Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation through Promoting 3′-End Antisense Transcription

Abstract

Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3'-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms.

Full text

Two Distinct Repressive Mechanisms for Histone 3 Lysine
4 Methylation through Promoting 39-End Antisense
Transcription
Thanasis Margaritis
1
, Vincent Oreal
2
, Nathalie Brabers
1
, Laetitia Maestroni
2
, Adeline Vitaliano-Prunier
3
,
Joris J. Benschop
1
, Sander van Hooff
1
, Dik van Leenen
1
, Catherine Dargemont
3.
*, Vincent Ge
´li
2.
*,
Frank C. P. Holstege
1.
*
1Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands, 2Marseille Cancer Research Center (CRCM), U1068 Inserm, UMR7258 CNRS, Aix-
Marseille Universite
´, Institut Paoli-Calmettes, Marseille, France, 3Institut Jacques Monod, Universite
´Paris Diderot, CNRS, Paris, France
Abstract
Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The
influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of
H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1
complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or
dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA
upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct
H3K4 methylation patterns, with enrichment of H3K4me3 at the 39-end, indicating that repression is coupled to 39-end
antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 39-end antisense
transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported
trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the
process of 39-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of
various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-
dependent 39-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark.
Removal of the 39-end antisense promoter also results in derepression of sense transcription and renders sense transcription
insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of
global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive
chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is
repression, achieved through promotion of 39-end antisense transcription to achieve specific rather than global effects
through two distinct mechanisms.
Citation: Margaritis T, Oreal V, Brabers N, Maestroni L, Vitaliano-Prunier A, et al. (2012) Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation
through Promoting 39-End Antisense Transcription. PLoS Genet 8(9): e1002952. doi:10.1371/journal.pgen.1002952
Editor: Hiten D. Madhani, University of California San Francisco, United States of America
Received July 14, 2011; Accepted July 31, 2012; Published September 20, 2012
Copyright: ß2012 Margaritis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: FCPH’s laboratory is supported by the Netherlands Bioinformatics Centre (NBIC) and the Netherlands Organization of Scientific Research (NWO): grants
016108607, 81702015, 05071057, 91106009, and 70057407 (JJB). VG’s and CD’s laboratories are supported by ‘‘La Ligue contra le Cancer’’ (VG and CD e
´quipes
labellise
´es) and by the ‘‘Agence Nationale pour la Recherche’’ (programme blanc, UBIGENEX). VO is recipient of a fellowship from the LNNC, and AV-P is supported
by the Association pour la Recherche contre le Cancer (ARC). The funders had no role in study design, data collection and analysis, decision to publish,or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: dargemont.catherine@ijm.univ-paris-diderot.fr (CD); geli@ifr88.cnrs-mrs.fr (VG); f.c.p.holstege@umcutrecht.nl (FCPH)
.These authors contributed equally to this work.
Introduction
Packaging of eukaryotic DNA with histones has a generally
repressive effect on transcription [1]. Histones themselves are subject
to a variety of post-translational modifications, such as acetylation,
methylation and ubiquitinylation. These modifications correlate with
specific states of transcription, as well as with the activity of other DNA-
linked processes, such as chromosome segregation and DNA repair
[2,3]. Among the epigenetic marks, histone methylation has been
extensively associated with both activation and repression of genes in
euchromatic and heterochromatic regions respectively [4]. Methyla-
tion of histone H3 on lysine 4 (H3K4) for example, has been linked to
transcriptional activation in many eukaryotic species. Vertebrates
possess several H3K4 methyltransferases related to the SET domain of
yeast Set1 and Drosophila Trx (MLL family) [5]. These methyltrans-
ferases are responsible for mono- (H3K4me1), di- (H3K4me2) and
trimethylation (H3K4me3) of H3K4 [6]. Di- and trimethylation of
H3K4 is generally restricted to euchromatin and genome-wide studies
in metazoan cells have revealed high levels of histone acetylation and
H3K4 methylation in promoter regions of active genes [7,8,9,10,11].
H3K4me2 and H3K4me3 are thought to facilitate transcription
through the recruitment of general transcription factors [12] and
cofactors [13] or by preventing repressors from binding to chromatin
[14]. The precise mechanism through which the various H3K4
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methylation states contribute to control of gene expression are not fully
understood.
In Saccharomyces cerevisiae, H3K4 methylation is carried out by the
Set1 complex, COMPASS [15], which is composed of the catalytic
subunit Set1 and at least six other components (Swd1, Swd2,
Swd3, Bre2, Sdc1 and Spp1) [16,17,18,19]. Loss or inactivation of
individual subunits differentially affects the methylation state of
H3K4. Swd1, Swd2 and Swd3 are required for COMPASS
stability and their disruption affects all three H3K4 methylation
states. Bre2 and Sdc1 promote the efficient di- and trimethylation
of H3K4, while inactivation of Spp1 only affects H3K4
trimethylation [20,21,22]. In addition, monoubiquitylation of
Swd2 has recently been shown to mediate the trans-tail process
between H2B ubiquitylation and H3K4 trimethylation, by
controlling the recruitment of the Spp1 subunit [23]. Set1 has
been found to be predominantly associated with the coding regions
of highly transcribed RNA polymerase II genes and the presence
of trimethylated H3K4 correlates with Set1 occupancy [24] and
transcription rate [25]. Genome-wide studies in yeast indicate that
active transcription is characteristically accompanied by histone
H3K4 trimethylation at the 59-end of genes and by H3K4
dimethylation and monomethylation at nucleosomes positioned
further downstream in the transcription unit [25].
Although H3K4 trimethylation has been linked to transcription
initiation and elongation in yeast [6,21,26], its precise role in
transcription as well as the role of H3K4 mono- and dimethylation
remain poorly understood. This is in part because previous genome-
wide analyses of the effects of H3K4 methylation loss have yielded
conflicting results [6,27,28,29] [30]. While two studies suggested a
global reduction in transcription when H3K4 methylation is
abolished [27,28], a third study reported and focused on only 480
very marginally down-regulated genes, even though twice as many
genes were observably upregulated upon applying the same selection
criteria [6]. The most recent study also reported roughly 300 genes
up-regulated and 100 down-regulated [30]. A more statistically
stringent study that included adequate replicate experiments showed
that 200 genes become up-regulated upon loss of SET1, with
virtually no down-regulation observed [29], suggesting that H3K4
methylation may actually play a more prominent role in repression
than in activation of protein-coding genes.
