A role for noncoding transcription in activation of the yeast PHO5 gene.

Abstract

Noncoding, or intergenic, transcription by RNA polymerase II (RNAPII) is remarkably widespread in eukaryotic organisms, but the effects of such transcription remain poorly understood. Here we show that noncoding transcription plays a role in activation, but not repression, of the Saccharomyces cerevisiae PHO5 gene. Histone eviction from the PHO5 promoter during activation occurs with normal kinetics even in the absence of the PHO5 TATA box, showing that transcription of the gene itself is not required for promoter remodeling. Nevertheless, we find that mutations that impair transcript elongation by RNAPII affect the kinetics of histone eviction from the PHO5 promoter. Most dramatically, inactivation of RNAPII itself abolishes eviction completely. Under repressing conditions, an approximately 2.4-kb noncoding exosome-degraded transcript is detected that originates near the PHO5 termination site and is transcribed in the antisense direction. Abrogation of this transcript delays chromatin remodeling and subsequent RNAPII recruitment to PHO5 upon activation. We propose that noncoding transcription through positioned nucleosomes can enhance chromatin plasticity so that chromatin remodeling and activation of traversed genes occur in a timely manner.

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A role for noncoding transcription in activation
of the yeast PHO5 gene
Jay P. Uhler*, Christina Hertel†, and Jesper Q. Svejstrup*‡
*Mechanisms of Transcription Laboratory, Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD,
United Kingdom; and†Institut fu ¨r Physiologische Chemie, Universita ¨t Mu ¨nchen, Schillerstrasse 44, 80336 Munich, Germany
Communicated by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, March 21, 2007 (received for review January 9, 2007)
Noncoding, or intergenic, transcription by RNA polymerase II (RNA-
PII) is remarkably widespread in eukaryotic organisms, but the
effects of such transcription remain poorly understood. Here we
show that noncoding transcription plays a role in activation, but
not repression, of the Saccharomyces cerevisiae PHO5 gene. His-
tone eviction from the PHO5 promoter during activation occurs
with normal kinetics even in the absence of the PHO5 TATA box,
showing that transcription of the gene itself is not required for
promoter remodeling. Nevertheless, we find that mutations that
impairtranscriptelongationbyRNAPIIaffectthekineticsofhistone
eviction from the PHO5 promoter. Most dramatically, inactivation
of RNAPII itself abolishes eviction completely. Under repressing
conditions, an ?2.4-kb noncoding exosome-degraded transcript is
detected that originates near the PHO5 termination site and is
transcribed in the antisense direction. Abrogation of this transcript
delays chromatin remodeling and subsequent RNAPII recruitment
to PHO5 upon activation. We propose that noncoding transcription
through positioned nucleosomes can enhance chromatin plasticity
so that chromatin remodeling and activation of traversed genes
occur in a timely manner.
elongation ? intergenic transcription ? RNA polymerase II
I
known and poorly understood untranslated RNAs. Recent
genome-wide studies in several species reveal that such noncod-
ing transcription is much more extensive than previously thought
and that it occurs across intergenic regions, introns, and exons
(see, for example, refs. 1 and 2). Recently, genome-wide studies
in yeast have identified many cases of intergenic transcripts
associated with promoters (3–5), raising the question of whether
and how intergenic transcription across a promoter is used as a
means of regulating that gene’s transcription.
During our studies on elongation and RNA processing factors
in yeast, we discovered an intergenic transcript across the PHO5
promoter. This finding led us to investigate whether noncoding
transcription might play a role in regulating this gene. PHO5
encodes an acid phosphatase that is regulated by phosphate
availability (6). In high phosphate, four positioned nucleosomes
are associated with the PHO5 promoter region (7). During
phosphate starvation, the Pho4 activator translocates to the
nucleus (8) and binds to PHO5 upstream activation sequences
(UASp1 and UASp2) along with the Pho2 activator (9–11). This
leads to eviction of the four positioned nucleosomes, making a
600-bp region effectively fully accessible (7, 12–14). Promoter
remodeling is facilitated by, although not always absolutely
dependent on, several transcription factor complexes including
SAGA, Swi/Snf complex, INO80, and the Asf1 chaperone (14–
18). High phosphate causes Pho4 accumulation in the cytoplasm,
nucleosome reassembly on the promoter, and transcriptional
repression of the gene.
