Running Header: FUNCTIONAL ANALYSIS OF THE CONSERVED TRANSCRIPTION ELONGATION FACTOR SPT5 FUNCTIONAL ANALYSIS OF THE CONSERVED TRANSCRIPTION ELONGATION FACTOR SPT5

Running Header: FUNCTIONAL ANALYSIS OF THE CONSERVED TRANSCRIPTION ELONGATION FACTOR SPT5

FUNCTIONAL ANALYSIS OF THE CONSERVED TRANSCRIPTION ELONGATION

FACTOR SPT5

Emily Schwenger

McGill University

Author Note

This paper was prepared under the expert supervision of Dr. Jason Tanny of the Department of Pharmacology and Therapeutics at McGill University.

FUNCTIONAL ANALYSIS OF THE CONSERVED TRANSCRIPTION ELONGATION FACTOR SPT5

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ABSTRACT INTRODUCTION: The passage of RNA polymerase II (RNAPII) during transcript elongation is

dependent upon accessory proteins, histone modifications, and large enzymatic complexes that

communicate with each other to regulate transcription rate and processivity. The elongation step

is a critical target for regulation at many genes in metazoans, and aberrant elongation is

associated with a variety of diseases, including cancer, HIV, and cardiac hypertrophy (Cherrier et

al. 2013, Le Douce et al., 2012). The transcription elongation factor Spt5 is directly bound to

RNAPII and is important for coupling chromatin modification states and RNA processing to

RNAPII elongation. Spt5 is phosphorylated by Cdk9, the kinase subunit of the positive

elongation factor (P-TEFb). Phosphorylation of Spt5 is critical for its functions in elongation but

the relevant downstream pathways have not been elucidated. Our lab has provided genetic

evidence that Spt5 phosphorylation (Spt5-P) has a close functional relationship with co-

transcriptional histone modifications such as monoubiquitylation of histone H2B (H2Bub1) and

methylation of lysine 4 of histone H3 (H3K4) (Sanso et al., 2012). METHODS: Here we further

extend our genetic analysis of the role of Spt5 in transcript elongation through genome-wide

synthetic genetic analysis in the model eukaryote Schizosaccharomyces pombe . Observed

growth defects, or synthetic lethal and synthetic sick (SSL) genetic interactions, were followed

up with phenotypic analysis by fluorescence microscopy. RESULTS: Spt5 mutants exhibited

genetic interactions with mutants affecting the histone variant H2A.Z, its chaperone the SNF2-

related helicase Swr1 complex comprised of Swr1, Swc2, and Msc1, and nucleoporin Mex67. In

particular, deletion of H2A.Z+ and msc1+ combined with mutation of the phosphorylation site of

Spt5’s CTR created a striking synthetic interaction bordering on lethality. CONCLUSION:

Taking into account that Spt5 is one of RNAPII’s only accessory proteins that is universally


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conserved across all domains of life, an implied overlapping function with a non-canonical

histone variant could be translated into humans and manipulated into an effective and specific

therapy for diseases involving aberrant gene expression, such as cancer (Hartzog et al., 2013).

INTRODUCTION

Eukaryotes have evolved means to regulate gene expression for a wide range of purposes,

including cell maintenance, delineation, and development (Campos et al., 2009). Central to gene

regulation is the nucleosome, composed of a histone octamer, 147 base pairs of DNA, and a

linker histone. While the typical histone core consists of two H2A-H2B dimers flanked by two

H3-H4 dimers, a variety of histone variants exist that can substitute in for any of the canonical

histones, serving functions such as centromeric silencing, activation of highly transcribed genes,

and DNA repair (Chen et al., 2014).

In addition to the central nucleosome, layers of protein-DNA and protein-protein

interactions form a complex network that oversees the regulation and processing of the mRNA

transcript. Paramount to this regulation is the recruitment and passage of RNA polymerase II

(RNAPII), which is associated with highly conserved patterns of histone modifications. These

patterns include, but are not limited to, methylation of histone H3 Lys4 (H3K4me) and

acetylation of histones H3 and H4 at 5’ ends, methylation of histone H3 Lys36 (H3K36me)

towards 3′ ends, and mono-ubiquitylation of histone H2B at a conserved site in the carboxyl-

terminus (H2Bub1) throughout coding regions of genes (Sanso et al., 2012). While some of these

modifications exert their main effects on transcription by altering the intrinsic structure of

chromatin, such as the charge neutralizing effects of histone acetylation, many function


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predominantly through the creation or elimination of binding sites. Though much is known about

how chromatin modifications regulate recruitment of RNA polymerase II and general

transcription factors to the promoter region during transcription initiation, their roles in transcript

elongation and the precise spatial and temporal arrangement of the overlapping processes of

generating a mature mRNA, such as capping and splicing, are poorly understood.