Recently, a form of RNA-mediated transcriptional repression
has been reported in S. cerevisiae, that is independent of the RNAi
machinery which is absent from budding yeast. Ty1, PHO84 and
GAL1/10 expression have been shown to be regulated by antisense
RNA transcription [31,32,33]. For PHO84, it was found that
expression of PHO84 antisense RNA from an ectopic PHO84 gene
copy was able to trigger silencing of the endogenous PHO84 gene
[34]. Production of the PHO84 antisense RNA was found to be
positively regulated by Set1 [34] potentially linking H3K4
methylation to non-coding RNA (ncRNA) regulation. Genome-
wide analysis has recently revealed the existence of hundreds of
previously uncharacterized ncRNAs in mammals [35,36,37,38]
and in yeast [39,40], that either stably exist or are rapidly
degraded by the RNA surveillance pathway. Strikingly, most of
these newly identified transcripts initiate from nucleosome-free
regions associated with bidirectional promoters of protein-coding
genes or regions in the body or close to the 39-ends of protein-
coding genes [40]. Regulation of ncRNAs is far from understood.
Here we present an extensive genome-wide analysis that
discriminates between the roles of the different H3K4 methylation
states. While preventing H3K4 trimethylation on its own has no
effect on mRNA expression of coding genes, 1% of coding genes
are derepressed upon combined loss of di- and trimethylation.
Further analyses indicate distinct roles for these two marks in
repression of coding genes through mechanisms that are mediated
through 39-end antisense transcription.
Results
Loss of H3K4 dimethylation correlates with increased
expression of a subset of genes
Previous genome-wide analyses of the effects of losing H3K4
methylation [6,27,28,29] [30] focused on loss of all three H3K4
methylation states simultaneously, either through deletion of the
gene that codes for the H3K4 methyltransferase, SET1 or through
substitution of H3K4 with alanine or arginine. To investigate
whether there are separate roles for H3K4 mono-, di- and
trimethylation, we made use of the fact that mutating different
components of the Set1 complex, COMPASS, results in different
methylation states. First, the methylation status of H3K4 was
assessed in strains with deletions of the non-essential members of
the complex, in the single genetic background used for this study
(BY4741). An additional strain was included that carries a
mutation that prevents monoubiquitylation of the essential subunit
Swd2 (swd2K68,69R), resulting in a severe reduction of
H3K4me3 [23]. Histones were purified from each strain and
their H3K4 methylation status was checked with antibodies
specific for each methylated state (Figure 1A). As expected from
previous results (see the introduction), deletion of SET1,SWD1 or
SWD3 abolishes mono-, di- and trimethylation of H3K4. Deletion
of BRE2 or SDC1 results in a complete loss of H3K4me3, a
significant decrease of H3K4me2 but no change in H3K4me1,
while inactivation of SPP1 or mutating SWD2 (swd2K68,69R)
results in a severe and specific decrease of H3K4me3 (Figure 1A).
The same strains were analyzed in parallel by long oligo DNA
microarray expression-profiling, targeting the coding strand of
virtually all yeast genes. Throughout this study all microarray
analyses were performed with four replicates (two independent
cultures, each measured in duplicate, Materials and Methods). In
addition, controls were included that allow detection of global
changes in the entire mRNA population [41]. Such global changes
were not detected. In agreement with the most recent studies of
Author Summary
In eukaryotes, DNA is packaged together with histones
into nucleosomes. This packaging has a repressive role on
gene expression. The N-termini of histones are subject to
multiple modifications that affect DNA–dependent pro-
cesses. The histone modification that has been predom-
inantly linked with active transcription in all eukaryotes is
histone H3 lysine 4 (H3K4) methylation. Here we investi-
gate the functional effects of each H3K4 methylation state
on transcription. Removal of the mark that is most
characteristic for transcription, H3K4 trimethylation, has
no effect on coding gene expression, in steady-state or
dynamically changing conditions. Combined loss of H3K4
tri- and di-methylation does have an effect and leads to
loss of repression of specific genes, the opposite of what is
expected for global marks of active genes. The affected
genes have antisense transcription. We show that there are
two separate mechanisms through which H3K4 methyla-
tion represses transcription of protein-coding genes, one
through antisense transcripts and one through the process
of antisense transcription. In summary, we show how a
general mark of active transcription can have specific
repressive effects that are themselves also linked to
repression through nucleosomes.
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SET1 deletion on its own [29] [30], expression of only a minority of
genes is affected in the different COMPASS mutants. Within the
entire set of deletion mutants, 89 genes changed significantly in at
least two mutants (p-value lower than 0.01 and fold-change versus
wild-type more than 1.7), with 69 genes showing increased expression
and only 20 exhibiting decrease (Figure 1B). Deletion of any of the
five core subunits Set1, Swd1, Swd3, Bre2 and Sdc1 leads essentially
to the same expression profile (Figure 1B and Figure S1).
It is interesting to compare the changes in gene expression to the
H3K4 methylation states observed in the different mutants.
Virtually no significant changes in gene expression are observed in
spp1Dor in the swd2K68,69R mutant (Figure 1B) that both show a
specific and severe decrease of H3K4 trimethylation (Figure 1A).
Changes in gene expression are observed in bre2Dand sdc1D,
where H3K4 dimethylation is significantly diminished on top of
the loss of trimethylation, (Figure 1A, 1B). The additional loss of
H3K4 monomethylation, as observed in set1D,swd1Dor swd3D
(Figure 1A), does not lead to additional changes in gene expression
(Figure 1B). Because of the correlation between their location and
transcription rates [25], H3K4 methylation marks in yeast have
generally been associated with transcription activation. The main
effect of mutating COMPASS components in S. cerevisiae is
nevertheless derepression (Figure 1B). Furthermore, the effect is
only strong upon loss of dimethylation on top of trimethylation
loss, which on its own has little effect.