Here we show that intergenic transcription plays a role in the
kinetics of PHO5 promoter remodeling.
n addition to transcribing all protein-encoding genes, RNA
polymerase II (RNAPII) also transcribes a large group of less
Results
An Intergenic Transcript Across the PHO5 Promoter. We initially
noticed the appearance of an additional PHO5 RNA species
during our characterization of transcription in rrp6 mutants
(J.P.U., unpublished data), which lack a functional nuclear RNA
exosome and accumulate intergenic transcripts (ref. 4 and
references therein). However, detection of this transcript is
possible even in wild-type cells in which noncoding RNAs are
otherwise unstable. It is ?2.4 kb in size and is observed only in
cells grown in high-phosphate (repressing) conditions (Fig. 1A).
Using strand- and promoter-specific RT-PCR on total RNA
from wild-type and rrp6 cells (Fig. 1B), we deduced that inter-
genic transcription is antisense relative to PHO5 mRNA.
A series of probes across the PHO5 locus was hybridized with
RNA isolated from wild type and rrp6, respectively. All probes
that spanned the ?2.4-kb region from 950 bp upstream of the
PHO5 transcription start site to the 3? end of the PHO5 ORF
hybridized to the intergenic transcript, but the transcript was not
detected with flanking probes 1 and 7 (Fig. 1C).
A strain carrying a temperature-sensitive allele of the largest
subunit of RNAPII (rpb1-1) (19) was used to establish that the
intergenic transcript is produced by RNAPII and that RNAPII can
indeed be detected in the upstream PHO5 promoter by ChIP, even
in repressing conditions [supporting information (SI) Fig. 6].
Together, the above results suggest that RNAPII actively
transcribes across the uninduced PHO5 gene and its promoter,
producing an unstable, noncoding, antisense RNA.
Intergenic Transcription Is Not Important for PHO5 Repression. Be-
cause some previous studies of PHO5 chromatin structure and
activation were conducted with versions of the PHO5 gene that did
not contain the region in which the intergenic transcript originates
(20,21),aneffectofintergenictranscriptiononthecapabilityofthe
PHO5 gene to be turned on or off was not expected to be absolute.
Indeed, it seemed reasonable to expect that the effect, if any, might
be reminiscent of that observed upon mutation of the histone
acetyltransferaseGCN5,whichaffectsthekinetics,butnotthefinal
level, of activated PHO5 transcription (15).
We first examined whether transcription in general is required
to establish or maintain histones at the repressed PHO5 pro-
moter. If not, a role for the intergenic transcript (representing
only a fraction of general transcription) would be highly unlikely.
For this purpose, wild-type and rpb1-1 cells were grown in high
phosphate at 23°C and then shifted to 37°C. Comparison of
histone H3 levels by ChIP assays revealed no significant differ-
ence in histone density at the promoter between wild type and
rpb1-1 cells, indicating that ongoing transcription is not required
Author contributions: J.P.U. and J.Q.S. designed research; J.P.U. and C.H. performed
research; J.P.U. contributed new reagents/analytic tools; J.P.U. and J.Q.S. analyzed data;
and J.P.U. and J.Q.S. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Abbreviations: RNAPII, RNA polymerase II; 6AU, 6-azauracil; YPD, yeast extract/peptone/
dextrose.
‡To whom correspondence should be addressed. E-mail: j.svejstrup@cancer.org.uk.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0702431104/DC1.
© 2007 by The National Academy of Sciences of the USA
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to maintain histones at the promoter during repression (Fig. 2A).
WealsofoundthatgeneralRNAPIItranscriptionisnotrequired
for establishment of repression (Fig. 2B). In this experiment,
wild-type and rpb1-1 cells were grown at 23°C in phosphate-free
medium and then shifted to 37°C for 30 min before adding
phosphate. H3C ChIP analysis showed that, upon adding phos-
phate, histones were rapidly deposited onto the PHO5 promoter
also in the transcription-defective rpb1-1 cells (Fig. 2B), indi-
cating that transcription is not required for a normal rate of
promoter closing either.