Transcription elongation factors Spt4 and Spt5 are bound directly to RNAPII and are its

only accessory proteins universally conserved across all domains of life. Spt5 has been shown to

have an essential function in the activation of transcription elongation and its deletion is lethal to

both budding and fission yeast (Hartzog et al., 2013). Importantly, Spt5 plays a vital role in

relieving RNAPII from its transcriptionally engaged but paused state just downstream of the

promoter. This process of promoter-proximal halting was originally thought to have unique

involvement in rapid activation of stress-induced heat shock proteins (HSPs) in Drosophila, but

is now known to take place across the genome (Gilchrist et al., 2012). Transcription machinery

and transcription factors are pre-assembled at a promoter-proximal region, thus making it

possible for elongation to be activated almost instantaneously in response to transcription

activators such as c-Myc (Rahl et al., 2010). Moreover, pausing facilitates recruitment of

mRNA-processing factors, and can be rate-determining for expression of tightly regulated genes

(Sanso et al., 2012, Core et al., 2008).

Spt5 is known to associate with RNAPII until its release near the 3’ end of the gene,

therefore, release from pausing does not encompass its full function. Spt5 has been shown to

interact with the RNA polymerase II associated factor 1 (Paf1) complex which itself associates

with elongating RNAPII and coordinates a variety of histone modifications (Squazzo et al.,

2002). Furthermore, it has known functional interactions with H3K4me3, H3K36me3, H2Bub1,


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and subunits of the Rpd3S histone deacetylase complex, whose function in occluding cryptic

promoters is dependent on H3K36 methylation (Quan et al., 2010).

Spt5 is phosphorylated by Cdk9, the kinase subunit of the essential positive elongation

factor P-TEFb, at threonine 1 of its CTR. This residue was mutated to create two of the query

strains used in this study, spt5T1A and spt5T1E, constituting ablation and constitutive activation

of the Cdk9 phosphorylation site, respectively. The CTD of RNAPII is similarly phosphorylated

by Cdk9 and other cyclin-dependent kinases that regulate transcription. These phosphorylations

are critical for RNAPII elongation, pre-mRNA-processing, histone modifications, and the release

from promoter-proximal pausing described above. Cdk9 activity is required for generation of co-

transcriptional H2Bub1, H3K4me, and H3K36me, although the molecular mechanisms have yet

to be determined.

Synthetic Genetic Analysis and the Cdk9-spt5-H2Bub1 Pathway

We have recently shown that Cdk9 and phosphorylated Spt5 (Spt5-P) have an intimate

functional connection with co-transcriptional histone modifications in the fission yeast S. pombe

through establishment of a positive feedback loop with H2Bub1. Our study also suggests that

Spt5-P and H3K4me may have a shared function comprising that of H2Bub1. We sought to gain

further insight into this function by collaborating with the lab of Dr. Nevan Krogan (University

of California, San Francisco) who had previously developed a high-throughput screen designed

for epistatic mapping of the entire S. pombe genome. The screen consisted of a system of genetic

manipulations combined with introduction of binary combinations of double mutants through

genetic crossing (Roguev et al., 2007). Selection strategies were four-fold in order to provide a

robust and efficient way of selecting for double mutants amongst a mixture of parent cells,

diploids, and recombinant cells. Their strategies included anti-diploid selection, mating type


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selection, and antibiotic and auxotrophic markers for mutant selection. Spt5T1A, spt5T1E, and

spt5∆C were the query strains of interest, and candidate mutants inducing observable growth

defects when mated with any of the query strains were further investigated through tetrad

analysis.

This report describes the characterization of a selection of genetic interactions identified

by the high-throughput screen. These analyses validated some key interactions and suggested

potentially novel functions for Spt5 in regulation of gene expression.

MATERIALS & METHODS

Schizosaccharomyces Pombe

The S. pombe strains used are listed in Table 1 below.

Table 1. List of S. pombe strains used

Strain Name Strain Information Reference Wild type ade6-M216 ura4+ leu+ Tanny et al., 2007 h2b-K119R htb1-K119R::kanMX6; ade6; ura+; h+ Tanny et al., 2007 h2b-K119R htb1-K119R::hphMX6; ade6; ura+; h+ This study spt5-WT spt5-WT(7)::ura4+ ade6-M210 leu1-32 ura4-D18

his3-D1 h- Fisher et al., 2006

spt5-T1A spt5-T1A(7)::ura4+ ade6-M210 leu1-32 ura4-D18 his3-D1 h-

Fisher et al., 2006

spt5-T1E spt5-T1E(7)::ura4+ ade6-M210 leu1-32 ura4-D18 his3-D1 h-

Fisher et al., 2006

spt5∆C

spt5∆C::ura4+ ade6-M210 leu1-32 ura4-D18 his3-D1 h-

Fisher et al., 2006

spt5-WT spt5-WT(7)::natMX6 (3 isolates) ade6-M210 leu1-32 ura4D-18 mat1_m-cyhS smt0 rpl42::cyhR (sP56Q) h-