To distinguish whether the repressive effect of COMPASS is
related to H3K4 methylation or is due to an unidentified methylation
target of Set1, a H3K4 point mutant was analyzed. The predominant
effect is up-regulation (Figure 1C) and the overlap with the
COMPASS-repressed genes is highly significant (p-value 3.1*10
227
,
hypergeometric test). An apparently lower number of genes is
derepressed in the H3K4 point mutant. As analyzed later, this is likely
related to the H3/H4 histone dosage effect of the strain used to
generate the point mutant. To nevertheless investigate the possibility
that Set1 repression is mediated by a target other than H3K4, SET1
was deleted in the H3K4 point mutant strain. DNA microarray
analysis of the double mutant shows a completely epistatic re-
lationship with no additional effect of deleting SET1 in the H3K4
point mutant strain (Figure S2). This confirms that the repressive
effect of COMPASS observed here is mediated through H3K4.
It has been previously shown that H3K4 di- and tri-, but not
monomethylation states are controlled by the Rad6/Bre1-mediated
monoubiquitylation of histone H2BK123 via a trans-tail pathway
involving ubiquitylation of Swd2 [23,42,43,44,45]. To investigate
whether the repressive effects of H3K4 methylation are mediated
by this pathway, a bre1Dstrain was analyzed. Changes in gene expres-
sion in bre1Dmatches the COMPASS mutants profiles with a
highly significant overlap (p-value of 1.0*10
237
, hypergeometric test)
(Figure 1C). The repressive effects observed here therefore correspond
to the action of the entire pathway starting from ubiquitylation of
histone H2B and leading to di- and trimethylation of H3K4.
Repression dynamics are subtly affected by loss of H3K4
methylation
Since the experiments described above deal with steady-state
changes in mRNA levels, we next asked whether the absence of
H3K4 methylation would affect the kinetics of gene expression
changes. This is based on the proposal that H3K4me3 may have a
memory function, bookmarking genes that require rapid induction
under specific growth conditions, both in mammals [46] and yeast
[24]. For this purpose, wild-type (wt), set1D(absence of all three
H3K4 methylation states) and spp1D(lack of H3K4 trimethylation
only) were expression-profiled at multiple time-points during the
transition from post-diauxic shift to early log phase, a transition
during which a large number of genes change expression levels [47].
During this transition, expression of approximately 3400 genes
change significantly in wt cells, covering a broad range of gene
expression dynamics (Figure 2A). No major differences in the
transcription kinetics between wt and the two mutant strains are
observed. This indicates that disruption of H3K4 methylation or
H3K4 trimethylation on its own does not have a global effect on the
dynamics of transcription (Figure 2A), even though most active
genes exhibit H3K4me3 marks [6,11,27]. These results also agree
Figure 1. Loss of H3K4 di- and trimethylation results in
upregulation of a subset of genes. (A) Commassie-stained gel of
purified histones from the indicated strains (top) and western blots with
antibodies directed against H3 carboxy-terminus and the different H3K4
methylation states (bottom). (B) Hierarchical clustering of all genes with
significantly changed mRNA expression (p-value less than 0.01 and fold-
change versus wild-type more than 1.7) in at least two COMPASS
mutants. Fold-change of mRNA expression in mutant versus wild-type is
indicated by the colour bar as log
2
values. Number of genes below each
heatmap correspond to the genes called significant in each mutant. (C)
Genes depicted in the same order as in B for the H3K4R point mutant
and bre1D.
doi:10.1371/journal.pgen.1002952.g001
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Figure 2. Loss of H3K4 di- and trimethylation leads to a delay in repression kinetics for a subset of genes. (A) Hierarchical clustering of
all genes with significant changes in mRNA expression during the shift from low to high glucose in any of the wt, spp1Dand set1Dtime-courses. The
log
2
values correspond to the difference with the zero time point of each time-course. (B) Hierarchical clustering of genes with delayed repression
compared to wt. These genes were identified based on statistically significant differences between the mutant and wt time-courses (Materials and
Methods). The first three panels show the differences in expression versus the wt zero time point. The last two panels (spp1Dvs wt) and (set1Dvs wt)
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with the lack of a global effect after removing the H3K4me3 mark
under steady-state conditions (Figure 1B).
A detailed statistical analysis for genes showing differences in their
induction or repression kinetics in the mutants was also performed
among the 3400 genes that change significantly during the time-
course experiment. In the set1D(loss of all three H3K4 methylation
states) time-course, 220 genes show statistically significant differences
in their expression kinetics compared to the corresponding time
points in wt (compare Figure 2B, 2C first and third panel). The vast
majority of these (194 genes - Figure 2B) exhibit defective repression,
observed as delayed repression or faster activation. Only aminority of
genes exhibit an activation defect (26 genes - Figure 2C). To facilitate
visualization of these mostly quite subtle changes, the wt time-course
was subtracted from each mutant time-course. This results in the
right-hand panels of Figure 2B and 2C, showing for each time-point,
the difference in expression levels for each mutant relative to the wt at
the same time point. For spp1D(loss of H3K4me3), only 15 genes
exhibit any differences in their expression kinetics (Figure 2B, 2C,
second panel). These all belong to the 220 genes with slightly altered
kinetics in the set1Dtime-course. In agreement with the steady-state
analysis, the effects detected in the time-course experiments are thus
virtually all attributable to the complete loss of methylation observed
in set1D, rather than to the specific loss of H3K4me3 observed in
spp1D. The results concur with a repressive role for COMPASS on
mRNA expression of a subset of genes, as observed in the steady-state
experiments too (Figure 1) with an extremely significant overlap
between the affected genes (p-value 6*10
235
), as expected.
Unconventional methylation patterns at the 39-end of
COMPASS-repressed genes
We next investigated whether there are any particular character-
istics shared by the set of genes upregulated upon mutation of
COMPASS components (Figure 1B). In agreement with a recent
analysis of set1D[29], statistically significant enrichment for location
close to telomeres is observed (Figure S3). Among the 69 COMPASS-
repressed genes, 10 are telomere-proximal (within 15 kb) Figure S3
and Table S1). Although this enrichment is significant, in most cases
the expression of adjacent genes was not found to be affected by the
deletion of COMPASS subunits. For instance, PHO11,SNO4,
MCH2,SOR2,YGL258W-A and PHO12, that are located between 4
and 10 kb from the telomeric DNA on different chromosomes (Table
S1) are all flanked by genes that are not affected by the absence of
Set1. This, as well as the small number of all telomere- proximal
genes being derepressed in the COMPASS mutants makes it unlikely
that the observed derepression of telomere-proximal genes is only
caused by loss of the Sir-dependent telomeric position effect
[19,48,49,50,51,52].
