Supplemental Inventory

Deyter S1

Supplemental Figures

Figures S1-S5

Supplemental Materials and Methods

Supplemental Tables

Table S1. Supplemental Strain Table

Table S2. Supplemental Plasmid Table

Supplemental References [Type text] [Type text] [Type text]2

Figure S1.

Nhp6 does

not regulate

stability.

(A) The

Odyssey

imaging

system was

used to

quantify Cse4

levels in

spt16-aid

(SBY11268)

cells treated as in Figure 1B. CHX= cycloheximide; -NAA= control; +NAA= Spt16-depleted cells. (B) pGAL-3Flag-CSE4 was induced in wild-type (SBY8851) and nhp6∆

(SBY11293) cells for 1 hr with galactose and then cycloheximide was added to inhibit protein synthesis at time zero. Corresponding lysates were monitored for

Flag-Cse4 levels with -Flag antibodies at the indicated time points. Pgk1 is a loading control. Part of Figure 1B has been duplicated and boxed on the right to serve as a comparison. (C) Five-fold serial dilutions of the indicated strains

(SBY3, SBY8851, SBY8903, SBY10626, SBY11293) were plated on glucose or

2 Deyter S3 galactose media at 23°C. (D) spt16-aid (SBY11268) cells containing pGAL-

3Flag-CSE4 were treated with vehicle (-NAA) or auxin (+NAA) to degrade Spt16 for 1 hr, followed by a 1 hr galactose induction of 3Flag-Cse4. 3Flag-Cse4 was induced in psh1∆ (SBY8903) cells by a 1 hr galactose induction. Whole cell extracts (WCE) were fractionated into soluble and chromatin fractions. Cse4 levels were monitored in each fraction with  antibodies. Pgk1 and H2B are markers of the soluble and chromatin fractions, respectively. [Type text] [Type text] [Type text]4

Figure S2. Psh1 binds to the Spt16-M domain.

(A) Diagram of Spt16 domains as previously defined (VanDemark et al. 2006).

Spt16 contains four domains including Spt16-D, the region that mediates dimerization, and Spt16-M, a region that has a double pleckstrin domain structure that interacts with histones H3/H4 (Kemble et al. 2013). A C-terminal region of Spt16-M called the U-turn motif (~ residues 913-964; not depicted in diagram) binds H2A/H2B (Hondele et al. 2013) (B) Glutathione-Sepharose resin bound to GST, GST-Psh1, or GST-Pob3 was incubated with MBP or the indicated MBP-Spt16 fusion proteins. The sepharose was washed and eluted, and the presence of the MBP and GST-tagged proteins was determined with -

4 Deyter S5

MBP and -GST antibodies. (C) Lysates were obtained from cells expressing either Psh1-3Flag (SBY7125) or Spt16-3Flag (SBY5647).  antibodies were used to analyze the levels of the Flag-tagged proteins and Pgk1 served as a loading control. The relative levels of Psh1 and Spt16 were quantified using the Odyssey imaging system and determined that Spt16 is approximately 35-fold more abundant than Psh1. [Type text] [Type text] [Type text]6

Figure S3. Cse4 ubiquitylation is decreased in psh1∆ C cells.

WT (SBY8851), psh1∆ (SBY8903), and psh1∆ C-13Myc (SBY10921) cells expressing pGAL-3Flag-CSE4 were grown in galactose and Cse4 was

immunoprecipitated with -Flag antibodies. Cse4 ubiquitin conjugates (Ubn) were detected on immunoblots by probing with -ubiquitin (-Ub) antibodies.

The blots were probed with -Flag antibodies to determine the level of unmodified Cse4.

6 Deyter S7

Figure S4. Analysis of Cse4 mononucleosomes.

The material obtained after step-salt gradient dialysis (prior to immobilization on

Streptavidin dynabeads; see Materials and Methods) was run on 5% nondenaturing PAGE, and the gel was stained with ethidium bromide to detect DNA species. [Type text] [Type text] [Type text]8

Figure S5. FACT inhibits Psh1 in vitro ubiquitylation reactions.

(A) V5-Cse4 octamers were incubated in the absence (-) or presence (+) of the ubiquitylation cocktail, GST-Psh1, and the indicated amounts of FACT.