Roguev et al., 2007

spt5-T1A spt5-T1A::natMX6 (3 isolates) ade6-M210 leu1-32 ura4D-18 mat1_m-cyhS smt0 rpl42::cyhR (sP56Q) h-

Roguev et al., 2007

spt5-T1E spt5-T1E::natMX6 (3 isolates) ade6-M210 leu1-32 ura4D-18 mat1_m-cyhS smt0 rpl42::cyhR

Roguev et al., 2007


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(sP56Q) h- pht1∆ pht1∆::hphMX4; ade6 ura4-D18 h- Tanny et al., 2007 msc1∆ msc1∆::kanMX6; leu1-32; ura- h- Tanny et al., 2007 sir2∆ sir2∆::kanMX6 h- Ekwall et al., 2005

clr3∆ clr3∆::kanMX h- Ekwall et al., 2005

pmt3∆ pmt3∆::hphMX6; ade6 h- Schneider et al., 2010 hos2∆ hos2∆::leu2; leu1-32 h- Ekwall et al., 2005

rtt109∆ rtt109∆::kanMX6 h- Schneider et al., 2010 msc1∆::kanR; leu1-32; h+ Ahmed et al., 2004

pht1∆ pht1∆::hphMX4; ade6; h+ Tanny et al., 2007 swr1∆ swr1∆::kanMX4; ade6-M216; ura4-D18; leu1-32;

h- Bioneer, Inc.

swc2∆ swc2∆::kanMX4; ade6-M216; ura4-D18; leu1-32; h-

Bioneer, Inc.

nrc1∆ nrc1∆::kanMX4; ade6-M216; ura4-D18; leu1-32; h-

Bioneer, Inc.

nto1∆ nto1∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. nup132∆ nup132∆::kanMX4; ade6-M216; ura4-D18; leu1-

32 Bioneer, Inc.

mst2∆ mst2∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. epe1∆ epe1∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. sgf29∆ sgf29∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. mug183∆ mug183∆::kanMX4; ade6-M216; ura4-D18; leu1-

32 Bioneer, Inc.

png2∆ png2∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. mex67∆ mex67∆::kanMX4; ade6-M216; ura4-D18; leu1-

32 Bioneer, Inc.

ulp2∆ ulp2∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. elp1∆ elp1∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. cxr1∆ cxr1∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. spt2∆ spt2∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. dbl8∆ dbl8∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. kap114∆ kap114∆::kanMX4; ade6-M216; ura4-D18; leu1-

32 Bioneer, Inc.

nap2∆ nap2∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. clr2∆ clr2∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc. lsg1∆ lsg1∆::kanMX4; ade6-M216; ura4-D18; leu1-32 Bioneer, Inc.


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Tetrad Dissection

S. pombe frozen glycerol stocks were streaked onto agar plates with yeast extract

supplement (YES) containing 5 g/L yeast extract, 30g/L glucose, and 250mg/L each of histidine,

leucine, adenine and uracil. These were grown at 30˚C in a dry box incubator until colonies were

visible, usually overnight for one to two days.

Two strains of opposite mating type, one h+ and the other h-, were subsequently mated.

A colony from each strain was spread, one on top of the other, onto a small patch on a standard

SPA mating plate (Moreno et al., 1991). The plates were then kept at room temperature for two

to four days, during which sporulation was induced through means of nitrogen starvation, as per

standard S. pombe mating protocol (Moreno et al., 1991).

Prior to preparing the tetrad dissection, the patch of mixed cells was inspected for the

presence of tetrads under a standard compound microscope at a magnification of 40x. If tetrads

were present, a swab of cells was diluted in 1 mL distilled water with thorough mixing. 20 µL

was subsequently plated in a bead near the edge of an agar YES plate, which was gently tilted to

form a line of cells down the middle of the plate.

After allowing five minutes to dry, the plate was placed upside-down on a staging

platform above a mounted micromanipulator, all part of the Zeiss Axioskop 40 tetrad dissection

microscope. 14 tetrads were selected and placed in a vertical line parallel to the central line of

cells, 7 on each side. They were then left at 37˚C to encourage breakdown of the ascus wall and

checked every few hours. Tetrads with broken down ascii were subsequently dissected, with

each of the four individual cells placed 3 mm apart in a horizontal line.

