1) When the elongation rate of RNA polymerase II is decreased via a mutation in the Rpb2 subunit, levels of the histone methyltransferase Set2 and methylation of histone H3 at lysine 36 (H3K36) both increase. 2) Genetic interactions between set2 and other genes change depending on whether the wild type or mutant form of Rpb2 is present. 3) Deletion of the SRI domain of Set2, but not mutations affecting its catalytic activity, is necessary for the synthetic sick growth phenotype seen when set2 is deleted in the Rpb2 mutant strain. This provides a novel view of Set2's functions.

1. Unstable Transcription: A Novel View of Set2 and
H3K36 Methylation
Brandon A. Boone and Dr. Brian D. Strahl
Biology Program, McNair Scholars Program, The University of North Carolina at Chapel Hill
Abstract
Background
Results Results
Conclusions
When the elongation rate of RNA Polymerase II is decreased by mutating the Rpb2
subunit Set2 levels become increased as well as H3K36 methylation. Whether the
methylation increase is caused directly by the increase of Set2, by the decreased rate of
elongation allowing Set2 to bind more serine repeats of the CTD and therefore catalyze
more methylations, or possibly another mechanism is still to be determined. These
increases accompany subtle changes in the genetic relationships set2 has with rco1, asf1,
ioc4, and pdp3 in nutrient stress and transcriptional stress. Even more, the discovery that
the SRI domain deletion is necessary for the synthetic sickness of the rpb2-10 phenotype
and not the catalytic site is a completely novel view of Set2 and its possible functions.
These findings bring questions to mind regarding the relationship between the elongation
rate of RNA Polymerase II , epigenetics, and the extent to which the kinetics and proteins
of transcription play in the overall establishment and maintenance of epigenetic pathways.
30o C
SC YPD SC YPD
37o C
RPB2
rpb2-10
RPB2 set2Δ
rpb2-10 set2Δ
RPB2 pdp3Δ
rpb2-10 pdp3Δ
RPB2 rco1Δ
rpb2-10 rco1Δ
3’5’
5’
5’
3’
3’
30o C 37o C
SC-Ura
SC-Ura +
100 μg/mL
6AU
SET SRI
Set2 Histone Methyltransferase Protein Domains and Mutations:
αH3K36me1
αH3K36me2
αH3K36 me3
αH3K36 me1(Long Exposure)
αSet2
αG6PDH
αSet2 (long exposure)
αG6PDH (long exposure)
SC-Ura
SC-Ura +
100 μg/mL
6AU
Set2 levels are increased in rpb2-10 background compared to wild type. Mono-, di-, and
tri-methylation of H3K36 are also increased in the rpb2-10 background. G6PDH is used
as a loading control for this experiment.
When transcriptional stress, via 6-azauracil, is placed on the strains at 30oC and 37oC a bypass phenotype
becomes evident in RPB2 setΔ. Whereas set2Δ has little to no effect on the rpb2-10 phenotype and does not
demonstrate a bypass characteristic. rco1Δ phenocopies set2Δ in both wild type and rpb2-10 backgrounds
demonstrating and epistatic relationship for both backgrounds. ioc4Δ and pdp3Δ phenocopy each other with
the addition of 6-AU. asf1Δ phenocopies wild type RPB2 and rpb2-10 in both 30oC and 37oC conditions.
Rpb1
Rpb2
Set2
Set2
Comparing RPB2, set2Δ and rpb2-10, set2Δ reveals a synthetic sick phenotype in the later in either 30o C or
37o C. YPD conditions lead to increased growth across all strains. The increase in nutrients leads to decreased
intensity of synthetic sick phenotypes in all backgrounds except asf1Δ. asf1Δ is severely sick in both WT and
rpb2-10 background whereas rpb2-10 ioc4Δ phenocopies the rpb2-10 background.
Rpb1
Rpb2
Rpb1
Rpb2
Elongation
120 237 623 713
Serines 5 and 2 of the Carboxyl terminal domain (CTD), extending from
the Rpb1 subunit, become phosphorylated during transcription
H3K36 is methylated by Set2, which binds to
phosphorylated Serine 5/2 of the CTD
The methylation tags allow other proteins to bind
and cause further alterations to Histones and/or
DNA.
3’5’ Rpb1
rpb2-10
Set2
Decrease in Elongation Rate of
RNA Polymerase II via rpb2-10
If the Elongation Rate of
RNA Polymerase II is increased?