Transcription Is Required for Normal Kinetics of Promoter Remodel-
ing.Wenextaskedwhethergeneraltranscriptionmightsomehow
facilitate histone eviction from the PHO5 promoter during
activation. Wild-type and rpb1-1 cells were grown at the per-
missive temperature and then shifted to 37°C for 30 min before
phosphate starvation. By 6 h of induction, wild-type cells had
?80% fewer histones at the promoter than in high phosphate
(Fig. 3A). In sharp contrast, histones were not evicted at all upon
phosphate starvation in the rpb1-1 strain, suggesting that tran-
scription might indeed play a role in PHO5 promoter opening.
To check that the inability to remodel in rpb1-1 was not simply
due to a defect in PHO signaling, we monitored Pho4 localiza-
tion. Pho4 translocates to the nucleus upon successful signal
transduction (8). Using GFP-tagged Pho4, we found that at 37°C
the majority of Pho4 is in fact found in the nucleus by 1.5 h of
phosphate starvation in both wild-type and rpb1-1 cells (Fig. 3B).
Furthermore, the PHO induction cascade was bypassed alto-
gether by using a PHO5 promoter derivative (PHO5v33), where
the Pho4 binding sites are replaced by Gal4 binding sites (15).
This promoter responds to the addition of galactose in a manner
virtually identical to the response of the normal PHO5 promoter
during phosphate starvation (ref. 15 and references therein).
Also using this construct, histone eviction was negligible at the
restrictive temperature in rpb1-1, whereas histone density was
reduced to very low levels in wild type (Fig. 3C).
The TATA Box Is Not Required for Normal Kinetics of Promoter
Remodeling. Previous experiments by Ho ¨rz and coworkers (22)
with promoter derivatives lacking a TATA box have argued
against transcription of the PHO5 gene being an absolute
requirement for its chromatin remodeling, but to address the
possibility that it might affect the kinetics of chromatin remod-
eling we mutated the PHO5 TATA box at its native genomic site
from TATATAA to CCTAGGA, asking whether this mutation
affected histone eviction from the promoter during activation.
RNAPII ChIP showed that, as expected, polymerase recruit-
ment to the TATA-less promoter in response to activation was
virtually abolished as a consequence of this mutation, but the
kinetics of histone eviction during PHO5 induction was largely
Fig. 1.
promoter. (A) Northern blots of total RNA hybridized with PHO5 promoter-
specific probe (intergenic) and an ORF probe (PHO5). (Left) PHO5 induction.
(Right) PHO5 shutoff. 18S is shown as loading control. (B) Two-step reverse
transcriptase,real-timePCRwasperformedontotalRNAfromwild-type(WT)
or rrp6 cells grown in YPD using PHO5 promoter-specific primers (across
UASp2;seeCUpper).Theinitialreversetranscriptasestepwasperformedwith
a primer that was either complementary to (antisense) or on the same strand
as (sense) the PHO5 transcript. y axis indicates relative RNA levels (arbitrary
units).Theresultshownisrepresentativeoftwoindependentexperiments.(C
Upper) Schematic drawing of the PHO5 region (?1 indicates the first base of
the ORF; ?1,404 indicates the last) and the probes (numbered) used to detect
theintergenictranscript.ThepositionsofUASp1and-2andtheTATAboxare
also indicated. (C Lower) Northern blots on wild-type and rrp6 RNA from cells
grown in YPD using the probes indicated by numbers below. The intergenic
transcript is detected with probes 2–6 (and stronger in rrp6).
An intergenic transcript is detected across the repressed PHO5
Fig. 2.
promoter chromatin. (A) Relative histone H3 occupancy (H3C ChIP) at the
PHO5 promoter in WT and rpb1-1 cells grown in YPD at 23°C or 37°C, as
indicated. y axis indicates relative histone H3 levels (arbitrary units). (B)
RelativehistoneH3density(H3CChIP)atthePHO5promoterinWTandrpb1-1
cells during the establishment of phosphate repression. Measurements from
cellsgrowninhighphosphate(YPD),intheabsenceofphosphate(?Pi,time?