If only a minority of asci broke down before the end of the day, the plate was left

overnight at room temperature or in a 20˚C dry box incubator and checked for broken down


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tetrads the following morning. Leaving the plate at 37˚C for more than 5 hours is discouraged

due to the possibility of cell doubling, which would make it impossible to accurately isolate the

cells into the four individual haploid products of meiosis.

This cycling of incubation and dissection was repeated until the majority of the 14

selected tetrads were successfully dissected. The plate was then incubated at 30˚C for a few days

until colonies grew to about 1 mm in diameter.

Thereafter, the tetrad colonies were replica plated onto selective media according to the

respective antibiotic resistance or auxotrophic markers of the parent cells and incubated

overnight at 30˚C. Agar plates were made with 200 mg/L geneticin (G418, Sigma-Aldrich), 200

mg/L hygromycin (Sigma-Aldrich), 200 mg/L nourseothricin (clonNAT, Werner BioAgents),

and EMM –ura, as per standard protocol (Moreno et al., 1991).

Microscopy

S. pombe cells were fixed and stained with diamino-phenylindole (DAPI) and calcofluor as

described previously (Viladevall et al., 2009). Cells were viewed using a Leica DM5000b

microscope and photographed with a CCD camera. Images were processed using Volocity

software.

Creation of h2b1-K119R::hygromycin Mutant Strain

To introduce a hygromycin marker in place of a G418 marker into a G418R h2b1-K119R

mutant, the primer sequences 5’-CGGATCCCCGGGTTAATTAAGGCG-3’ and 5’-

GAATTCGAGCTCGTTTAAACACTGGATGGCGGCGTTAGTATCG-3’ were used to

amplify a hygromycin B (hygB) cassette with flanking homology regions from plasmid pFA6a-


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hphMX6 (Sato et al., 2005). Synthesis was catalyzed by Taq DNA Polymerase (Invitrogen) in

reactions containing PCR Reaction Buffer (Invitrogen), MgCl2 (Invitrogen),

deoxyribonucleotides (Invitrogen) as per standard PCR protocol (Azuma et al., 1991).

10 mL liquid cultures were grown overnight, spun down in a Thermo tabletop centrifuge

at 3000 rpm for two minutes and washed with water and a solution of 100 mM lithium acetate,

10 mM Tris, and 1 mM EDTA (LiAc/TE), consecutively. They were then resuspended in the

LiAc/TE at a concentration of 5 x 10exp8 cells per mL. 100 µL cells was mixed with 1 µL

salmon sperm DNA (Invitrogen) and 2 µg transforming DNA, and the mixture was left to rest at

room temperature for ten minutes. A solution of LiAc/TE and Polyethylene glycol was added

prior to a one hour incubation at 30˚C, followed by addition of 43 µL dimethyl sulfoxide and 20

minute heat shock in a 42˚C water bath. Cells were recovered by incubating on non-selective

YES plates and replica plated onto hygromycin B media when colonies were easily observable.

Western Immunoblotting

S. pombe cells were grown as per standard procedure (Moreno et al., 1991). 10 mL

cultures were grown in a shaking incubator at 30˚C until they reached an optical density between

0.4 and 0.6, indicative of logarithmic growth. These were then spun down, washed in 5-10 ml of

20% trichloroacetic acid (TCA), and stored at -80˚C.

Cells were lysed through agitation with cold-acid-washed glass beads (Sigma, 425-600

microns) using the MiniBeadBeater-16 (Biospec) for four thirty-second intervals with one

minute breaks in between. 1 mL 5% TCA was added and the liquid retrieved and spun down for

ten minutes at maximum speed using a standard tabletop centrifuge. The supernatant was


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discarded, samples were resuspended in Laemmli buffer (50µM Tris pH 6.8, 2% SDS, 10%

glycerol, 5% β-mercaptoethanol, bromophenol blue), and neutralized with 2M Tris base.

15% acrylamide (BioRad) gels were set and samples run at a voltage of 120 V for fifteen

minutes, followed by 180V until the front of the loading dye ran into the buffer. The running

buffer used contained 3 g/L of Tris base, 20g/L glycine, and 0.1% SDS.

Transfer was performed at a constant current of 200 mA on ice in cold transfer buffer

containing 3.6g/L Tris, 15g/L Glycine, 0.4g/L of sodium dodecyl sulfate (SDS) and 2%

methanol. Nitrocellulose membranes were blocked for one hour on a shaking platform with 5%

skim milk powder dissolved in Tris-buffered saline with Tween-20 (TBST). Primary antibody

sources and concentrations can be found in Table 2. Membranes were incubated overnight at 4˚C

in primary antibody followed by one hour with anti-rabbit antibody conjugated with HRP (GE

Healthcare UK Limited) at a dilution of 1 to 4000 in TBST, with three five minute washes in

TBST after each incubation. Finally, the ECL Prime detection kit (BioRad) procedure was

followed, and the nitrocellulose membrane exposed on film.

