Set1 mono-methylation of H3K4 in S.

cerevisiae is advantageous
for survival under histidine starvation conditions
in the presence of 3-Amino-1,2,4-triazole
Alex Griffith, Dr. Michelle Pozzi, Sreya Bose, Amber Wadle
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA

Abstract:
Set1 is a highly conserved histone H3 lysine 4 (H3K4) methyltransferase that operates as part of
the multiprotein complex COMPASS in S. cerevisiae (Miller et al. 2001). The MLL1 protein in
humans, a homolog of Set1, also supports mono-, di- and tri- methylation activity specific to
H3K4 and has been tied to an aggressive form leukemia (Milne et al. 2002). In this study, it has
been determined that Set1 methylation of H3K4 is required for survival under histidine
starvation in the presence of 3AT, with mono-methylation being required for optimal growth. In
order to come to this conclusion, strains of S. cerevisiae were constructed using data provided by
Williamson et al to have varying levels of Set1 methylation capabilities (Williamson et al. 2013).
Strains expressing different SET1/set1 genes including SET1+, set1, Y967F-set1, Y967A-set1,
H1017A-set1 and R1013H-set1 were then spot platted on a specialized media lacking histidine
and containing 10mM 3-Amino-1,2,4-triazole (sc-his+3AT). 3AT is an inducer of the
transcriptional activator Gcn4, which is known to cause extensive nucleosome rearrangements
around the coding regions of genes (Cui et al. 2012). In the instance of HIS3, 3AT induction
results in the shift in nucleosome spacing and thereby reducing HIS3 gene expression under
starvation conditions (Cui et al. 2012). It is now believed that Set1 methylation of H3K4 can
reverse this disruption in nucleosome arrangement; specifically, mono-methylation of H3K4 by
Set1 directs the chromatin remodelers RSC and SWR-C to stress response genes (Nadal-Ribelles

et al. 2015). This cooperation between nucleosome modifying and chromatin remodeling
enzymes exhibits the dynamic relationship of events leading to gene activation or repression and
the mobilization of various signal transduction pathways in response to stress.

Introduction:
DNA replication is a highly regulated process in eukaryotic organisms that initiates at
distinct loci known as origins of replication. Individual origins vary in the likelihood that they
will initiate replication for any given replication cycle depending on neighboring sequence
elements and the accessibility of the origin (Weinreich et al. 2004). Eukaryotic DNA is packaged
into chromatin by wrapping its double helix structure around an octameric core of proteins called
histones (H2A, H2B, H3 and H4). While DNA is highly condensed to minimize the space it
occupies within the cell and to protect it from environmental dangers like oxidative damage, it
must still be accessible for transcription. Histone modifiers are enzymes that are capable of
carrying out reactions in which chemical groups, such as acetyl or methyl groups are covalently
added to N-terminal histone proteins, altering DNA accessibility and thereby enhancing or
reducing gene expression. While DNA sequence elements can be necessary, it is clear that
chromatin structure surrounding origins play an essential role in transcription activity.
Mixed-lineage Leukemia (MLL) family proteins (MLL1, MLL2, MLL3, MLL4, MLL5,
Set1A and Set1B) are histone methyltransferases (HMT) in humans that play a critical role in
gene activation and epigenetics (Southall et al. 2009). These proteins possess a highly conserved
SET domain, which supports mono-, di- and tri- methylation of histone H3 lysine 4 (H3K4)
specific activity (Milne et al. 2002). Mixed-lineage leukemia is contributed to MLL gene
translocations at locus 11q23 as a result of nonhomologous end joining (NHEJ) repair processes
(Morse et al. 1982). This aggressive form of blood cancer is primarily found in infants and has a