0),andatdifferenttimesaftershiftingto37°Candintophosphate-containing
medium (?Pi, different time points). Density in YPD was set to 1, and all other
values are expressed relative to that.
RNAPII activity is not required to maintain or establish repressed
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unaffected by this defect in polymerase recruitment (SI Fig. 7),
confirming and extending the conclusion from previous studies
of TATA-less PHO5 plasmid constructs (22): RNAPII recruit-
ment to the PHO5 promoter is not required for normal histone
eviction.
Defects in Transcriptional Elongation Affect the Kinetics of Promoter
Remodeling at PHO5. To further test the idea that intergenic
transcription helps condition the PHO5 promoter for remodel-
ing, we specifically targeted the elongating form of RNAPII
using strains or conditions causing defects in this process, namely
rpb2-10 (carrying an elongation-impairing mutation in the Rpb2
subunit) or dst1? (lacking the gene encoding TFIIS) strains and
the elongation inhibitor 6-azauracil (6AU). Although, as ex-
pected, the results were much less dramatic than upon complete
disruption of RNAPII transcription, the effect on PHO5 pro-
moter histone eviction of these phenotypically innocuous mu-
tations and 6AU was nevertheless significant and highly repro-
ducible. H3C ChIP revealed that 6AU treatment led to a slower
loss of histones (Fig. 4A). Significantly, RNAPII recruitment to
the PHO5 TATA box was also both delayed and decreased under
these elongation-prohibiting conditions, this effect being more
dramatic in dst1? cells (Fig. 4B). Similar results were also
obtained here by using the Gal-responsive PHO5 promoter
derivative (PHO5v33) used in Fig. 3C (data not shown).
Similarly, in the rpb2-10 strain (SI Fig. 8), an almost 2-fold-
higher level of histones remained at the promoter (both at
UASp2 and TATA) after 1.5 h of phosphate starvation com-
pared with wild type. Only at later time points did the level of
histone eviction reach wild-type levels, and there was an accom-
panying delay in transcriptional activation of PHO5, as revealed
by delayed RNAPII recruitment to the PHO5 TATA box.
Together, these data further support the idea that efficient
transcript elongation across the PHO5 gene under repressing
conditions is required for its rapid activation.
Deletion of the 3? End of the PHO5 ORF Affects Histone Eviction from
the Promoter. We finally sought to more specifically block inter-
genic transcription across the PHO5 promoter, to investigate
whether it is relevant for PHO5 promoter remodeling. Because
Fig. 3.
(A) Relative histone H3 density (H3C ChIP) in the PHO5 promoter in WT and
rpb1-1 cells during phosphate starvation at 37°C. (B) WT and rpb1-1 cells
expressingaPho4-GFPfusionproteinweregrowninhighphosphateandthen
transferred to medium lacking phosphate at 37°C. GFP and DAPI fluorescence
are shown. (C) Relative histone H3 density (H3C ChIP) in the indicated regions
of the galactose-regulated PHO5v33 promoter in WT and rpb1-1 cells during
galactoseinductionat37°C.InbothAandC,densityattime?0(23°C)wasset
to 1 and all other values are expressed relative to that. The schematic in C
indicates location of PCR products used to measure histone H3 density in the
PHO5v33 promoter. Small gray spheres indicate the position of Gal4 binding
sites (replacing UASp1 and 2).
PHO5 promoter remodeling is abolished when RNAPII is inactivated.
Fig. 4.
impaired. (A) Relative histone H3 density (H3C ChIP) in the indicated regions
of the PHO5 promoter (UASp2 and TATA) in WT and dst1 cells during phos-
phate starvation, with or without 6AU treatment to inhibit RNAPII transcript
elongation. (B) As in A, but RNAPII recruitment to the TATA box (4H8 ChIP).
Densityattime?0wassetto1,andothervaluesareexpressedrelativetothat.