Table 2. Western Blotting Conditions

Proteins Primary Antibody Concentration

Source of Primary Antibody

!-H3

H3K4me3

H3K36me3

1:5000

1:3000

1:2000

Abcam

Abcam

Abcam


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RESULTS

In order to further elucidate the mechanisms behind

the cdk9-spt5-H2Bub1 pathway in governing

chromatin states and transcription, we analyzed

putative SSL interactions from a high-throughput

screen involving genome-wide epistasis mapping.

The screen was carried out by our collaborator Dr.

Nevan Krogan (UCSF, Roguev et al., 2007). We

sought to confirm genetic interactions from this screen of candidate knockout mutants

queried against htb1-K119R and three mutant spt5 strains.

Two of the spt5 strains used are characterized by point mutations at the phosphoacceptor

threonine residue (Thr1) in the CTR. Spt5∆C has its entire CTR deleted, and therefore exhibits a

wider array of genetic interactions than the point mutants. To conduct the screen we re-

constructed the spt5T1A and spt5T1E mutants, as well as a wild-type spt5+ control, in the

standard strain used for screening by the Krogan lab (Roguev et al., 2007). DNA sequences

encoding the variant Spt5 C-termini were introduced into yeast and recombinants were selected

using a linked antibiotics resistance marker. PCR analysis of the transformants confirmed correct

integration of the new spt5 sequences (see Figure 1).

Tetrad dissection is a powerful genetic technique unique to yeast and was our primary

follow-up to the genetic interactions displayed in the screen. It is based on the premise that

diploid cells undergo meiosis and package the four haploid nuclei into spores, with each haploid

cell containing a distinct genotype (Moreno et al., 1991). The most common segregation pattern

ura4+ -‐2.7 kb

NATMX6 -‐2.8 kb Figure 1. Confirmation of spt5 CTR by PCR Spt5 CTR was amplified. The two sets of transformants exhibit correct size of PCR product, representing the length of the CTR added to the flanking marker, ura4+ or clonNAT.

spt5-‐W

T

spt5-‐T1A

spt5-‐T1E


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is tetratype, with two cells identical to each of the single mutant parent cells, and two

recombinant cells, one wild-type and one double mutant. The four cells are manually separated

and the double mutant is isolated after antibiotic and/or auxotrophic selection. This acts as a

robust and efficient strategy for performing genetic manipulations and observing synthetic

interactions that provide useful insight into protein functionality, and was used to validate the

results from the high-throughput screen (summarized in Table 3).

We confirmed a subset of strong negative and positive interactions identified by the

screen. We crossed previously analyzed spt5 mutant strains (marked with ura4+ conferring

uracil prototrophy) with individual gene knockout strains provided by the Krogan lab (marked

with a G418R cassette). We also tested interactions with the spt5∆C mutant and the functionally

related htb1-K119R mutant, both of which display a stronger growth phenotype than the

phosphoacceptor mutants. We reasoned these crosses might more sensitively reveal genetic

interactions. Tetrads were dissected and plated on selective media containing G418, hygB,

and/or EMM-ura to isolate the double mutants. Observed growth patterns are summarized in

Table 3.

Tetrad dissections with the spt5 mutants revealed six novel genetic interactions. The most

striking of these were pht1+ and msc1+ knockout mutants, which produced synthetic interactions

bordering on lethality when crossed with all three spt5 mutants (Figure 4). The interactions with

spt5T1A and spt5T1E are indicative of a specific, shared function of both Pht1 and Msc2 with

Cdk9, the upstream kinase responsible for phosphorylating the substituted threonine residue.


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Pht1 is the S. pombe homolog of the histone variant H2A.Z, which plays an important

role in governing boundaries between chromatin states (Campos et al., 2009). Interestingly,