Immunoblots were probed with -V5 antibodies to analyze V5-Cse4 ubiquitylation (Ubn). (B) GST-tagged Full-length (FL) or the C-terminal Psh1 mutant (∆ C) were incubated in the presence (+) or absence (-) of the ubquitylation (Ub) cocktail and FACT. The blot was probed with -GST antibodies to analyze Psh1 autoubiquitylation. Notice that in the presence of the

Ub cocktail, the unmodified full length (FL) and Psh1∆ C ( ∆ C) bands decrease and higher migrating species of autoubiquitylated Psh1 appear. However, these upper bands of Psh1 decrease and the levels of unmodified FL and ∆ C species

8 Deyter S9 increase when FACT is added to the reactions. These results show that FACT binding to Psh1 is not required for the inhibition since GST-Psh1∆ C, which has a decreased interaction with FACT, is inhibited by FACT similarly to GST-Psh1. [Type text] [Type text] [Type text]10

Supplemental Materials and Methods

Plasmid Construction

The pGAL-3FLAG-CSE4 (pSB1665), GST-Psh1 (pSB1535) and GST-

Psh1 (C45S, C50S) (pSB1541) constructs used were previously published

(Ranjitkar et al. 2010). pSB1067 was constructed as follows: the CSE4 promoter was amplified with primers SB1278 and SB1279 with engineered KpnI and XhoI sites. The GAL promoter was cut out of an integrating plasmid that contained pGAL-3FLAG-CSE4 (pSB1066) by digestion with the same enzymes, and the

CSE4 promoter was cloned into the same sites to generate pCSE4-3FLAG-

CSE4. MBP-Nhp6A (pSB1767) was constructed by amplifying genomic NHP6A using primers SB2936 and SB2937 with engineered SalI and PstI sites and cloned into the same sites of the pMAL-C2X vector (NEB). MBP-Pob3

(pSB1887) was constructed by amplifying POB3 from pJW21 (gift from T.

Formosa) using primers SB3006 and SB2941 with engineered EcoRI and HindIII sites and cloned into the same sites of the pMAL-C2X vector. MBP-Spt16

(pSB1888) was constructed by amplifying SPT16 from pJW22 (gift from T.

Formosa) using primers SB2942 and SB2943 with engineered BamHI and PstI sites and cloned into the same sites of the pMAL-C2X vector. GST-Psh1 (1-350)

(pSB2165) was generated by using pSB1535 as a template for PCR amplification with primers SB2248 and SB3446 with engineered BamHI and EcoRI sites and cloned into the same sites of the pGEX2T vector (GE Healthcare). GST-Psh1 (1-

325) (pSB2166) was generated by using pSB1535 as a template for PCR amplification with primers SB2248 and SB3447 with engineered BamHI and

10 Deyter S 11

EcoRI sites and cloned into the same sites of the pGEX2T vector. GST-Psh1 (1-

300) (pSB2167) was generated by using pSB1535 as a template for PCR amplification with primers SB2248 and SB3448 with engineered BamHI and

EcoRI sites and cloned into the same sites of the pGEX2T vector. MBP-Spt16

(485-804) (pSB2170) was constructed by amplifying SPT16 from pSB1888 using primers SB2994 and SB2995 with engineered BamHI and PstI sites and cloned into the same sites of the pMAL-C2X vector. MBP-Spt16 (485-642) (pSB2171) was constructed by amplifying SPT16 from pSB1888 using primers SB2994 and

SB3043 with engineered BamHI and PstI sites and cloned into the same sites of the pMAL-C2X vector. MBP-Spt16 (643-804) (pSB2172) was constructed by amplifying SPT16 from pSB1888 using primers SB3044 and SB2995 with engineered BamHI and PstI sites and cloned into the same sites of the pMAL-

C2X vector. pSB2176 was constructed as follows: pSB2173 (CSE4 in pET28) served as a template for mutagenesis PCR with SB3902 and SB3903 to eliminate the XbaI site in the pET28 vector backbone, making the XbaI site in

CSE4 unique. A linker containing two V5 epitopes (2V5) flanked by XbaI sites

(generated by annealing primers SB3917 and SB3918 to each other) was then cloned into the XbaI site in CSE4. Mutagenesis PCR was then performed with

SB4010 and SB4011 to put the 2V5 sequences in frame with CSE4. GST-Psh1

(301-406) (pSB2234) was generated by using pSB1535 as a template for PCR amplification with primers SB4191 and SB2249 with engineered BamHI and

EcoRI sites and cloned into the same sites of the pGEX2T vector. All oligo sequences are available upon request. [Type text] [Type text] [Type text]12