PHO5 promoter remodeling is delayed when RNAPII elongation is
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abrogation of RNAPII transcriptional initiation can be achieved
only by removing the TATA box or initiator, we initially looked
for potential TATA boxes near the end of the PHO5 ORF. No
potential TATA boxes inside the PHO5 ORF were detected by
sequence searching, but in the region immediately downstream
from it, two fairly conserved cryptic sites were found. Three
different approaches were then pursued to block intergenic
transcription. First, we attempted to block usage of the potential
TATA sites, either by inserting a URA3 marker right after the
PHO5STOPcodonorbyreplacingtheentiredownstreamregion
between PHO5 and PHO3 with a bidirectional terminator se-
quence (normally found between FBA1 and YKL061W), which
has no discernable TATA sequences. Neither of these ap-
proaches halted intergenic transcription, nor did they affect
PHO5 promoter activation (data not shown). This suggests that
intergenic transcription is initiated by (non-TATA) sequence
elements near the 3? end of the PHO5 ORF itself. Second, we
inserted the bidirectional terminator at a position 500 bp into the
PHO5 ORF, hoping to thereby block intergenic transcription
through the PHO5 promoter region. Interestingly, although this
approach resulted in the disappearance of a stable, detectable
intergenic transcript in wild-type cells, a slightly longer inter-
genic transcript could still be detected across the PHO5 pro-
moter in rrp6 cells (SI Fig. 9), supporting the idea that RNAPII
continued to transcribe through the PHO5 promoter region (SI
Fig. 10). Accordingly, insertion of the terminator at this position
also failed to affect PHO5 promoter activation (data not shown).
Given that the above approaches did not halt intergenic
transcription, the region containing the intergenic transcription
start site region was deleted by substitution with a URA3 marker
(PHO5-3??) orientated in the sense direction, in the hope that
anypotentialtranscriptionthroughthePHO5promoterfromthe
inserted marker gene itself, as well as from initiation region(s)
downstream from the site of insertion, could be abrogated (the
marker replaced PHO5 ORF sequence from ?751 to ?1,404;
Fig. 5A Upper). The intergenic transcript was indeed absent from
wild-type and, importantly, also from rrp6? cells carrying this
modification (Fig. 5A Lower and data not shown), although
RNAPII ChIP analysis suggested that transcription through the
promoterhadstillnotbeenreducedtobackgroundlevels(SIFig.
10), pointing to some ‘‘sporadic,’’ noncoding transcription still
occurring across the locus.
Nevertheless, this specific block clearly led to slower PHO5
promoter remodeling, with H3C ChIP revealing a significant
delay in histone loss compared with wild-type cells (Fig. 5B).
Thus, considering that four nucleosomes are being evicted from
the PHO5 promoter during remodeling (13, 14), the wild-type
cell population lost nucleosomes at a rate of ?2.2 nucleosomes
per hour, whereas cells lacking the intergenic transcript did so at
a rate of ?1.1 nucleosomes per hour. A corresponding delay in
RNAPII recruitment to the PHO5 regulatory region upon
activation was also observed in the absence of the intergenic
transcript (Fig. 5C). Conversely, we obtained further evidence
that the intergenic transcript does not play a role in maintaining
a closed promoter and repressing PHO5 transcription under
noninducing conditions (data not shown).
Taken together, these data support the idea that noncoding
transcription through the PHO5 promoter affects the speed of
histone remodeling during transcriptional activation, but not
repression, of the gene.
Discussion
HereweidentifiedanintergenicRNAPII-generatedtranscriptthat
initiates in the region around the end of the PHO5 ORF and is
transcribed in the antisense direction up to and across the PHO5
promoter.Thistranscriptwasalsorecentlyindependentlyidentified
by whole-genome microarray studies (3–5). By employing various
mutations (and the compound 6AU) affecting RNAPII transcript
elongation and generation of the intergenic transcript, we uncov-
ered evidence for the idea that the kinetics of activation, but not
repression, of PHO5 is affected by intergenic transcription. Below
we argue that transcription across the PHO5 promoter somehow
contributes positively to chromatin plasticity, enabling rapid nu-
cleosome disassembly upon activation.