Msc1 is a key regulator in the Swr1 complex, the chaperone responsible for loading H2A.Z. By

htb1-‐K119R x HTS

Result Tetrad Analysis Result

pmt3∆ (-‐) N/IT hos2∆ (-‐) N/IT clr3∆ (-‐) N/IT sir2∆ (-‐) C rtt109∆ (-‐) C

spt5WT spt5T1A spt5T1E spt5∆C x HTS

Result Tetrad Analysis Result

HTS Result

Tetrad Analysis Result

HTS Result


HTS Result


pht1∆ (-‐) C (-‐) SSL (-‐) SSL SSL pht1∆ C C (-‐) (-‐) (-‐) (-‐) n/a (-‐) msc1∆ C C (-‐) (-‐) (-‐) (-‐) n/a (-‐) swr1∆ C C (-‐) C (-‐) C n/a (-‐) swc2∆ C C (-‐) C (-‐) C n/a (-‐) nrc1∆ C N (-‐) N (-‐) N/IT n/a (-‐) nto1∆ C N/IT (-‐) N/IT (-‐) N/IT n/a N/IT nup132∆ C C (-‐) C (-‐) C n/a N/IT mst2∆ C N/IT (-‐) N/IT (-‐) N/IT n/a N/IT epe1∆ C C (-‐) N/IT (-‐) N/IT n/a N/IT sgf29∆ C C (-‐) N (-‐) N n/a N mug183∆ C C (-‐) N (-‐) N/IT n/a N/IT png2∆ C C (-‐) C (-‐) C n/a C mex67∆ C C (-‐) C (-‐) C n/a (-‐) ulp2∆ C C (-‐) N (-‐) N n/a N/IT elp1∆ C N/IT (-‐) N/IT (-‐) N/IT n/a N/IT cxr1∆ C C (-‐) C (-‐) C n/a C spt2∆ C C (+) C (+) C n/a C dbl8∆ C C (+) C (+) C n/a C kap114∆ C C (+) C (+) C n/a C nap2∆ C C (+) C (+) C n/a C clr2∆ C N/IT (+) N/IT (+) N/IT n/a N/IT lsg1∆ C C (+) N (+) N n/a N

Table 3. Results of Tetrad Analyses Candidate strains were crossed with either htb1-K119R or spt5 mutant query strains. Double mutant progeny were examined for growth defects or enhancements after plating on EMM-ura G418, and/or HygB selective media; N=no pattern; IT=incomplete tetrads; C=consistent colony sizes for all four haploids; (-)=synthetic sick or synthetic lethal; (+)=double mutants are observably healthier than single mutants. N/A=


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displaying similar patterns in growth defects, Msc1 corroborates our finding that H2A.Z

genetically interacts with Spt5 and Cdk9.

Staining with DAPI and calcofluor revealed wild-type phenotypes across all double

mutants with the exception of the pht1∆spt5∆C mutant, which exhibited marked septation

defects resembling those observed in the htb1-K119R single mutant (Figure 5). This implies a

greater functional overlap of pht1 with the truncation mutant over the phosphorylation site

mutants, which can be attributed to the added functions of other residues along Spt5’s CTR that

may serve as docking sites for various transcriptional regulators and chromatin remodelers.

However, it is important to note that spt5 mutants lacking a CTR display some minor septation

defects on their own (data not shown).

We further studied the

genetic interaction between

Spt5 and H2A.Z by performing

tetrad dissections of the histone

exchange chaperones Swr1 and

Swc2, responsible for

facilitating incorporation of the

histone variant H2A.Z. Both swr1∆ and swc2∆, of the swr1 complex, formed pronounced

synthetic sick interactions with spt5∆C, but not with either of the spt5 point mutants.

Accordingly, this reaffirms a shared function between H2A.Z and Spt5. Despite this, it appears

that the Swr1 complex lacks a specific interaction with Cdk9 and, rather, interacts with another

facet of Spt5.

pht1∆

msc1∆

spt5WT spt5T1A spt5T1E spt5∆C Figure 2. Effects of different mutations in the CTR of spt5 Pht1 and msc1 deletion mutants were crossed with spt5 query strains. The smallest colonies represent the double mutants, as confirmed by plating on minimal media lacking uracil and G418.


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Furthermore, nrc1∆ and mex67∆ exhibited synthetic interactions with the spt5 truncation

mutant. However, it is important to note that the wild-type control for the nrc1∆ cross did not

show four healthy colonies, as expected. For this reason, this cross should be repeated to reaffirm

swr1 x spt5C swc1 x spt5C nrc1 x spt5C mex67 x spt5C Figure 3. Four confirmed genetic interactions with spt5C Tetrad analysis of four candidate strains. In all four crosses, the double mutant is represented by the smallest colony or no colony due to synthetic lethality.

set1spt5-‐T1A

sir2h2b1-‐K119R

wt

pht1spt5-‐T1A pht1spt5-‐T1E pht1spt5C

rtt109h2b1-‐K119R

Figure 4. Phenotypic Analysis of Septation Defects Micrographs of putative enhancers of spt5 and h2b-K119R mutants. Samples of cells were fixed and stained with DAPI and calcofluor to visualize DNA and division septa, respectively. The pht1∆spt5∆C mutants appear to have slightly enhanced septation defects, indicated by the arrows.

set1spt5T1A

clr3h2b1-‐K119R


17

that the observed growth defects in the truncation mutant are a result of a synthetic genetic

interaction, and not the effects of the nrc1+ knockout alone.

