Supplemental Inventory

Supplemental Figures

Figures S1-S5

Supplemental Materials and Methods

Supplemental Tables

Table S1. Supplemental Strain Table

Table S2. Supplemental Plasmid Table

Supplemental References

Deyter S1

Figure S1. Nhp6 does not regulate Cse4 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

Deyter S2

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 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.

Deyter S3

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 -

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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.

Deyter S5

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.

Deyter S6

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% non-

denaturing PAGE, and the gel was stained with ethidium bromide to detect DNA

species.

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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

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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.

Deyter S9

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

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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.

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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 μg of the dialyzed GST

fusion proteins onto 100 μl 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 μl of the beads (~ 1.5 μg protein) were incubated with ~ 1.5 μg of the

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indicated MBP proteins in a reaction volume of 50 μl (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 μl sample buffer, and boiled for 4 min.

To purify the FACT complex, 12 L of yeast cells in log phase (OD600 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 μg/ml leupeptin, 10 μg/ml

pepstatin, 10 μg/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 μl 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

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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 μl of protein G dynabeads

(Invitrogen) coupled to 0.75 μl of M2 anti-Flag (Sigma) for 3 hr at 4°C. For TAP

tag purifications, lysates were incubated with 30 μl 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

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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 μg DNA and 8 μg Cse4 octamers were

incubated at 25°C for 30 min in a 400 μl 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

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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 μl 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

4°C.

To generate polynucleosomes, 2 μg 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 μl of 0.4 mM biotin-14-dATP (Invitrogen),

and 0.6 μl of 10 mM dTTP, dGTP, and dCTP in a 40 μl 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 μg) was

immobilized onto 100 μl Dynabeads M-280 Streptavidin (Invitrogen) overnight at

4°C. In a typical nucleosome assembly reaction, Dynabeads coupled with 2 μg

of DNA were incubated with 2 μg of recombinant yeast histone octamers and

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approximately 9 μg 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 μl zymolase (5 mg/ml stock) and 10 μl of 1 M DTT.

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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 μl 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 μl EBX, twice with 500 μl (EBX + 150 mM NaCl), and

once with 500 μl 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 μl 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

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were then analyzed by immunoblotting or by immunoprecipitating the fractions as

described above.

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

Strain GenotypeSBY3 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1SBY5647 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100

bar1::LEU2, SPT16-3FLAG::KANMXSBY6006 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1

spt16-22SBY6423 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1

PSH1-13MYC::HIS3SBY6439 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100

bar1::LEU2 PSH1-13MYC::HIS3 SPT16-3FLAG::KANMXSBY7125 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1

PSH1-3FLAG::TRP1SBY8336 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1-1

psh1::KANMXSBY8851 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::KANMXSBY9977 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100

bar1::LEU2 PSH1-13MYC::HIS3 SPT16-TAP::TRP1SBY9978 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

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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

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Table S2. Plasmids used in this study.

Plasmid DescriptionpSB8 pGEX2T GST empty vectorpSB283 pBluescript SK(-)pSB1067 pCSE4-3FLAG-CSE4 + 500 bp downstream, URA3 (integrating)pSB1535 GST-PSH1 in pGEX2TpSB1541 GST-PSH1 C45S C50S in pGEX2TpSB1665 pGAL-3FLAG-CSE4 + 500 bp downstream, URA3 (integrating)pSB1721 pMAL-C2X MBP empty vectorpSB1767 MBP-Nhp6A in pMAL-C2XpSB1843 X. borealis 5S gene in pUC18 (also known as pXP-10)pSB1887 MBP-Pob3 in pMAL-C2XpSB1888 MBP-Spt16 in pMAL-C2XpSB2165 GST-PSH1(1-350) in pGEX2TpSB2166 GST-PSH1(1-325) in pGEX2TpSB2167 GST-PSH1(1-300) in pGEX2TpSB2168 His-Nap1 in pET28pSB2169 GST-Pob3 in pGEX2TpSB2170 MBP-Spt16(485-804) in pMAL-C2XpSB2171 MBP-Spt16(485-642) in pMAL-C2XpSB2172 MBP-Spt16(643-804) in pMAL-C2XpSB2173 CSE4 in pET28pSB2174 H4 in pET22pSB2175 H2A/H2B in pCDFDuetpSB2176 2V5-CSE4 in pET28pSB2234 GST-PSH1(301-406) in pGEX2T

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