences of the primers used for PCR analysis are listed in Table S3.

Synchronization conditions Logarithmically growing cdc10-V5 mutant cells (OD600≈0.25) were arrested in G1/S by incubating for 4 h at 36°C. Cells were released into the cell cycle by rapid cooling to 25°C (time 0) and samples were taken as indicated.

Peptide Synthesis and coupling Peptides corresponding to amino-acid residues 1–21 of histone H3 coupled at their C terminus to Biotin were synthesized at >96% purity. The competing H3K9me2 peptide was not biotinylated. The biotinylated peptides were bound to MyOneä Streptavidin T1 Dynabeads® (Invitrogen) using 1 mg of each peptide for 100 µl of beads in a total volume of 500 µl of TBS-N (Tris-buffered saline, 0.1% NP-40) for 2h at 4°C. The beads were washed 3 times with 1 ml of TBS-N and resuspended in 1ml of TBS-N.

Peptide competition assays H3K9me2 and H3K4acK9me2 biotinylated peptides were coupled as above to MyOneä Streptavidin T1 Dynabeads® (Invitrogen). 200 µl of beads were incubated with 50 or 100 µg of the indicated GST purified chromodomains in a total volume of 1 ml of TBS-N for 1 h at 4°C to achieve saturated binding. The beads were washed 3 times with 1 ml of TBS-N and resuspended in 400 µl of TBS-N. 50 µl of these beads were rapidly distributed to 7 tubes containing 0, 0.25, 0.5, 1, 2, 4 and 8 µg of free un-biotinylated H3K9me2 peptide in a total volume of 200 µl of TBS-N and incubated for 1h at 4°C. The beads were washed once with 500 µl of TBS-N, resuspended in 25 µl of Laemmli buffer and heated for 5 min at 95°C. The resulting supernatants, as well as known quantities of the appropriate chromodomains were separated in a 12% SDS-PAGE gel, stained with Coomassie and destained until the background was transparent. The bound levels from each sample were quantified with Odyssey® Infrared Imager (LI-COR) at 700nm, using standards to control for a linear signal. The levels of bound chromodomain were plotted as a ratio to the sample that was not competed with free H3K9me2.


Supplemental References

Alfa C., F.P., Hyams J., McLeod M. and Warbrick E. 1993. Experiments with fission yeast.

Buhler, M., Verdel, A., and Moazed, D. 2006. Tethering RITS to a nascent transcript initiates RNAi- and\ heterochromatin-dependent gene silencing. Cell\ 125\(5\): 873-886\.

Driscoll, R., Hudson, A., and Jackson, S.P. 2007. Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56. Science 315(5812): 649-652.

Lyne, R., Burns, G., Mata, J., Penkett, C.J., Rustici, G., Chen, D., Langford, C., Vetrie, D., and Bahler, J. 2003. Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data. BMC Genomics 4(1): 27.

Nakayama, J., Klar, A.J., and Grewal, S.I. 2000. A chromodomain protein, Swi6, performs imprinting functions in fission yeast during mitosis and meiosis. Cell 101(3): 307-317.

Nicolas, E., Yamada, T., Cam, H.P., Fitzgerald, P.C., Kobayashi, R., and Grewal, S.I. 2007. Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection. Nat Struct Mol Biol 14(5): 372-380.

Partridge, J.F., Scott, K.S., Bannister, A.J., Kouzarides, T., and Allshire, R.C. 2002. cis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site. Curr Biol 12(19): 1652-1660.

Xhemalce, B., Riising, E.M., Baumann, P., Dejean, A., Arcangioli, B., and Seeler, J.S. 2007. Role of SUMO in the dynamics of telomere maintenance in fission yeast. Proc Natl Acad Sci U S A 104(3): 893-898.


Supplemental Figure Legends

Fig. S1. DNA damage sensitivity assay. Unlike set1∆, H3K4R mutant cells display increased sensitivity to S-phase specific DNA damage mediated by camptothecin.

Fig. S2. Telomere length analysis by Southern Blot. H3K4R mutant has a mean telomere length similar to set1∆.

Fig. S3. Validation of our anti H3K4ac antibody by Dot Blot. 5 fold dilutions of the indicated H3 peptides (aa1-21) were spotted onto PVDF membrane and blotted with the anti H3K4ac antibody.

Fig. S4. Validation of our anti H3K4ac antibody by Western Blot. WCE extracts from logarithmically growing fission yeast cells of the indicated genotypes were Western blotted with the anti H3K4ac antibody. An anti H3 Western Blot and a Ponceau staining of the membrane are shown as loading control. The H3K4ac antibody recognizes a single band running at the level of the histone H3 and its intensity is greatly reduced in all H3K4R mutants.

Fig. S5. Validation of our anti H3K4ac antibody by Fluorescence microscopy. Logarithmically growing WT and H3K4R mutant fission yeast cells were analyzed by immuno-fluorescence with the anti H3K4ac antibody. The nuclear signal obtained by this antibody in WT cells disappears in the H3K4R mutant.