Protein Purification, Binding Assays, Immunoprecipitation, and

Immunoblotting

Plasmids containing either GST alone (pSB8), GST-Psh1 (pSB1535),

GST-Psh1 RING mutant (pSB1541), GST-Psh1(1-350) (pSB2165), GST-Psh1(1-

325) (pSB2166), GST-Psh1 (1-300) (pSB2167), GST-Psh1 (301-406)

(pSB2234), GST-Pob3 (pSB2169, gift from Richard Singer), MBP alone

(pSB1721), MBP-Nhp6A (pSB1767), MBP-Pob3 (pSB1887), MBP-Spt16

(pSB1888), MBP-Spt16 (485-804) (pSB2170), MBP-Spt16 (485-642) (pSB2171),

MBP-Spt16 (643-804) (pSB2172) or His-Nap1 (pSB2168, gift from T. Tsukiyama) were transformed into BL-21 codon-optimized cells. Proteins were purified from

2 L of cells after inducing expression for 2 hr at 37°C with 0.5 mM IPTG. GST,

MBP, and HIS-tag purifications were carried out using Glutathione Sepharose

(GE Healthcare), Amylose resin (New England Biolabs), and His-Select nickel affinity gel (Sigma), respectively, as described (Kellogg et al. 1995) or as per manufacturer’s instructions. After elution, proteins were dialyzed overnight in

PBS + 30% glycerol. His-Nap1 was kept in elution buffer (50 mM sodium phosphate (pH 8.0), 0.3 M sodium chloride, 250 mM imidazole) since dialysis caused it to precipitate out of solution.

Binding assays were performed by immobilizing 5 μ g of the dialyzed GST fusion proteins onto 100 μ l Glutathione Sepharose (taken from a 50/50 slurry) for

1 hr at 4°C. Resin was washed several times with PBST (PBS + 0.1% Tween-

20). 30 μ l of the beads (~ 1.5 μ g protein) were incubated with ~ 1.5 μ g of the

12 Deyter S 13 indicated MBP proteins in a reaction volume of 50 μ l (in PBST) for 30 min at

25°C with constant rotation. Beads were washed three times with 1 ml PBST

(Figure 2A) or 1 ml PBST + 250 mM NaCl (Figure 2D and 2E) for 3 min each at

RT, resuspended in 50 μ l sample buffer, and boiled for 4 min.

To purify the FACT complex, 12 L of yeast cells in log phase (OD 600 of

~1.5) expressing Spt16-Flag at the endogenous locus were harvested. Cellular lysate was obtained as previously described for Dsn1-Flag immunoprecipitations

(Akiyoshi et al. 2010) with the following modifications. The cleared lysate was incubated with 1 ml of anti-Flag M2 Affinity Gel resin (Sigma) for 3 hr at 4°C followed by seven washes in buffer H/1.0 M KCl (25 mM HEPES (pH 8.0), 2 mM

MgCl2, 0.1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 0.1% NP-40, 15% glycerol, and 1 M KCl) supplemented with protease (10 μ g/ml leupeptin, 10 μ g/ml pepstatin, 10 μ g/ml chymostatin, and 1 mM PMSF) and phosphatase (1 mM sodium pyrophosphate, 2 mM Na-beta-glycerophosphate, 0.1 mM NA3VO4, 5 mM NaF) inhibitors. The resin was eluted in 450 μ l elution buffer (20 mM Tris-

HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5 mM β -mercaptoethanol, 5% glycerol, and Flag peptide (final concentration 0.5 mg/ml). The eluate was dialyzed overnight at 4°C in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM

EDTA, 0.5 mM β -mercaptoethanol, 5% glycerol and stored at -80°C.

For immunoprecipitations, 50 ml cultures were harvested at mid-log phase and lysates were prepared as described in buffer H/0.15 M KCl (Akiyoshi et al.

2010) except that cell extracts were made by beating cells with glass beads in a bead beater (Biospec Products) three times for 30 s with 1 min on ice in [Type text] [Type text] [Type text]14 between. The lysates were then centrifuged for 30 min at 13,000 rpm at 4°C.

For experiments analyzing ubiquitin conjugates of Cse4, NEM (N- ethylmaleimide) was added to the lysis and wash buffers to a final concentration of 5 mM. The supernatant was incubated with 15 μ l of protein G dynabeads

(Invitrogen) coupled to 0.75 μ l of M2 anti-Flag (Sigma) for 3 hr at 4°C. For TAP tag purifications, lysates were incubated with 30 μ l of IgG Sepharose (GE

Healthcare) for 2 hr at 4°C. The beads were washed four times in lysis buffer, and the immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting.