Intergenic Transcription and Its Consequences. Data published over
the last few years indicate that intergenic transcription is much
more widespread than had previously been expected. However,
studies addressing the putative role(s) of such transcription are
few (23–26). Previous data in yeast demonstrated a repressive
role for intergenic transcription in the regulation of SER3,
activated when serine is limiting (25). RNAPII generating this
noncoding transcript (called SRG1) represses transcription by
transcriptional interference, i.e., by inhibiting binding of activa-
tors to the SER3 UAS, and of TBP to its TATA box. The
situation at PHO5 is fundamentally different from that at
SER3/SRG1: in contrast to SRG1, the intergenic transcript
across PHO5 is transcribed at low levels. Second, the SRG1
Fig. 5.
remodeling and RNAPII recruitment to the PHO5 TATA box. (A Upper) Sche-
maticofthePHO5locusinWTandPHO5-3??.URA3replacesPHO5?751–1,404
(toendofgene).(ALower)Northernblot(usingprobe3fromFig.1C)showing
absence of intergenic transcription in the rrp6 version of the strain is shown.
Note that only RRP6 cells were used in B and C and other experiments
addressing the functional consequences of intergenic transcription. (B) Rela-
tive histone H3 occupancy (H3C ChIP) in the indicated regions of the PHO5
promoterinWTandPHO5-3??cellsduringthecourseofphosphatestarvation.
(C) As in B, but RNAPII recruitment to the TATA box (4H8 ChIP). Density at
time ? 0 was set to 1, and other values are expressed relative to that. Histone
density in the two strains at time ? 0 was similar.
Abolishing the intergenic transcript leads to slower PHO5 promoter
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transcript initiates upstream of SER3, and on the same strand,
whereas the PHO5 (antisense) intergenic transcript is initiated
?1,400 bases downstream from the PHO5 TATA box. Tran-
scription across the PHO5 promoter does not result in transcrip-
tional interference, but instead seems to allow efficient histone
eviction upon activation, promoting timely recruitment of
RNAPII to the PHO5 TATA box and transcription of the gene.
Possible Mechanism Underlying the Effect of PHO5 Intergenic Tran-
scription. Intergenic transcription could in theory affect histone–
DNA interactions at PHO5 in at least three different ways. First,
the RNA transcript itself could facilitate more rapid activation,
perhaps by acting as a histone acceptor/chaperone, as RNA has
been shown to do in vitro (27). Second, physical movement of
RNAPII through the promoter might increase the level of
histone modification, such as acetylation and methylation, or
increase insertion of the histone H2A variant Htz1. Finally,
RNAPII movement through the region might cause temporary
histone/nucleosome displacement, which could be required for
or increase histone loss at the locus upon activation. Indeed,
RNAPII-generated changes in chromatin integrity are well
documented in a number of experimental systems (28–31).
IfthepresenceoftheRNAtranscriptitselfwereimportant,we
reasoned that a higher level of the intergenic transcript near the
PHO5locusmightfacilitatepromoterremodeling.Therrp6stain
allowed a test of these conditions because it has significantly
higher levels of the intergenic transcript. In turn, nucleosomes
might be evicted faster in rrp6 cells than in wild type. However,
we found that histone eviction in rrp6 cells is similar to that of
wild-type cells (data not shown). Likewise, expressing the inter-
genic transcript in trans from a plasmid failed to suppress the
slower histone eviction and RNAPII TATA box recruitment
observed in PHO5-3?? (data not shown). Finally, insertion of a
terminator ?500 bp into PHO5 did not affect the kinetics of
PHO5 activation, although it resulted in a dramatic decrease of
stable intergenic transcript. Together, these results argue
against, but do not rule out, a positive role for the intergenic
RNA transcript itself.
We also used the rpb1-1 and PHO5-3?? strains to test whether
transcription across the PHO5 promoter affects the acetylation
(H3-K9, H3-K27, H3-K18, H4-K5, and H4-K12) or methylation
(H3-diMetK4, H3-diMetK36, and triMetK4) level of histones, or
density of the histone variant Htz1, but ChIP assays failed to
detect major changes in these histone characteristics (after
normalizing to histone density) when transcription was inacti-
vated (data not shown). Although these data in themselves do
not rule out the possibility that RNAPII-mediated changes in
histone modification play a role in PHO5 histone eviction, they
do argue against this possibility. The PHO5-3?? mutation also
does not result in a measurable change in the accessibility of
promoter chromatin to restriction enzymes (data not shown),
suggesting that, as expected, nucleosome positioning is not
dramatically altered by intergenic transcription.