As the effect of COMPASS deletions is attributable to H3K4
methylation (Figure 1C), the H3K4 methylation patterns of
COMPASS-repressed genes were examined using chromatin
immunoprecipitation data from a wt strain from the same genetic
background, grown under similar conditions [53]. Intriguingly, the
di- and trimethylation patterns of the 69 COMPASS-repressed
genes (Figure 3A) deviate from the average gene which has
enrichment of H3K4me3 around the transcription start site
(Figure S4) [25,53]. Instead, the majority of COMPASS-repressed
genes show enrichment of H3K4me3 at the 39-end or in the body
of the gene. In the minority of cases where 59-end enrichment is
observed, this is accompanied by a second trimethylation peak at
the 39end. To exclude that the deviating localization of peaks is
not due to measurement noise or signal originating from
neighbouring genes, the methylation profiles are averaged in
Figure 3B only for those genes that have a greater than 2-fold
enrichment of H3K4 methylation on any portion of the gene. This
average pattern for COMPASS-repressed genes shows a clear
enrichment of H3K4me3 at the 39end, followed by H3K4me2
enrichment in the gene body, which is in turn followed by
H3K4me1 further towards the 59-end. Genes repressed by
COMPASS therefore show abberant H3K4 methylation patterns
that are characterized by a reversed orientation of the normal
H3K4 methylation pattern observed for active genes [3].
depict the differences between mutant and wt for each different time point, by subtracting the log base 2 gene expression ratios of the wt time-
course from the mutant time-course. (C) Hierarchical clustering of genes that show delay in activation.
doi:10.1371/journal.pgen.1002952.g002
Figure 3. COMPASS-repressed genes have aberrant H3K4
methylation patterns, indicative of 39-end antisense transcrip-
tion. (A) Heatmaps of enrichment of H3K4me2(left) and H3K4me3(right)
over H3 in the gene body and flanking regions of the 69 COMPASS
repressed genes, based on [53]. The enrichments are rescaled for each
individual gene with blue and white corresponding to the highest and
lowest enrichment, respectively. PHO84 is marked by P. (B) The average
enrichment of H3K4me1 (blue), H3K4me2 (red) and H3K4me3 (grey) over
H3, for the set of 47 COMPASS-repressed genes that show at least a two-
fold enrichment of H3K4me2 or H3K4me3 somewhere across the gene or
flanking region [53].
doi:10.1371/journal.pgen.1002952.g003
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Promotion of 39-end antisense transcription by Set1
contributes to repression on coding genes through two
distinct mechanisms
A plausible explanation for the H3K4 di- and trimethylation
peaks at the 39-ends of COMPASS-repressed genes is the presence
of antisense transcription initiation at the 39-end of the coding
region, leading to non-coding transcription over the same genomic
location but in the opposite direction of the sense transcription. The
DNA microarrays used in the previous experiments are coding
strand-specific and do not detect anti-sense transcripts. However,
two recent genome-wide surveys of non-coding transcripts [39,40],
do detect antisense RNAs for more than 85% of the COMPASS-
repressed genes (Table S2). Interestingly, PHO84 belongs to the
group of COMPASS-repressed genes identified here (Figure 3A,
marked with P) and has been shown to be regulated by antisense
RNA transcripts originating from its 39-end both in cis and in trans
[32,34]. We therefore investigated the manner in which 39-end
antisense transcription may be involved in Set1-mediated repres-
sion.
One hallmark of the mechanism of repression of PHO84 is the
contribution of the antisense transcript itself rather than only the
process of antisense transcription. Stabilization of the antisense
transcript by deletion of the exosome component RRP6 [32] is
sufficient to repress sense PHO84 transcription. To test whether
COMPASS repression is mediated by 39-end antisense tran-
scripts, an rrp6Dprofile was generated and compared to set1D.
Deletion of RRP6 affects expression of 117 coding genes in total
(p,0.01, fold-change.1.7) and does not have a general effect on
all COMPASS-repressed genes (Figure 4A). In agreement with
previous studies however, a significant down-regulation of PHO84
is observed (marked P in Figure 4A). Lack of down-regulation
of the other COMPASS-repressed genes in rrp6Dmay be simply
due to an already repressed state in wt. Since these genes are
derepressed in set1D, the possible involvement of antisense tran-
scripts in repressing all COMPASS-affected genes was further
tested by analysis of an rrp6Dset1Ddouble mutant (Figure 4A).
The double mutant expression-profile reveals two classes of
COMPASS-repressed genes. On the smaller group of genes
(Figure 4A, marked with a black bar), that includes PHO84 as
well as several other phosphate-related genes, an epistatic effect is
observed in rrp6Dset1D, whereby the upregulation in set1Dis lost
in the double mutant. This implies that the antisense transcript
mediated repression of sense genes is not unique for PHO84,but
is shared with functionally related genes. Such genes are the
exception however. The largest group of Set1-repressed genes
behaves in a different manner, still showing derepression in the
double mutant, similar to their behaviour upon deletion of SET1
on its own. This therefore likely represents a distinct mechanism
of COMPASS repression.
In order to understand the mechanism by which COMPASS
represses coding transcription in an exosome-independent man-
ner, five representative genes from this group, AMS1,YGR110W,
ARG1,SPR3 and OYE3 (indicated by 1 to 5 in Figure 4A), were
analyzed in greater detail. These genes represent different
functional categories, different telomeric proximities and different
types of antisense transcripts, as suggested by the genome-wide
datasets. The first three genes contain antisense stable unannotat-
ed transcripts (SUTs), while the other two have antisense cryptic
unstable transcripts (CUTs) [40]. The location of H3K4 methyl-
ation patterns [53] corresponds to the location of the transcription
initiation sites of these antisense transcription units (Figure S5).
The effects of different COMPASS mutants on both sense and
antisense transcription of these genes were analyzed by quantita-
tive RT-PCR using strand-specific primers (Figure 4B).