For immunoblotting, protein extracts were made and analyzed as described (Minshull et al. 1996). 9E10 anti-Myc antibodies were obtained from

Covance (Richmond, CA) and used at a 1:5,000 dilution. Pgk1 antibodies obtained from Invitrogen were used at 1:25,000. Flag M2 antibodies (Sigma) were used at 1:3,000. Spt16 antibodies (gift from Richard Singer, Dalhousie

University) were used at 1:5,000. H2B antibodies from Active Motif were used at

1:3,000. GST antibodies from Santa Cruz Biotechnology were used at 1:1,000.

HRP-conjugated MBP antibodies from New England Biolabs were used at

1:25,000. V5 antibodies from Invitrogen were used at 1:5,000. Cse4 antibodies

(gift from Carl Wu, National Institutes of Health) were used at 1:2,000. H3K4me2 antibodies were obtained from Millipore and used at 1:5,000. Ubiquitin antibodies from Zymed were used at 1:500. Ponceau S was used at a final concentration of 1mg/ml. Quantitative immunoblotting was performed with IRDye secondary antibodies from LI-COR at a 1:15,000 dilution. The immunoblots were

14 Deyter S 15 imaged on a LI-COR imaging system and the protein levels were quantified using the ImageJ program.

Cse4 octamer and nucleosome generation

Cse4 octamers were prepared as described (Kingston et al. 2011). Cse4 in pET28 vector (pSB2173) or V5-Cse4 in pET28 (pSB2176), histone H4 in pET22 (pSB2174), and H2A/H2B in pCDFDuet (pSB2175), all gifts from Martin

Singleton (except pSB2176), were transformed into BL21 (DE3)-RIL cells

(Agilent Technologies). Cse4 octamers were purified from 3 L cells grown in

2xTY supplemented with the appropriate antibiotics as described (Kingston et al.

2011). Fractions were run on SDS-PAGE, and those that contained Cse4 octamers (visualized by coomassie staining) were pooled and stored at 4°C.

To generate mononucleosomes, primers SB3040 and SB3211 were used to PCR a 146 bp fragment that has a strong nucleosome positioning sequence

(NPS) from a plasmid (pSB1843) that contains the X. borealis 5S rRNA sequence. Oligonucleotide SB3211 has a 5’ biotin modification and adds an additional 33 bp of linker DNA between the biotin moiety and the beginning of the

146 bp sequence. PCR reactions were gel purified using QIAquick Gel

Extraction columns (Qiagen). ~8 μ g DNA and 8 μ g Cse4 octamers were incubated at 25°C for 30 min in a 400 μ l reconstitution mixture containing 2 M

NaCl (final volume) and TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH

8.0)). The reconstitution mixture was placed into a Slide-A-Lyzer Dialysis

Cassette (7,000 MWCO, Thermo Scientific) and dialyzed in 1 L of TE buffer (pH [Type text] [Type text] [Type text]16

8.0) containing 1.2 M NaCl for 2 hr at 4°C. Step salt dialysis was performed by placing the cassette successively, each time for 2 hr at 4°C, in TE buffer containing 1.0 M, 0.8 M, and 0.6 M NaCl. The reconstitution mixture was then dialyzed overnight at 4°C in TE buffer without NaCl. To confirm the generation of mononucleosomes prior to immobilization, an aliquot of the dialyzed material was removed, glycerol was added to a final concentration of 4.5%, and the sample was run on a 5% nondenaturing polyacrylamide gel that was subsequently stained with ethidium bromide. To immobilize the nucleosomes, the dialyzed sample was added to 200 μ l Streptavidin Dynabeads M-280 (Invitrogen) and incubated overnight at 4°C with constant rotation. The immobilized nucleosomes were washed three times with TE buffer and stored in TE buffer + 50% glycerol at

To generate polynucleosomes, 2 μ g of pBlueScript SK(-) (pSB283) template was linearized by EcoRI and Cla1 digestion and recovered by purification with a Bio-Spin P-30 column (Biorad) as previously described

(Gelbart et al. 2001). The DNA was incubated with 2.5 units of Klenow polymerase (NEB), NEB Buffer 2, 3.75 μ l of 0.4 mM biotin-14-dATP (Invitrogen), and 0.6 μ l of 10 mM dTTP, dGTP, and dCTP in a 40 μ l reaction volume for 2 hr at 37°C to generate the biotinylated 3 Kb linearized DNA template. After Bio-