relatively low survival rate (Kosaka et al. 2004). After the discovery of MLL1s correlation to
leukemia it has been the focus of multiple structural studies (Li et al. 2014). However, it is
difficult to study due to its similarity to at least six other proteins in its complex and its large size
of 3,000 amino acid residues (Shilatifard 2008).
Set1 protein is a histone methyltransferase in Saccharomyces cerevisiae (S. cerevisiae)
and is a homolog of the MLL1 protein (Lee et al. 2005). Like MLL1, it contains a highly
conserved domain with four sequence motifs (I-IV) that support the H3K4 specific methyl
transfer activity (Figure 1). The Set1 protein is also a member of a multiprotein complex known
as COMPASS (complex proteins associated with Set1) and is capable of mono-, di- and tri levels
of H3K4 methylation via a S-adenosyl methionine (AdoMet) methyl donor (Miller et al. 2001).
For the transfer of a methyl group from AdoMet to a lysine residue, deprotonation of the epsilon
amino group of lysine is necessary (Xhiao et al. 2003). Two separate mechanisms have been
proposed for this process, one involving the active site base tyrosine and another involving
deprotonation through an active site water channel (Xhiao et al. 2003). It is widely accepted that
tri-methylation of H3K4 is associated with gene activation whereas mono-methylation is
commonly linked to silenced regions of the genome like in the ribosomal DNA (rDNA), HM
loci, and the telomeres (Cosgrove et al. 2010). While there are at least 60 SET domain proteins in
humans, there are only 11 identified in S. cerevisiae (Martin et al. 2003). This, along with Set1
protein being the only histone methyltransferase in S. cerevisiae, has proven it to be an excellent
model for study and further understanding the role of MLL1 in leukemia (Noma et al. 2002).

Figure 1. Conserved sequence motifs (I-IV) of SET domain. Sequences of SET domain proteins from
S. cerevisiae Set1 (Set1_Sc), Homo sapiens MLL1, Neurospora crassa Dim-5, and Homo sapiens Set7/9
aligned to illustrate conserved residues. (Williamson et al., 2013)

In a previous study conducted by Williamson et al single amino acid substitutions of

several conserved residues in the SET domain established the importance of the Y967 residue in
motif II of the Set1. Two mutants were made, first by replacing the tyrosine with phenylalanine
(Y967F-Set1) and then an alanine (Y967A-Set1) (Williamson et al. 2013). Results showed that
Y967F-Set1 had H3K4 mono-methylation activity while Y967A-Set1 was not capable of any
methylation (Williamson et al. 2013). To assess the phenotypic growth differences of these two
mutants, the reporter gene HIS3 was inserted into the heterochromatic region (rDNA) of
chromosome XII of S. cerevisiae (Simpson et al. 2014). Strains were grown on a specialized schis media containing 10mM 3-Amino-1,2,4-triazole (3-AT), an inhibitor of the HIS3 gene
product imidazole glycerophosphate dehydratase (Simpson et al., 2014). This enzyme catalyzes a

critical step in the histidine biosynthesis pathway and allows growth, even at very low levels. By
adding 3-AT, only strains producing high levels of genes involved in histidine biosynthesis
should be able to grow, allowing for discernible differences in growth. Strains with a functional
SET1 protein were expected to have low growth on sc-his+3AT due to methylation of H3K4 and
thereby suppression of the HIS3 gene in the rDNA, whereas strains lacking functional SET1
would allow for greater expression of HIS3 resulting in higher levels of growth. It was found
that growth patterns of Y967F-Set1 mutants were similar to WT-Set1 strains, even though
Y967F-Set1 showed better growth, indicating that mono-methylation of H3K4 by Set1 is
sufficient for HIS3 gene activation and survival. Unexpectedly, the Y967A-Set1 and set1
mutants exhibited low levels of growth on sc-his+3AT despite reduced gene silencing (Simpson
et al. 2014). This finding was not expected since lacking a functional Set1 protein should result
in an absence of H3K4 methylation and therefore less gene silencing in the rDNA and higher
expression of HIS3. To determine if the low levels of growth were due to the unpredictable
nature of rDNA, HIS3 was moved to the euchromatic region of chromosome XII. Despite
movement of HIS3, the same lack of growth was observed showing that growth differences are
primarily attributed to Set1 activity and not so much HIS3 gene location (Moehlman et al. 2014).
These results also suggest that mono-methylation activity is more advantageous to gene
activation and cell survival under the harsh conditions provided by sc-his+3AT media.
To further verify the significance of Set1 to cell growth on sc-his+3AT Nguyen et al
returned HIS3 to its endogenous location on chromosome XV (Nguyen et al. 2015). Again,
diminished growth was observed demonstrating that Set1 activity is required for healthy growth
under stressful sc-his+3AT conditions. From this point it was necessary to confirm that
methylation of H3K4 by Set1, and not an alternate substrate of Set1, was indeed causing the