Amino Acids:
Protrusion Lobe Protrusion Fork
External 2/
External 1
Wall
Rpb2 Protein Domains and Mutation:
Hybrid
Binding
ClampAnchor
44 218 219 405 406 465 466 547 548 750 751 852 853 973 974 1127-8 1151-2 1224
Amino
Acids:
Organisms, from the budding yeast Saccharomyces cereviciae to Homo sapiens, require highly complex and
well-tuned transcriptional processes in order for cellular function to occur properly. Transcription is
accomplished via the holoenzyme RNA polymerase II (RNAP II). In accordance with other proteins,
RNAP II transcribes DNA into mRNA, the necessary template for protein production and cellular function.
One protein that interacts with RNAP II is Set2, a histone methyltransferase, which binds to a
carboxy-terminal Domain (CTD) that extends from the major subunit of RNAP II, Rpb1. In S. cereviciae the
CTD is composed of 26 repeats of the heptapeptide: YSPTSPS. Set2 binds to phosphorylated serine 2 and/or
serine 5 residues of the heptapeptide repeat. As RNAP II transcribes DNA, Set2 methylates lysine 36 on the
tail of Histone 3 (H3K36 methylation). Histones are protein complexes composed of eight subunits, 2 copies
each of the proteins H3, H4, H2A, and H2B, each of which contains a tail that extends away from the
histone. These discoveries lead to the role of Set2 being hypothesized as a negative regulator of
transcription because of the histone deacteylase complex, Rpd3S, that binds to H3K36 methylation, enhancing
the interaction between histone tails and DNA essentially inhibiting transcription. Past publications revealed
the mutant allele rpb2-10 (P1018S) that increases polymerase stalling and lowers the transcription efficiency
of RNAP II. Using rpb2-10 mutant strains, we asked if Set2, domains of complexes that bind to H3K36
methylation (Ioc4, Pdp3, and Rco1), and nucleosome assembly factor Asf1 show phenotypic differences in
strains expressing the unstable rpb2-10 mutant. Even more we sought to understand the implication decreased
elongation rate has on H3K36 methylation. Using temperature and transcriptional stress, via 6-azurauracil,
a synthetic sick phenotype was discovered in the rpb2-10 set2Δ strain that contrasted with the bypass phenotype
of the set2Δ in the rpb2 wild type background. This synthetic sick phenotype implies that in the presence of
an unstable RNAP II set2 has a positive effect on transcription instead of a negative effect as would be
expected and that was evident in the wild type strain. Further, novel genetic interactions were discovered
between the asf1Δ, rco1Δ, pdp3Δ, ioc4Δ, and set2Δ strains depending on whether wild type RPB2 or rpb2-10
was expressed. These findings begin to reveal a more complex relationship between Set2, RNA polymerase II,
and H3K36 methylation interacting proteins during transcription.
How is Set2, H3K36 methylation, and the relationship between the
proteins that interact with that methylation affected when RNA
Polymerase II elongation rate and efficiency is decreased?
AWS
63 118
WW CC
475 507 584 621
Hybrid
Binding
Background
RPB2
rpb2-10
SC-Leu Control
rpb2-10 empty vector
rpb2-10 set2H199L
rpb2-10 set2R193C
rpb2-10 set21-618
rpb2-10 set2FL
30o C 37o C
SC-Leu SC-Leu
rpb2-10 empty vector represents the set2Δ phenotype. Comparing H199L (catalytically
dead), R195C (no H3K36me3), and 1-618 (No SRI domain) with the empty vector
demonstrates that only the 1-618 vector phenocopies the rpb2-10 set2Δ synthetic sick
phenotype. H199L, R195C, and the full length Set2 all phenocopy RPB2.
How does the SRI domain of Set2
facilitate the rpb2-10 set2Δ phenotype?
RPB2
rpb2-10
RPB2 set2Δ
rpb2-10 set2Δ
RPB2 ioc4Δ
rpb2-10 ioc4Δ
RPB2 asf1Δ
rpb2-10 asf1Δ
RPB2
rpb2-10
RPB2 set2Δ
rpb2-10 set2Δ
RPB2 pdp3Δ
rpb2-10 pdp3Δ
RPB2 rco1Δ
rpb2-10 rco1Δ
RPB2
rpb2-10
RPB2 set2Δ
rpb2-10 set2Δ
RPB2 asf1Δ
rpb2-10 asf1Δ
RPB2 ioc4Δ
rpb2-10 ioc4Δ