Spin P-30 column purification, the recovered biotinylated DNA (~ 2 μ g) was immobilized onto 100 μ l Dynabeads M-280 Streptavidin (Invitrogen) overnight at

4°C. In a typical nucleosome assembly reaction, Dynabeads coupled with 2 μ g of DNA were incubated with 2 μ g of recombinant yeast histone octamers and

16 Deyter S 17 approximately 9 μ g of purified recombinant His-Nap1 in 10 mM HEPES-KOH (pH

7.6), 50 mM KCI, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol for 4 h at 30°C with constant mixing. The beads were washed five times with 1 ml of 25 mM HEPES-

KOH (pH 7.6), 600 mM KCI, 0.1 mM EDTA, 0.5 mM EGTA, 5 mM MgCl2, 20% glycerol to remove unincorporated octamers and His-Nap1 from the immobilized nucleosomes. To confirm the loading of histones on DNA, histones were eluted from the immobilized templates with sample buffer and analyzed by SDS-PAGE and silver staining.

Chromatin Fractionation Assays

Chromatin fractionations were performed similar to (Keogh et al. 2006) with the following modifications. 50 ml cells were grown in YEP + lactic acid media to OD600 ~ 0.7, and galactose was added to a final concentration of 2% for

1 hr. For the Spt16 degradation experiments, NAA was added 1 hr before galactose induction. In Figure 1E, cycloheximide was added for 30 minutes after galactose induction. Sodium azide was added to 0.1% prior to centrifugation.

The pellets were washed once with 10 ml ddH2O and then once with 10 ml SB buffer (1 M Sorbitol, 20 mM Tris-HCl (pH 7.4)). Pellets were flash frozen in liquid nitrogen and stored at -80°C. Pellets were thawed on ice and washed once with

1.5 ml PSB (20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 100 mM NaCl, 10 mM β - mercaptoethanol) and then once with 1.5 ml SB, spinning at 9,000 rpm for 1 min to collect cells. Spheroplasting was performed by resuspending cells in 1 ml SB and adding 10 μ l zymolase (5 mg/ml stock) and 10 μ l of 1 M DTT. [Type text] [Type text] [Type text]18

Spheroplasting efficiency was monitored by adding cells to 1% SDS and monitoring the decrease in OD600 until it was ~10% of its initial value. 1 ml of SB was added and spheroplasts were centrifuged for 7 min at 2,000 rpm at 4°C.

The 1 ml SB wash was repeated, followed by resuspension of spheroplasts in

500 μ l EBX (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.25% Triton X-100, 15 mM

β-mercaptoethanol) supplemented with protease and phosphatase inhibitors.

Additional Triton X-100 was added (0.5% final) to the resuspended cells to lyse the cell membrane and the tubes were incubated on ice for 10 min with intermittent inversion of tubes during the incubation period. A WCE (whole cell extract) sample was taken, and the remaining cellular solution was layered over

1 ml NIB (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1.2 M Sucrose, 15 mM β - mercaptoethanol) supplemented with protease and phosphatase inhibitors.

Tubes were centrifuged for 15 min at 12,000 rpm at 4°C and the supernatant was collected (soluble fraction). The crude nuclear pellets were resuspended in 500

μl EBX, additional Triton X-100 was added (1% final) to lyse the nuclear membrane, and tubes were placed on ice for 10 min as before. Tubes were centrifuged for 10 min at 13,000 rpm at 4°C. The crude chromatin pellets were washed four times with 500 μ l EBX, twice with 500 μ l (EBX + 150 mM NaCl), and once with 500 μ l EBX to remove residual soluble protein contaminants, spinning for 1 min at 12,000 rpm each time to collect chromatin. Pellets were resuspended in 500 μ l EBX and sonicated five times for 10 sec each on ice to release chromatin and its associated factors. Tubes were spun for 2 min at

13,000 rpm and the supernatant was collected (chromatin fraction). Samples

18 Deyter S 19 were then analyzed by immunoblotting or by immunoprecipitating the fractions as described above. [Type text] [Type text] [Type text]20

Table S1. Yeast strains used in this study. All strains are isogenic with the W303 background. Plasmids are indicated in brackets