observed phenotypic growth differences. Set1 also has the capability to methylate Dam1, a nonhistone substrate that promotes proper chromosome segregation and cell viability (Zhang et al.
2005). To prove that H3K4 methylation by Set1 was the cause of the observable phenotypic
differences rather than another substrate of Set1, Ghirmay et al constructed strains by mutating
lysine 4 of H3 to an arginine so that it would no longer be a target for Set1 methylation (Ghirmay
et al. 2015). With all other variables from the Nguyen et al experiment kept constant it was
observed that all mutant strains died out on sc-his+3AT media, even at low dilutions (Ghirmay et
al. 2015). These results clearly provide evidence that the observed growth differences on schis+3AT are solely due to Set1 methylation of H3K4 and not some other substrate.
To investigate if mono-methylation of H3K4 is truly advantageous for growth on schis+3AT media two new mutant strains R1013H-Set1 which has mono- and di-methylation
capabilities, and H1017A-Set1 which has the ability to mono-, di- and tri-methylate H3K4 at
hyper levels, will be constructed (Williamson et al. 2013). Phenotypic growth of these new
mutants will be compared to that of strains previously constructed by Nguyen et al to determine
if mono-methylation of H3K4 is in fact correlated to gene activation.

Methodology:
Media, Plasmids and Strains
The media used throughout this study were ordered from MP Biomedicals and were used
according to vendor instructions. Yeast cells were growth on YPDT and three separate types of
synthetic complete (sc) media: synthetic complete lacking uracil (sc-ura), synthetic complete
lacking histidine (sc-his), and synthetic complete lacking histidine media containing 3-amino1,2,4-triazole (sc-his+3AT). The mutant plasmids pRS406-set1-R1013H and pRS406-set1H1017A along with the parent strain (MBY1590) were obtained from Dr. Bryks lab in

Department of Biochemistry at TAMU. This parent strain was designed to have a set1 and a
nonfunctioning URA3 gene, ura3-52. The strains MBY2992, MBY2998, MBY3000 and
MBY2994 were all previously constructed (Table 1) (Nguyen et al. 2015).
Table I: Strains constructed by previous and current semesters along with relevant gene
information for S. cerevisiae

HIS3 Location

Endogenous
HIS3

Possible Levels of

Strain

SET1 Type

MBY1590**

ura3-52 set1 (parent)

MBY2992*

set1

MBY2994*

SET1+

1, 2 & 3

MBY2996

R1013H-set1

1&2

MBY2998*

Y967A-set1

MBY3000*

Y967F-set1

MBY3002

H1017A-set1

1, 2 & 3

Methylation

*Strains constructed by Nguyen et al. (Nguyen et al. 2015)

**Strains donated by Dr. Mary Bryks lab

Strain Construction for set1 mutant strains

A lithium acetate transformation was completed using StuI digested pRS406-set1R1013H and pRS406-set1-H1017A plasmids using an adapted protocol from the Bryk and
Peterson labs. These plasmids contained a functional URA3 gene that served as both the site of
homologous recombination for integration of a mutant set1 gene and as a future marker check for
successful transformation. For the transformation to take place, MBY1590 cells were lysed using
40% PEG 3350/1xTE/0.1 M LiAc buffer followed by the addition of plasmid DNA and carrier
DNA. The reaction was heat shocked at 42oC for 15 minutes to facilitate the uptake of the
plasmid. A 100 L sample from each transformant and the negative control were plated on scura. These plates were then incubated at 30oC for 48hrs to obtain colonies of R1013H and
H1017A strains.