Sense transcript upregulation of the five genes is observed in
set1D(Figure 4B), that exhibits loss of all H3K4 methylation marks
(Figure 1A), in bre2D(Figure 4B), that exhibits loss of all H3K4me3
and most H3K4me2 (Figure 1A) and in set1Dcombined with rrp6D
(Figure 4B), all in agreement with the sense-specific microarray
results (Figure 1B, Figure 4A). In bre2Dand set1D, upregulation of
Figure 4. COMPASS repression is mediated through 39-end
antisense transcriptional gene silencing. (A) Hierarchical cluster-
ing of the 69 COMPASS-repressed genes in the rrp6D,set1Dand the
set1Drrp6Dstrains. PHO84 is marked by P and AMS1,YGR110W,ARG1,
SPR3 and OYE3 are marked by 1 to 5, respectively. The black bar marks
the subset of genes where the two mutations are epistatic. Three
quarters of these genes are related to phosphate metabolism. (B) Sense
and antisense RNA levels analyzed by qPCR in indicated backgrounds.
The schematic representation of the follow-up genes shows the relative
positions of the primers used for strand-specific reverse transcription
reactions in arrows, while the black box indicates the location of the
DNA fragment produced during the qPCR. Error bars reflect standard
deviations of an average signal obtained from at least two independent
experiments. The significance of the difference in expression changes
observed between the mutant cells and the corresponding background
strain (rrp6Din the case of spp1Drrp6Dand set1Drrp6D,wt for the
others), was evaluated using Student’s t-test (
*
P0.01–0.05;
**
P0.001–
0.01;
***
P,0.001).
doi:10.1371/journal.pgen.1002952.g004
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sense transcription is accompanied by a decrease in antisense
transcription (Figure 4B, panels 1–3). As expected, changes in
antisense CUT transcription are not evident without prior
stabilization by the exosome deletion (Figure 4B, panels 4,5). For
all five genes, stabilisation of antisense transcripts does occur in
rrp6D(Figure 4B), but these increased antisense levels do not
necessarily result in more repression of sense transcription
(Figure 4B) as is clearly the case for PHO84 ([32] and Figure 4A).
This confirms that an increase in antisense transcript levels
through rrp6D-dependent stabilisation is not the mechanism of
COMPASS repression for these genes. Rather, the data suggest
that it is the process of antisense transcription itself that represses
the sense transcription.
The Set1 repressive effect is mediated through 39-end
antisense transcription
Because sense and 39end antisense transcription seem coupled
[54], it is difficult to distinguish whether the increased sense
transcription in COMPASS mutants is caused, or is followed, by a
decrease in 39-end antisense transcription. One way of addressing
this directly is to eliminate 39-end antisense transcription by other
means than through disruption of COMPASS. For this purpose
strong terminator sequences were introduced downstream of the
five model genes analyzed in Figure 4B, either as insertions
between antisense promoters and the end of the ORF, or as
replacement of complete intergenic sequences. Neither approach
resulted in loss of 39-end antisense transcription, which agrees with
the recent finding that terminators can function as promoters [55]
. Disruption of 39-end antisense transcription was then attempted
by removal of all, or a significant part of the intergenic region.
Complete loss of all antisense transcription was only observed for
the YGR110W intergenic deletion mutant, which we further
analyzed in depth (YGR110W-ingdel, Figure 5).
Strand-specific Northern blot analysis of YGR110W-ingdel
shows that loss of antisense transcription (Figure 5B, asYGR110W,
lane 1 versus lane 3), is accompanied by derepression of sense
transcription (Figure 5B, sYGR110W). This demonstrates that 39-
end antisense transcription results in repression of sense transcrip-
tion. Furthermore, introduction of SET1 deletion into the
YGR110W-ingdel strain, does not result in significant further
derepression as is observed in the presence of 39-end antisense
transcription (Figure 5B, lanes 1 and 2 versus lanes 3 and 4). This
agrees with the proposal that the repressive effect of COMPASS on
coding genes is a result of promoting 39-end antisense transcription.
H3K4me3 promotes 39-end antisense transcription and
H3K4me2 contributes to coding gene repression
The results presented in Figure 4B and Figure 5B imply a
positive role for Set1 on antisense transcription. SET1 deletion
results in loss of H3K4me1, me2 and me3 (Figure 1A). SPP1
deletion (loss of H3K4me3 only), has little effect on sense transcript
levels (Figure 1, Figure 2, Figure 4B). SPP1 deletion does result in
decreased antisense transcripts as observed either in the presence
or absence of RRP6 (Figure 4B). Our results indicate that H3K4
trimethylation, which is found at the 39-end of these genes, has a
role in promoting 39-end antisense transcription. This effect is not
absolute however. Antisense transcripts are reduced in the SET1
RRP6 double deletion compared to rrp6D, but are not completely
absent. This indicates that antisense transcription is promoted by,
but not fully dependent on, H3K4me3. Since spp1Dstill exhibits
wt levels of H3K4me2 (Figure 1A) and virtually no derepression of
sense transcription (Figure 1B and Figure 4B), this indicates that it
is the H3K4me2 mark which is most important for repression of
sense transcription on these genes. Together, the results of these
experiments are consistent with a model, whereby the majority of
COMPASS-repressed genes are maintained in an inactive state
through 39-end antisense transcription that is in part promoted
through H3K4me3 at the 39-end, and in turn deposits a repressive
H3K4me2 mark further into the body of the gene.
COMPASS repression is mimicked by reducing
nucleosome levels
We next asked what determines the specificity of the effects
observed upon mutation of COMPASS. H3K4 methylation marks
all active genes and approximately one third of all genes exhibit
Figure 5. Set1 represses sense transcription through promo-
tion of antisense transcription. (A) Scheme showing YGR110W and
YGR111W genes before (YGR) and after (YGR-ingdel) deletion of their
intergenic region. The sense YGR110W transcript (sYGR110W), as well as
the longer sense transcript in the YGR-ingdel strain are shown in dark
grey, while the antisense transcript (SUT557) is shown in light grey. The
position and 59-39direction of the strand-specific probes used to detect
the transcripts are also shown. (B) Autoradiographs of Northern blots
hybridized with the strand-specific DNA probes designed to detect the
sense (sYGR110W) or antisense (asYGR110W) transcripts of YGR110W in
the YGR and YGR-ingdel strains with wild-type (SET1) or deleted SET1
(set1D). An autoradiograph of the same blot hybridized with a tubulin
probe (TUB1) was used as loading control. Quantitation of the bands are
shown below each panel relative to the wt (SET1 YGR) strain for TUB1
and relative to the wt (SET1 YGR) strain and the loading control for the
sYGR110W and asYGR110W panels.
doi:10.1371/journal.pgen.1002952.g005
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antisense transcripts [40], yet only a subset are affected by deleting
COMPASS subunits (Figure 1). It has recently been proposed that
the transcription factor Reb1 may drive non-coding transcription,
either from neighbouring genes [33] or from the promoter of the
antisense transcript itself [56]. Reb1 binding sites are found
downstream of only three of the 69 COMPASS-repressed genes.