Strain Genotype SBY3 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 SBY5647 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1::LEU2, SPT16-3FLAG::KANMX SBY6006 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 spt16-22 SBY6423 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 PSH1-13MYC::HIS3 SBY6439 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1::LEU2 PSH1-13MYC::HIS3 SPT16-3FLAG::KANMX SBY7125 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 PSH1-3FLAG::TRP1 SBY8336 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 psh1::KANMX SBY8851 MAT leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3- 1::pGAL-3FLAG-CSE4::URA3 (pSB1665) SBY8903 MATa leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3- 1::pGAL-3FLAG-CSE4::URA3 (pSB1665) psh1::KANMX SBY9977 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1::LEU2 PSH1-13MYC::HIS3 SPT16-TAP::TRP1 SBY9978 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1::LEU2 PSH1-13MYC::HIS3 POB3-TAP::TRP1 SPT16- 3FLAG::KANMX SBY10626 MAT ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 NHP6A::TRP1 NHP6B::TRP1 SBY10917 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1::LEU2 PSH1(1-300)-13MYC::HIS3 SPT16-3FLAG::KANMX SBY10919 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 PSH1(1-300)-13MYC::HIS3 SBY10920 MAT leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3- 1::pGAL-3FLAG-CSE4::URA3 (pSB1665) PSH1(1-300)- 13MYC::HIS3 SBY10921 MATa leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3- 1::pGAL-3FLAG-CSE4::URA3 (pSB1665) PSH1(1-300)- 13MYC::HIS3 SBY11090 MATa leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3- 1::pGAL-3FLAG-CSE4::URA3 (pSB1665) PSH1-13MYC::HIS3 SBY11102 MATa leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3-1:: pCSE4-3FLAG-CSE4::URA3 (pSB1067) CSE4::KANMX PSH1- 13MYC::HIS3 SBY11104 MATa leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3-1:: pCSE4-3FLAG-CSE4::URA3 (pSB1067) CSE4::KANMX psh1(1- 300)-13MYC::HIS3 20 Deyter S 21

SBY11186 MATa leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3- 1::pGAL-3FLAG-CSE4::URA3 (pSB1665) spt16-22 SBY11265 MATa ura3-1 leu2,3-112 trp1-1 ade2-1 can1-100 bar1-1 his3- 11::pADH1-OsTIR1-9MYC::HIS (pSB1934) SPT16-AID::KANMX SBY11268 MATa leu2,3-112 trp1-1 ade2-1 can1-100 bar1-1 ura3-1::pGAL- 3FLAG-CSE4::URA(pSB1665) his3-11::pADH1-OsTIR1-9MYC::HIS (pSB1934) SPT16-AID::KANMX SBY11293 MAT leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3- 1::pGAL-3FLAG-CSE4::URA3 (pSB1665) NHP6A::TRP1 NHP6B::TRP1 SBY11295 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 pob3-L78R SBY11296 MATa leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1 ura3- 1::pGAL-3FLAG-CSE4::URA3 (pSB1665) pob3-L78R [Type text] [Type text] [Type text]22

Table S2. Plasmids used in this study.

Plasmid Description pSB8 pGEX2T GST empty vector pSB283 pBluescript SK(-) pSB1067 pCSE4-3FLAG-CSE4 + 500 bp downstream, URA3 (integrating) pSB1535 GST-PSH1 in pGEX2T pSB1541 GST-PSH1 C45S C50S in pGEX2T pSB1665 pGAL-3FLAG-CSE4 + 500 bp downstream, URA3 (integrating) pSB1721 pMAL-C2X MBP empty vector pSB1767 MBP-Nhp6A in pMAL-C2X pSB1843 X. borealis 5S gene in pUC18 (also known as pXP-10) pSB1887 MBP-Pob3 in pMAL-C2X pSB1888 MBP-Spt16 in pMAL-C2X pSB2165 GST-PSH1(1-350) in pGEX2T pSB2166 GST-PSH1(1-325) in pGEX2T pSB2167 GST-PSH1(1-300) in pGEX2T pSB2168 His-Nap1 in pET28 pSB2169 GST-Pob3 in pGEX2T pSB2170 MBP-Spt16(485-804) in pMAL-C2X pSB2171 MBP-Spt16(485-642) in pMAL-C2X pSB2172 MBP-Spt16(643-804) in pMAL-C2X pSB2173 CSE4 in pET28 pSB2174 H4 in pET22 pSB2175 H2A/H2B in pCDFDuet pSB2176 2V5-CSE4 in pET28 pSB2234 GST-PSH1(301-406) in pGEX2T

Supplemental References

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Akiyoshi B, Sarangapani KK, Powers AF, Nelson CR, Reichow SL, Arellano-

Santoyo H, Gonen T, Ranish JA, Asbury CL, Biggins S. 2010. Tension

directly stabilizes reconstituted kinetochore-microtubule attachments.

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