Verification of set1 mutant strains

Following lithium acetate transformation, single colony purification was conducted to
ensure the strains are properly constructed and free from contaminants. This was completed by
plating two isolated colonies from each transformation, R1013H-set1 and H1017A-set1, on scura. Single colony purified strains of MBY1590 (set1), MBY2998 (Y967A-set1), MBY3000
(Y967F-set1), MBY2994 (SET1), MBY2992 (set1), MBY2996 (R1013H-set1-1&2) and
MBY3002 (H1017A-set1-1&2) were patched on YPDT to obtain dense growth. From this patch
plate strains were replica plated on sc-ura, verifying the transformation of the linearized pRS406
plasmid and thus a functional URA3 gene, and on sc-his to test for histidine biosynthesis and
ensure that any growth differences were not due to strain construction. MBY1590, which lacks
the pRS406 plasmid, was used as a negative control while MBY2994 was used as a positive
control. Lastly, colonies were replica plated onto YPDT media to create a master plate of fresh
cells to be used for colony PCR.
Fresh cells (~24 hours old) were obtained and used to conduct a colony PCR in order to
further verify the construction of R1013H-set1 and H1017A-set1. A small but visible amount of
cells from each colony was transferred to a labeled PCR tube containing 10 l of 0.02 N NaOH
to lyse the cells and release the DNA. To be sure that the DNA was accessible for PCR
amplification, the reactions were incubated at 99oC for 10 minutes. During incubation, a PCR
master mix containing 87.5 l of GoTaq Green master mix (Promega), 17.5 l of dH2O, 17.5 l
of 10 M ura3-52 forward primer, and 17.5 l of 10 M set1 reverse primer was prepared. The
forward primer sequence in ura3-52 was 5-GAG AAG ATG CGG CCA GCA AAA C-3 while
the reverse primer for set1 was 5-GGT TAT AAC GTC GAC GTT G- 3. These primers were
chosen since PCR amplification of this region would verify the transformation of the pRS406

plasmid, and therefore a functional set1 gene. Following incubation, 40 l of dH2O was added to
each reaction making a 5-fold dilution. From the diluted samples 5 l were added to the
appropriately labeled PCR tubes containing the master mix making them ready for amplification.
Colony PCR amplification was conducted by denaturing the DNA at 94oC for 10 minutes
followed by 30 cycles of denaturation at 94oC for 30 seconds, annealing at 54oC for 30 seconds
and extension at 72oC for 1.5 minutes. The reactions were then incubated at 72oC for 10 minutes
before they were held indefinitely at 4oC. Samples were then loaded onto a 0.7% agarose gel
containing Gel Red (Phenix) allowing the visualization of DNA fragment location. A 1Kb ladder
(Promega) was also loaded onto the gel as a reference marker. The gel electrophoresis was set to
75 V for ~45 minutes before it was analyzed for a PCR product fragment of approximately
1500bp, which is the relative length of DNA between the designed forward and reverse primers.
Spot Plate Assay
Spot plate assays were performed to determine phenotypic growth differences between S.
cerevisiae strains capable of varying levels of SET1 methylation activity. Prior to platting, 5 mL
of YPDT liquid culture was inoculated for each of the six strains: MBY2992, MBY2994,
MBY2996, MBY2996, MBY2998, MBY3000, and MBY30002. Cultures were grown overnight
at 30oC to ensure saturation. A series of serial dilutions, using a dilution factor of ten from 10-1 to
10-6, were then completed for each of the six starter cultures. Afterwards, 5 l of each strain
dilution was spot plated on sc-complete and sc-his+3AT. During incubation at 30oC, images
were taken at 24 hrs and 48 hrs for both plates, and again at 60 hrs for sc-his+3AT to analyze for
growth differences.

Results:
Strain Construction and Verification
Following lithium acetate transformation of parent strain MBY1590 using StuI digested
pRS406-set1-R1013H and pRS406-set1-H1017A plasmids, S. cerevisiae strains were plated on
sc-ura media to ensure integration. All strains used in this experiment (Table I) were then replica
plated on both sc-ura and sc-his to verify auxotrophies. All strains showed growth on sc-his,
confirming the presence of a functioning HIS3 gene on chromosome XV and histidine
biosynthesis (the ability to synthesize histidine). On sc-ura, growth was observed for all strains
except the MBY1590 parent strain verifying plasmid transformation and the presence of a
functional URA3 gene. Lack of growth for MBY1590, which served as the negative control, was
expected since it was not transformed with the pRS406 plasmid and therefore only contained a
nonfunctional ura3-52 gene.
Colony PCR was used to further verify plasmid integration by amplifying a region
approximately 1500 bp in length between ura3-52 and set1 genes. PCR products were observed
using gel electrophoresis. After imaging, a single band of roughly 1500 bp was detected for all
strains except for the MBY1590 parent strain, which lacks SET1/set1. These results, along with
the growth observed during replica plating, certify the accurate construction of all strains and
provide the confidence to proceed to the spot plate assay.
Spot Plate Assay
To examine phenotypic growth differences between S. cerevisiae strains with different
levels of SET1 methylation capabilities, a spot plate assay was performed. Newly constructed
strains MBY2996 and MBY3002, along with strains constructed by Nguyen et al (MBY2992,
MBY2994, MBY2998 and MBY3000) were plated on both sc-complete and sc-his+3AT. All