Fig. S6. H3K4 is a major target of Mst1 mediated acetylation in vitro. In vitro HAT assays with Mst1 using recombinant histone hH3.1 WT, H3K4R or H3K14R as substrate and radioactive [3H]-AcetylCoA as acetyl donor. (A) The incorporated radioactive acetyl moieties were quantified using a liquid scintillation counter as previously described (Driscoll et al. 2007). (B) A third of the reaction mixture from (A) was electrophoresed in a 15% SDS-PAGE gel, stained with Coomassie brilliant blue (bottom panel), treated with Enlightning (PerkinElmer), dried and exposed on film for 24h at -80°C (top panel).

Fig. S7. Mst1 is modestly enriched at the dh pericentric repeat. ChIP with anti HA antibody analyzed by Q-PCR with dh and adh1 primers from untagged and mst1-HA cells. The data are represented as mean ± SD, after normalization to CHIP levels in untagged cells.

Fig. S8. ChIP with anti Swi6 antibody analyzed by Q-PCR with dh and dg primers from WT, H3K4R and clr4∆ cells. The data are represented as mean ± SD, after normalization to the control adh1 euchromatic gene and CHIP levels in WT cells.


Table S1. List of S.pombe strains.

Strain / mat / his / ade / leu / ura / arg / other / Origin
SPBX5 / h+ / + / ade6-216 / leu1-32 / ura4-D18 / + / - / Lab stock
FY648 / h+ / + / ade6-210 / leu1-32 / ura4-DS/E / + / otr1R(Sph1)::ura4 / R. Allshire
FY4754 / h+ / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / H3.2::ura4+//otr1R(Sph1)::ade6+ / R. Allshire
FY4640 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / H3.2/H4.2WT::ura4 tag//H3.1/H4.1::his3+//
H3.3/H4.3::arg3+//otr1R(Sph1)::ade6+ / R. Allshire
LP3278 / h+ / hst4∆ / L. Pillus
SPG1013 / h+ / + / ade6-216 / leu1-32 / ura4-D18 / + / otr1R(Sph1)::ura4//clr6-1 (ts) / S. Grewal
FY2535 / h+ / + / ade6-210 / leu1+ / ura4-D18 / + / mst1∆::kanMX6//
leu1+::Pnmt1-mst1L344SL (ts) / S. Forsburg
SPBX93 / h+ / + / + / + / + / + / cdc10-V50 (ts) / J. Cooper
SPBX61 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / H3.2K4R//H3.1/H4.1::his3+//
H3.3/H4.3::arg3+//otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX111 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / set1∆-::KanMX6//H3.2/H4.2WT::ura4 tag//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX131 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / set1∆::kanMX6//H3.2K4R//
H3.1/H4.1::his3+// H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX125 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / sir2∆-::KanMX6//H3.2/H4.2WT::ura4 tag//
H3.1/H4.1::his3+// H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX133 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / sir2∆::kanMX6// H3.2K4R//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX126 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / mst2∆::kanMX6//H3.2/H4.2WT::ura4 tag//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX162 / h+ / + / ade6-210 / leu1-32 / ura4-D18 / + / hat1∆::kanMX6 / B. Xhemalce
SPBX164 / h+ / + / ade6-210 / leu1-32 / ura4-D18 / + / gcn5∆::kanMX6 / B. Xhemalce
SPBX126 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / rtt109∆::kanMX6//H3.2/H4.2WT::ura4 tag//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX120 / h+ / + / ade ? / leu1-32 / ura4-D18 / + / sir2∆-::kanMX4 / B. Xhemalce
SPBX121 / h+ / + / ade ? / leu1-32 / ura4-D18 / + / hst2∆-::kanMX4 / B. Xhemalce
SPBX122 / h+ / + / ade ? / leu1-32 / ura4-D18 / + / hda1∆-::kanMX4 / B. Xhemalce
SPBX123 / h+ / + / ade ? / leu1-32 / ura4-D18 / + / clr3∆-::kanMX4 / B. Xhemalce
SPBX128 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / mst1HA::kanMX6//H3.2/H4.2WT::ura4 tag//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX127 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / clr4∆::kanMX6//H3.2/H4.2WT::ura4 tag//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX186 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / Rad21_5xFlag::hphMX6//H3.2/H4.2WT::ura4 tag//H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX187 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / Rad21_5xFlag::hphMX6//H3.2K4R//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1RSph1ade6 / B. Xhemalce
SPBX213 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / Swi6_5xFlag::hphMX6//H3.2/H4.2WT::ura4 tag//H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX214 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / Swi6_5xFlag::hphMX6//H3.2K4R//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1RSph1ade6 / B. Xhemalce
SPBX217 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / Chp2_5xFlag::hphMX6//H3.2/H4.2WT::ura4 tag//H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX218 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / Chp2_5xFlag::hphMX6//H3.2K4R//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1RSph1ade6 / B. Xhemalce
SPBX219 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / Clr3_5xFlag::hphMX6//H3.2/H4.2WT::ura4 tag//H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1R(Sph1)::ade6+ / B. Xhemalce
SPBX220 / h- / his3-D1 / ade6-210 / leu1-32 / ura4-D18 / arg3-D4 / Clr3_5xFlag::hphMX6//H3.2K4R//
H3.1/H4.1::his3+//H3.3/H4.3::arg3+//
otr1RSph1ade6 / B. Xhemalce


Table S2. List of antibodies.