Mex67∆, on the other hand, crossed successfully with all strains, and, noteably, displayed

consistent SSL interactions with the spt5 truncation mutant. In line with all other crosses, the

double mutant was verified via plating on G418 and EMM -ura selective media, reaffirming that

Mex67 has a redundant function with Spt5, independent of Cdk9.

Many of the crosses exhibited consistent colony sizes, indicating that some of the

putative enhancers were false positives. Moreover, they appeared phenotypically wild-type after

staining with DAPI and

calcofluor (data not shown),

suggesting that no genetic

interaction exists and, as a result,

they do not share any appreciable

function with the transcription

elongation factor Spt5. It is

suggested that all tetrad analyses

with either no observable pattern

and/or incomplete tetrads are

repeated to attain a greater sample size and increase the likelihood of a discernible pattern.

We examined whether some of the knockouts that positively interact with the spt5

mutants might show a similar positive effect on the htb1-K119R phenotypes, which are more

severe. Crosses done with htb1-K119R produced markedly sick tetrads that were often

incomplete sets of two or three, making it difficult to identify any synthetic interactions. In order

H3K36me3 -‐15 kDa

H3K4me3 -‐15 kDa

!-‐H3 -‐15 kDa Figure 5. Comparison of methylation patterns in the spt5 CTR mutants The H3K4me3 displays an effect of phosphorylated Thr1 on lysine 4 trimethylation. As described previously, lysine 4 trimethylation is activated by Cdk9 phosphorylating Spt5. However, the phosphoacceptor Thr1 residue of Spt5 shows no significant effect on H3K36 trimethylation.

Wild ty

pe

H2b1

-‐K11

9R

Set2

Spt5-‐W

T

Spt5-‐T1A

Spt5-‐T1E

Spt5C


18

to discern a reliable pattern, these crosses should be repeated in larger numbers, as well. The

sir2+ and rtt109+ knockouts exhibited no added growth defects in combination with htb1-K119R.

Subsequent staining with DAPI and calcofluor revealed familiar septation defects in the double

mutants seen previously in the htb1-K119R alone, usually in the form of cells with multiple

division septa (Figure 5). This, combined with the lack of defect in growth, indicates that these

strains do not interact genetically.

Finally, immunoblot analysis was performed to characterize the dependency of

trimethylation of lysines 4 and 36 of H3 on Spt5 phosphorylation state. Previous knowledge that

Spt5 phosphorylation promotes H3K4me3 was reiterated, along with the absolute requirement of

H2Bub1 for H3K4me3. H3K36me3, on the other hand, does not require H2Bub1. Notably, there

is no apparent difference between H3K36me3 levels between the wild-type strain and the

spt5T1A mutant. Due to prior research showing that Cdk9 activates trimethylation of H3K36, we

suggest that a different substrate exists for Cdk9 that functions to promote H3K36me3. In

addition, H3K36me3 is less dependent on Cdk9 phosphorylation of the spt5 CTR, but appears to

rely upon other functions of the CTR, independent of Cdk9 action. Therefore, the downstream

effect of the Spt5 CTR on lysine 36 trimethylation and subsequent recruitment of histone

modulators provides a potential explanation for the genetic interactions described above

observed solely with the spt5 truncation mutant. However, it is important to note that the

H3K36me3 antibody did not bind efficiently, and the H3K36me3 immunoblot should be

repeated for more accurate results.


19

DISCUSSION

These results confirm the novel genetic interactions involving the histone variant H2A.Z,

its chaperones Msc1, Swr1, and Swc2, and Spt5 or its upstream modulator Cdk9. Previous

studies have shown that H2A.Z functions in enforcing heterochromatin boundaries. With the

facilitation of the Swr1 complex (Swr1C) as chaperone, a H2A.Z-H2B dimer can substitute into

the nucleosome and restrict chromatin states to their respective domains. Our data suggest that

H2A.Z, and the Swr1C components Msc1, Swr1 and Swc2 display redundancy with Spt5. Msc1

appears to exert a regulatory function on the chaperone complex, as previous work has shown

that H2A.Z is ectopically found in the inner centromere and in subtelomeric chromatin in msc1+

deletion strains (Buchanan et al., 2009). This demonstrates that Msc1 acts as a negative regulator

of H2A.Z loading in these regions, which normally lie past the heterochromatin boundary and

are not actively transcribed. On the other hand, ablation of either Swr1 or Swc2 disrupts the

ability of Swr1C to bind and load H2A.Z into promoter regions of euchromatin (Weber et al.,

2010).