strains plated on sc-complete displayed comparable growth for each dilution (Figure 2A). Since
there were not any discernible growth differences on sc-complete media, it was concluded that
the serial dilution and spotting techniques were correctly performed; and that it could be used as
a positive control when comparing growth on sc-his+3AT. This also indicated that any
phenotypic growth differences observed on sc-his+3AT would solely be due to set1 gene
activity.
At 60 hrs on sc-his+3AT growth was halted by the 10-2 dilution for MBY2992 (set1)
and MBY2998 (Y967A-set1), both of which had no methylation capability. A similar phenotype
was observed for MBY2994 (SET1+) and MBY3002 (H1017A-set1), essentially indicating that
tri-methylation levels have a negative impact on survivability (Figure 2B). The highest
survivability was observed for MBY3000 (Y967F-set1), followed closely by MBY2996
(R1013H-set1), both of which perform mono-methylation. MBY2996 is also capable of dimethylation; this presumably takes away from the amount of mono-methylation and thereby
slightly reduces survivability. The observations from the spot plate assay imply that monomethylation levels of H3K4 by SET1 are the most advantageous for survival when platted on the
stressful conditions of sc-his+3AT.

Figure 2. Spot plate assay S. cerevisiae strains with varying levels of Set1 activity. Six S.
cerevisiae mutant strains were plated and photographed on A) sc-complete at 48 hrs and B) schis+3AT at 60 hrs.

Discussion:
The results of this study clearly indicate the importance of Set1 methylation, particularly
mono-methylation, of the H3K4 target for survival under histidine starvation in the presence of
3AT. HIS3 encodes imidazoleglycerol-phosphate dehydratase (IGPD), which catalyzes the 6th
step in the histidine biosynthesis pathway (Struhl 1977). Since all strains have HIS3 gene
expression to a certain extent, 3-Amino-1,2,4-triazole (3AT) was added to the media. This
competitive inhibitor of IGPD prevents growth of strains with low levels of HIS3 expression.
The fact that strains capable of tri-methylation displayed a similar phenotype to the negative
control (MBY2992/set1) under these specified stress conditions indicates that tri-methylation is
not only less favorable but actually has negative effects on growth.
By placing the HIS3 reporter gene in the ribosomal DNA (rDNA) Simpson et al was able
to demonstrate that the observed phenotype on sc-his+3AT was not solely due to the competitive

inhibition of IGPD by 3AT, but also due to the activity of Set1 (Simpson et al. 2014). Prior
studies show that 3AT is an inducer of the transcriptional activator General Control
Nonderepressible 4 (Gcn4) which causes extensive nucleosome rearrangements around the
coding regions of genes (Cui et al. 2012). Gcn4p is considered a master regulator of gene
expression in S. cerevisiae, regulating the expression of nearly all genes encoding enzymes
involved in amino acid synthesis under starvation conditions (Vital-Lopez et al. 2013). In the
instance of HIS3, 3AT induction results in the rearrangement of two nucleosome position
clusters (D3 and D4) causing a shift in nucleosome spacing (Cui et al. 2012). This modification
reduces HIS3 gene expression under starvation conditions but it is now believed that Set1
methylation of H3K4 can reverse this disruption in nucleosome arrangement. Particularly, monomethylation of H3K4 by Set1 directs the chromatin remodelers RSC and SWR-C to stress
response genes (Nadal-Ribelles et al. 2015). This cooperation between nucleosome modifying
and chromatin remodeling enzymes exhibits the dynamic relationship of events leading to gene
activation or repression and the mobilization of various signal transduction pathways in response
to stress (Natarajan et al. 2001).
Previous studies established that the observed phenotypic differences of S. cerevisiae on
sc-his+3AT were related to Set1 activity. Specifically, the mono-methylation activity of Y967Fset1 (Williamson et al. 2013) strains displayed superior growth on sc-his+3AT despite the
location of the HIS3 reporter gene (Nguyen et al. 2015, Simpson et al 2014, Moehlman et al
2014). In a related study it was shown that the Set1 complex and H3K4 methylation were
required for resistance to the antifungal drug BrefeldinA (BFA) and maintaining cellular
ergosterol homeostasis (South et al. 2013). This study showed that Set1 methylation of H3K4 is
needed for the proper expression of genes involved in the ergosterol biosynthetic pathway such