There is also no statistically significant enrichment for Reb1
binding sites in the ORFs or flanking regions of genes up-regulated
in the COMPASS mutants. Both observations suggest that the
specificity of Set1 repressive effects is not generally linked to Reb1.
In addition, no other putative regulatory motifs could be detected
in these regions using different search algorithms [57].
An alternative explanation for the specificity of COMPASS
repressive effects is that specificity is dictated by increased
sensitivity of specific genes to a particular chromatin structure
which is influenced by H3K4 methylation. While profiling strains
with altered histone expression levels we noted an interesting
correlation with the collection of COMPASS mutants. To
investigate this, a strain bearing single copies of the histone H3
and H4 genes under control of their native promoters [58] was
analyzed. The two-fold reduction in mRNA levels of H3 and H4
in this strain (Figure 6B, marked HHT2 and HHF2)is
accompanied by slightly decreased H3 and H4 protein levels
(Figure 6A). Interestingly, this results in upregulation of a specific
subset of genes that strongly correspond to the genes upregulated
upon SET1 deletion (p-value 1.4*10
223
, Figure 6B). Although the
overlap is highly statistically significant, it is not complete and does
not extend to PHO84 for example, in agreement with the proposal
for a distinct repressive mechanism for such genes (Figure 4A).
SET1 deletion does not globally affect nucleosome levels (data not
shown), and antisense transcript levels are not reduced in the single
copy H3 H4 strain (Figure 6C). Besides antisense transcription, a
second common property of the genes affected by loss of
COMPASS function is therefore sensitivity to histone abundance.
Since histone abundance affects nucleosome density [59], this
suggests that Set1 may repress genes by effecting nucleosome
density. As is discussed below, one manner in which this may be
achieved is through the repressive H3K4me2 mark that is laid
down through 39-end antisense transcription.
Discussion
Repressive role of COMPASS in S. cerevisiae
The results presented here add to a number of reports that
indicate that the major non-redundant role of COMPASS in S.
cerevisiae is repression of coding genes [34,56]. Early genome-wide
analyses of set1Dyielded conflicting results, in two cases pointing to
global positive effects [27,28] and in one case ignoring the
prevalence of specific repressive effects [6]. Some of the differences
between these studies and the current one can in retrospect be
attributed to use of double-stranded cDNA arrays, less convenient
for discriminating between sense and ant-sense effects, as well as to
normalisation issues. The analyses presented here, using strand-
specific techniques, with replicate experiments for a variety of
different mutants under both steady-state and dynamic conditions,
indicates that removal of H3K4me3, a global mark of active
transcription, has no global effect. The repressive effects observed
on a specific subset of genes agree with the most recent other
genome-wide analyses of set1D[29] [30], as well as with the fact
that deletion of SET1 is not lethal. Gene Ontology analysis of the
affected genes reveals an overrepresentation of vitamin metabo-
lism (essentially thiamin biosynthesis) and spore wall assembly
(Table S3) in agreement with the cell wall and stationary phase
defects previously observed in set1Dcells [51].
COMPASS and antisense transcription
What is the mechanism of the observed repression? Despite the
fact that COMPASS-repressed genes show a significant enrich-
ment for telomeric-proximal localization (Figure S3 and [29]),
Set1-dependent repression of these genes due to a telomere
position effect can probably be ruled out since the derepression
observed in set1Donly affects a small percentage of individual
genes within these regions. Only a few of the affected genes are
close to telomeres and only few telomere-proximal genes are
affected. Analysis of methylation patterns (Figure 3 and [53]), non-
coding RNA maps (Table S2 and [39,40]) and the comparison of
mutants with different methylation states support a model whereby
COMPASS mediates repression of coding genes by promoting the
expression of 39-end antisense transcripts through deposition of
H3K4me3 at their 39-end.
Figure 6. COMPASS-repressed genes are derepressed upon
decrease in nucleosome content. (A) The cellular expression level of
H3 was analyzed by Western blotting in wt and hht1Dhhf1Dcells. (B)
Correlation of the effects, as measured by log base 2 ratios, of low
nucleosomes levels (hht1Dhhf1D) and SET1 deletion (set1D) on the
COMPASS-repressed genes versus wt cells. LYS2 is excluded as it is used
as an auxotrophic marker for the hht1Dhhf1Dstrain. PHO84 is marked,
as it is significantly repressed in the low nucleosome strain. The two
histone genes are also depicted verifying the expected 2-fold reduction
in their mRNA levels. (C) Strand-specific qPCR analysis, as in Figure 4B,
for the indicated backgrounds.
doi:10.1371/journal.pgen.1002952.g006
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An involvement of Set1 in promotion of 39-end antisense
transcription, resulting in a repressive effect on sense transcription
has been reported for PHO84, which is repressed through the
presence of antisense transcripts [34]. Our results are consistent
with a repressive role for Set1 on PHO84. The genome-wide
nature of our experiments indicates however that the majority of
Set1 affected genes are repressed through a different mechanism,
independent of the level of antisense RNA transcripts. Rather, for
the majority of Set1-regulated genes, repression is caused by the
process of antisense transcription itself. This mechanism is
therefore related to the recently reported attenuation in GAL10-
GAL1 activation which is also facilitated through cryptic
transcription [56]. One major difference is that for the mechanism
reported here, COMPASS is required to maintain antisense
transcription whereas this does not seem to be the case for the
cryptic transcription observed at the GAL10-GAL1 locus [56].