It is possible that aberrant loading of H2A.Z into condensed chromatin and consequent

transcription of inactive genes has more functional overlap with Cdk9 function than the loss of

H2A.Z observed in swr1∆ and swc2∆ mutants. This could account for the specific interaction

observed between the Msc1 and the strains mutated at the Cdk9 phosphorylation site on Spt5’s

CTR. However, this is paradoxical with the finding that loss of H2A.Z itself mimics the growth

defects of msc1∆. To better explain this discrepancy, experiments should be done to further

characterize the nature of the role of Spt5’s CTR in establishing boundaries between chromatin

states.


20

Generation of point mutations at other putative phosphoacceptor residues along the CTR

of Spt5 would further narrow down the functional overlap between H2A.Z and Spt5, and

minimize the likelihood of structural divergence induced by a truncation mutant. Moreover,

genetic analyses with pht1+ and cdk9+ knockouts would give some insight into what fraction of

the interaction, if any, can be attributed to Cdk9 activity upstream of Spt5 activity.

Furthermore, chromatin boundaries are marked by conserved consensus sequences,

therefore, transformation of a marker adjacent to a boundary consensus sequence makes it

possible to design chromatin boundary assays based on the aberrant spreading of

heterochromatin. Transforming the spt5 strains in this study in such a way and measuring

differences in expression of the marker gene could give insight into whether the CTR of Spt5

plays a role in separating euchromatin from heterochromatin (Weber et al., 2014).

The deletion of nuclear mRNA export receptor mex67 combined with the spt5∆C mutant

formed a distinct synthetic interaction independent of the four proteins described above. Mex67

acts primarily to prevent the export of unspliced pre-mRNAs until intron removal is completed to

avoid the expression of aberrant and potentially harmful proteins (Hackmann et al., 2014). This

loosely implies a shared function in mRNA quality control between the Spt5 CTR and Mex67.

Interestingly, Mex67 and the family of nucleoporins have been shown to interact with

transcription machinery. Certain export factors, such as TREX, bind co-transcriptionally to the

nascent mRNA and facilitate its packaging into a messenger ribonucleoprotein particle (mRNP,

Chanarat et al., 2012). This demonstrates that transcription and nuclear export are intrinsically

intertwined, and that the elaborate complexes forming the nuclear pore are spatially arranged

with nucleosomes in a highly organized manner. Future experiments investigating the exact

function and binding partners of Mex67 could perhaps unveil a role beyond that of mRNA


21

exporter for Mex67. The possibility of a role in chromatin boundary mechanisms for Mex67

should also be considered, given the consistent pattern in our findings.

Our results also displayed a pattern with relation to the high-throughput screen based on

the magnitude of the initial interactions (data not shown). The genetic interactions that we

successfully recapitulated showed very severe growth defects in the high-throughput screen,

whereas the double mutants that showed no difference in phenotype in the tetrad analysis

exhibited quite minor growth defects in the original screen. We attribute this to two possible

reasons. It is plausible that the high-throughput screen is more sensitive than tetrad analysis, due

to the competitive environment imposed upon the double mutants. In the initial screen, a pool of

cells were induced to mate and sequentially plated on three rounds of selective media, including

cycloheximide on EMM -ura, geneticin (G418), and nourseothricin (NAT). This requires the

double mutants to survive amongst healthier diploid cells and single mutants until their

respective selections, forcing them to contend for nutrients and space. Subtle growth defects may

be augmented as a result. On the other hand, the colonies plated from tetrad analyses are clones

of one cell, therefore no competition between strains exists.

It is, however, more likely that the succession of cycloheximide (a highly toxic protein

synthesis inhibitor) combined with minimal media and subsequent antibiotic treatment inflicted

excessive amounts of stress on the cells, contributing to the detrimental effects of the existing

mutations. This combined with potential interference between each selection is conducive to

generating false positives, not uncommon for a high-throughput screen of this nature (Wiren et

al., 2005).

This type of analysis provides a platform for in-depth investigation of a number of

different Spt5-dependent mechanisms. While much follow-up work is needed to substantiate


22

these suggested interactions, we have provided good evidence of significant functional overlap

with Spt5 and the five confirmed candidate mutants. Additional tetrad dissections should be

conducted to increase the sample size of the crosses that had incomplete tetrads and/or did not

exhibit a pattern with the aim of discerning a consistent pattern.

ACKNOWLEDGEMENTS

These studies were supported by the Canadian Institute for Health Research (CIHR). I

would like to thank Dr. Jason Tanny for his guidance, and for offering me this opportunity to

participate in this project, Dr. Jean Mbogning for his guidance and advice, and Viviane Pagé for

her technical support.


23

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