as ERG11, ERG4 and HMG1 and genes responsible for the repression of ergosterol uptake like
PDR11 and DAN1 (South et al. 2012). These results correspond with this current study to show a
direct link between Set1 methylation of H3K4 and cell survival under stressful conditions.
Although the results of this study clearly indicate the importance of Set1 methylation,
particularly mono-methylation, of the H3K4 target for survival under histidine starvation in the
presence of 3AT, it is unclear as to why exactly this occurs. One possibility is that Set1 monomethylation causes a nucleosome rearrangement such that Gcn4 transcription levels are
increased. Not only does Gcn4 regulate the expression of nearly all genes encoding enzymes
involved in amino acid synthesis under starvation conditions, it also activates a the gene
Aminotriazole Resistance 1 (ATR1). ATR1 is a multidrug resistance transport protein, localized
in the cell membrane, with both 3AT and boron efflux function (Uluisik et al. 2011). ATR1 and
Gcn4 gene expression are both upregulated under several stress conditions, including the
presence of 3AT under amino acid starvation conditions (Kaya et al. 2009).
To determine if this proposed pathway is correct, HPLC analysis of total cellular 3AT
should be conducted for the six S. cerevisiae strains used in this study. If strains with higher
survivability on sc-his+3AT also exhibit lower levels of cellular 3AT, then ATR1 plays a crucial
role in cell survival by pumping toxic levels of 3AT out of the cell. This would suggest that
mono-methylation of H3K4 by Set1 not only activates transcription of genes involved in amino
acid biosynthesis but also genes that are required to combat toxic environments.
In the future, MLL1 inhibition could be a powerful tool for stimulating cancerous growth
arrest through direct epigenetic regulation of proliferation-promoting genes (Capell et al 2016).
Loss of Set1 mediated H3K4 methylation causes changes in chromatin structure and genomic
instability, which not only triggers apoptosis, but also sensitizes yeast cells to apoptotic stimuli

such as exposure to H2O2 (Walter et al. 2014). For this reason, inhibitors that block Set1
methylation activity of H3K4 should be investigated. The first step would be to identify a small
molecule that inhibits SET1 HMT activity in vitro through disruption of its interaction with
COMPASS. Utilizing the same strains and growth media in this experiment, small wafer discs
coated in inhibitory proteins of interest could be placed on plates streaked with each yeast strain.
Following overnight incubation, the zone of inhibition (area around the disc where colonies were
unable to grow) could be measured to assess the relative effectiveness of each inhibitory
molecule. This protein-protein interaction for inhibition of the SET1 family of chromatinregulatory enzymes would be a potential starting point for the development of therapeutics
targeting the homologous MLL1 protein.
By focusing on the methyltransferase activity of the SET domain we may gain important
insight to how MLL1 functions to regulate the expression of its various gene targets. MLL1
directly regulates the expression of numerous critical proliferative cell cycle and cancer target
genes, therefore by performing a MLL1 knockdown it is possible to repress proliferative cell
cycle regulators required for DNA replication and suppress inflammatory gene expression in
cancer in vivo (Capell et al 2016).

Acknowledgements:
I would like to thank Dr. Mary Bryks lab from the Biochemistry and Biophysics
Department of TAMU for providing the linearized plasmids used in the construction of the S.
cerevisiae mutant strains, my lab partner Nika Westberg, and Dr. Michelle Henderson Pozzi
along with Sreya Bose and Amber Wadle for their direction and assistance throughout the
duration of this experiment.

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