Distinct roles for H3K4me2 and H3K4me3
The comparison of different COMPASS mutations carried out
here, facilitates distinguishing between the roles of the different
H3K4 methylation states. Mutants with grossly lowered or
completely absent H3K4me3 exhibit decreased antisense tran-
scription. However this only results in derepression of the coding
gene if H3K4me2 is also abolished. The positive role of H3K4me3
on antisense transcription fits with the correlation observed
between the presence of this mark and promoter activity of
coding genes [7,8,9,10,11,25]. Most non-coding RNAs originate
from nucleosome-free regions (NFRs) shared with protein-coding
transcripts [40]. This is also the case for three of the five
COMPASS-repressed genes analyzed here in detail (AMS1,ARG1
and OYE3). Interestingly, despite sharing a NFR with the
downstream protein-coding gene, loss of H3K4 trimethylation
causes reduction of antisense transcription without affecting
transcription of the flanking protein-coding gene in each case.
This fits with the observation that bidirectional transcription from
a single NFR, originates from two distinct preinitiation complex
recruitment sites [60]. This may indicate the presence of
redundant mechanisms for maintaining protein-coding gene
transcription in the absence of H3K4me3, which are lacking for
the antisense non-coding transcription originating from the same
NFR. Another, non mutually exclusive mechanism, can be that
lack of H3K4 trimethylation in the antisense transcription start site
increases the recruitment of corepressor complexes, such as Rpd3S
[61], that repress the expression of the non-coding transcript, but
not that of the coding gene [62].
The results also indicate a role for the H3K4me2 mark in
facilitating repression. This agrees with several recent studies
suggesting mechanisms through which H3K4me2 may play a
repressive role. For example, it has recently been reported that
H3K4me2 in the body of active genes is recognized by the Set3
complex, leading to histone deacetylation, a repressive chromatin
state [63]. A different histone deacetylase, Rpd3, has been
implicated in the repressive role involving Set1 on the GAL10-
GAL1 locus [56]. Furthermore, methylation of H3K4 protects
against an H3 tail endopeptidase recently described in S. cerevisiae
and humans that facilitates transcription initiation and precedes
histone eviction [64,65]. All these possible mechanisms fit with the
observation made here that globally reducing H3 and H4 levels
mimics the derepression of COMPASS mutants. The degree of
overlap between the COMPASS mutants’ profiles and the histone
depletion profile also give an explanation for the specificity of the
COMPASS repression. Genes repressed by COMPASS have
antisense transcription, but are also sensitive to nucleosome
density.
Sensitivity to histone depletion may not be the only reason for
the lack of genome-wide effects upon COMPASS mutation.
Functional redundancy may also contribute. One of the prevalent
ideas for a general role of H3K4 methylation in S. cerevisiae is that
transcription-associated H3K4 methylation, as well as deposition
of the histone variant H2A.Z, antagonizes the local spread of Sir-
dependent silent chromatin into adjacent euchromatic regions
[52,66,67]. It has recently been shown that H2A.Z deposition and
Set1 cooperate to prevent Sir-dependent repression of a large
number of genes located across the genome [29]. This functional
redundancy between H3K4 methylation and H2AZ deposition
may thus buffer transcription from changes in euchromatin,
thereby minimizing the observed effects of H3K4 methylation loss.
This work offers a plausible explanation for how a transcription
factor, previously thought to positively contribute to transcription,
can nevertheless exert a negative effect, through promoting
antisense transcription. The opposite has previously been shown
for regulation of IME4. Here a repressor complex binds to the
promoter of an antisense transcription unit in the 59-end of IME4
and by repressing the antisense transcription, facilitates the in cis
sense transcription activation [68]. Although further work is
required to pinpoint the mechanisms further downstream of
H3K4 di- and trimethylation, COMPASS exemplifies the growing
insight that the roles of histone modifications in gene expression
are non-linear [69] and context-dependent [70].
Materials and Methods
Microarray data is accessible through the public microarray
database ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) un-
der accession number E-TABM-486. The accession numbers
below refer to detailed protocols in ArrayExpress.
Strains and primers
Strains and primers used in this study are described in Tables S4
and S5 respectively. The YGR110W-ingdel strain was created by
first inserting a cassette containing the Sp HIS5 gene in reverse
orientation flanked by the Ag TEF promoter, terminator sequences
and loxP sites from plasmid pUG27 [71] to replace the intergenic
region between YGR110W and YGR111W using YGR110-
W_HIS5_F and YGR110W_HIS5_R primers. Subsequently the
cassette was floxed out by transforming the strain with the plasmid
pSH47 and expressing Cre recombinase as previously described
[72].
Histone purification and Western blotting
Histones were purified as described [42], subjected to 16%
SDS-polyacrylamide gel electrophoresis, and either Coomassie
Blue stained or transferred to 0.2 mm Protran
R
nitrocellulose.
Antibodies used to detect mono-, di- and trimethylated H3K4 and
histone H3 were from Abcam.
Cultures
Two independent colonies of each strain were first inoculated
and grown overnight in synthetic complete medium with 2%
glucose. For the mid-log/steady-state experiment, larger cultures
were inoculated the next day at an OD600 of 0.15 in fresh
medium, allowed to grow at 30uC and harvested at OD600 0.6,
(P-UMCU-36). For the time-course experiment, overnight cultures
were used to inoculate 50 ml cultures at an OD600 of 0.15. These
were allowed to deplete glucose by growing for 24 hours and were
used the next day to start 500 ml cultures at an OD600 of 0.15 in
fresh medium for the time-course sampling (P-UMCU-47).
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RNA isolation and amplification
Total RNA isolation was by hot acid phenol (P-UMCU-37) and
cleaned up using RNeasy (Qiagen). Before amplification, external
RNA controls were added to total RNA to check for global shifts
in mRNA levels [41]. cRNA amplification and labelling using
amino-allyl UTP was performed on a Caliper robot system (P-
UMCU-38).
Microarrays and hybridizations
Each sample was generated twice, as independent biological
replicates. These were hybridized in dye-swap against a common wt
reference RNA (P-UMCU-39) on oligo-arrays that represented
each gene twice (P-UMCU-34). After scanning (P-UMCU-40), raw
data were extracted with Imagene (Biodiscovery) (P-UMCU-42).
Data analysis
Since spike-in of external RNA controls revealed no global
changes in the mRNA population [41] for the mid-log experiment,
non-background corrected data were normalized with print-tip
LOESS [73] on gene probes with a span of 0.4 (P-UMCU-41). For
the time-course, all features, including negative and external
controls (EC) except EC 4, 6 and 8, were used for the estimation of
the LOESS curve (P-UMCU-46). Probes flagged as absent, or with
a nearly saturated signal were not used to estimate the LOESS
curve. For differential expression analysis, the LIMMA package
[74] was used. Mitochondrial-encoded genes and Ty elements
were excluded due to their high biological variation. Genes with
an FDR-adjusted p-value less than 0.01 and a fold-change of more
than 1.7 were considered significant. These thresholds are based
on systematic analyses of the variation observed in a collection of
more than 100 wt expression profiles [75]. For the time-course
experiment, changes were considered significant if they fulfilled
these criteria for two consecutive time-points. Hierarchical
clustering was by MeV [76], using standard correlation and
average linkage. Analysis of overlap between genelists of signifi-
cantly changing genes of two expression profiles was by
hypergeometric test. For the GO and transcription factor
enrichment analysis, a right-sided Fisher’s exact test was used
and the p-values were corrected for multiple testing using
Bonferroni. The GO annotations were obtained from SGD.
For the H3K4 methylation ChIP-chip analysis, the data are
from [53]. For each gene, a region corresponding to the ORF plus
500 bps in both directions was used. The ORF was divided into 30
bins of equal length and the flanking regions in 10 bins each. A
loess algorithm [77] with a span of 0.2 was used to estimate the
enrichment of the methylation marks for every bin.
Reverse Transcription and qPCR
cDNAs of sense RNA or antisense RNA were generated by
SuperScript III Reverse transcriptase (Invitrogen) from total RNAs
using gene and strand-specific primers. For each gene, cDNAs
obtained from the reverse transcription of sense or antisense RNA
were quantified by a real-time qPCR with gene-specific primers
corresponding to a 150 bp fragment (Figure 4B). The same
primers were used to quantify sense and antisense cDNA of each
gene. The position and the sequence of each primer are indicated
in Figure 4B and Table S5.
Northern blotting
The strand-specific DNA probes used to detect the presence of
sense and antisense transcripts of YGR110W are shown schemat-
ically in Figure 5A. First a cold PCR product template was
obtained using primers 39qYGR110W and 59qYGR110W.
Subsequently the hot ssDNA probes for detection of the sense
and antisense transcripts were generated from the template using
the first or the second primer, respectively, in linear PCR
reactions. Quantitation of the radioactive signal was performed
using ImageQuant (Molecular Dynamics).
Supporting Information
Figure S1 (A) Hierarchical clustering, as in Figure 1B, of all
genes with significantly changed mRNA expression in any
COMPASS mutant. Figure 1B depicts those genes that have
significantly changed expression in at least two mutants. (B) Genes
depicted in the same order as in A for the H3K4R point mutant
and bre1D.
(TIF)
Figure S2 The repressive effect of Set1 on transcription is
through H3K4 (A) Gene expression scatter plot of the average,
normalized fluorescent intensity values of each gene in set1D
compared to the wt strain. The 69 COMPASS-repressed genes are
represented by yellow dots. (B) As in A, but now for the set1D
H3K4R double mutant compared to the H3K4R point mutant.
The deleted gene is represented by a blue dot.
(TIF)
Figure S3 COMPASS-repressed genes are enriched near
telomeres. The histogram shows the genomic location of the 69
genes significantly upregulated in at least two COMPASS deletion
mutants. The bars represent the numbers of genes found in 5-kb
intervals from nearest chromosome end. The line represents the
log
10
p-value as a function of distance to the nearest chromosome
end. Note that the scale of log
10
p-values runs from 0 to -15 so that
the height of the line corresponds to higher significance.
(TIF)
Figure S4 Methylation patterns for all genes. The average
enrichment of H3K4me1 (blue), H3K4me2 (red) and H3K4me3
(grey) over H3, for the set of 5977 yeast genes that show at least a
two-fold enrichment of H3K4me2 or H3K4me3 somewhere
across the gene or flanking region [53].
(TIF)
Figure S5 H3K4 methylation patterns indicating antisense
transcription. Patterns of H3K4 methylation [53] on the five
model genes followed up in Figure 4B, expressed as log
2
of each
methylation mark over H3 (top panels). Mapping of coding
regions by SGD indicated in red and non-coding ones by [39],
indicated in blue and yellow for CUTs and SUTs, respectively
(bottom panels).
(TIF)
Table S1 The 69 COMPASS-repressed genes and their distance
from the nearest chromosome end.
(PDF)
Table S2 Evidence for the presence of ncRNAs in the
COMPASS-repressed genes that have H3K4me2/3 levels more
than 2-fold over H3. The evidence for non-coding transcription is
based on [39,40]. Three types of non-coding RNAs were reported:
antisense transcripts spanning the body of the gene (antisense),
transcripts in the promoter of the genes (promoter) and known
non-coding transcripts (SGD). ‘‘No data available’’ indicates cases
when the above studies didn’t include the regions of specific genes
in their results.
(PDF)
Table S3 The 69 COMPASS-repressed genes are enriched in
specific Gene Ontology functional categories and transcription
H3K4 Methylation and Antisense-Mediated Repression
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factor binding sites in their promoters as described by the Fraenkel
lab - MacIssac (2006) BMC Bioinformatics. The number of co-
occurences between functional categories and the repressed genes
(Hits), the corresponding genes (Annotated Genes), the number of
background hits, as well as the corresponding Bonferroni-
corrected p-values (Cor. p-val) are reported.
(PDF)
Table S4 Strains and plasmids used in this study.
(PDF)
Table S5 Primers used in this study.
(PDF)
Acknowledgments
We thank Maria Hobeika and members of A. Morillon’s and M. Timmers’
laboratories for helpful discussions and their input in this work.
Author Contributions
Conceived and designed the experiments: TM CD VG FCPH. Performed
the experiments: TM VO NB LM AV-P JJB DvL. Analyzed the data: TM
VO NB LM AV-P JJB SvH DvL CD VG FCPH. Contributed reagents/
materials/analysis tools: TM VO NB LM AV-P JJB SvH DvL CD VG
FCPH. Wrote the paper: TM CD VG FCPH.
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