We also considered the possibility that the result we obtained
with PHO5-3?? might be due to loss of putative promoter–
terminator contacts (‘‘gene looping’’), rather than loss of inter-
genic transcription. However, slower PHO5 activation was not
observed in strains where such looping would be disrupted
because the PHO5 terminator was removed (but intergenic
transcription still occurred) (data not shown), arguing against a
role for gene looping.
AModeltoExplaintheEffectofIntergenicandSporadicTranscription.
Considering the results described above, as well as previous data
from others on the disruptive effect of RNAPII transcription
through nucleosomes, we suggest that the actual movement of
RNAPII through the PHO5 promoter affects nucleosome evic-
tion by somehow increasing the local rate of nucleosome ex-
change/turnover. In support of this idea, more generally inhib-
iting or abrogating transcription (6AU and in particular rpb1-1)
within and outside the PHO5 locus reduced nucleosome eviction
to a much greater extent than specific abrogation of the inter-
genic transcript (PHO5-3??). Indeed, it is possible that noncod-
ing, sporadic transcription, i.e., random transcription originating
from multiple spurious initiation sites and terminating at ran-
dom, is as widespread as the intergenic transcription that gives
rise to detectable transcripts. The concept of widespread, spo-
radic transcription may well be of substantial general importance
in all eukaryotes, but it might play a particularly important role
in maintaining chromatin plasticity in an active genome such as
that of Saccharomyces cerevisiae.
The Kinetics of Gene Regulation. It is obvious that the speed at
which a cell responds to a stimulus by activating a set of genes
andrepressinganotherisofpivotalimportancetothefateofthat
cell. However, this aspect of gene regulation is often not
appreciated. Instead, the amplitude of regulation or the absolute
levels of expression are generally seen as the hallmarks of a
regulated gene. Here we have shown that the rate of activation
of the PHO5 promoter is affected by intergenic transcription
across it, whereas the final level of induction is not. Given that
intergenic transcripts are often found to be expressed in a tissue-
ortime-dependentmanner,theeffectofnoncodingtranscription
described here may be a general one, and one important
consequence of it could be to increase the rate of chromatin
remodeling and thereby the rate of gene induction, rather than
to affect the final steady-state levels of expression. Experiments
in higher cells suggest that intergenic transcription is generally
extremely widespread. Our findings thus have important impli-
cations for the approaches that should be taken to study the
effect of noncoding transcription also in metazoans.
Materials and Methods
Yeast Strains and Growth Conditions. Strains used are listed in SI
Text. For PHO5 activation, yeast strains were grown in yeast
extract/peptone/dextrose (YPD; high-phosphate conditions) to
mid-log phase, and then shifted to phosphate-free minimal media
in a final concentration of typically 0.5 ? 107cells per milliliter.
KH2PO4was added to 10 mM final concentration to cultures in
PHO5 shutoff experiments. For galactose induction (PHO5v33),
strainsweregrowninyeastextract/peptonemediumcontaining2%
raffinose. Galactose was added to 2% final concentration, and cells
weredilutedto0.5?107cellspermilliliter.rpb1-1anditswild-type
counterpart were grown at 23°C as the permissive temperature and
at 37°C as the restrictive temperature. Temperature shifts were
performed 30 min before phosphate starvation, phosphate addi-
tion,orgalactoseinduction.6AUwasaddedtoafinalconcentration
of50?g/ml,withcellsmadeURA3?bytransformationwithaCEN
plasmid, as required.
ChIPAssaysandReal-TimePCRAnalysis.ChIPassayswereperformed
essentially as previously described (32). 4H8 (anti-Rpb1 antibody)
was from Upstate Biotechnology (Lake Placid, NY), and the H3C
antibody was a gift from Alain Verreault (Institute for Research in
Immunology and Cancer, Montreal, QC, Canada). Coprecipitated
DNA was analyzed in triplicate by quantitative PCR using the ABI
Prism7000Sequencedetectionsystem(AppliedBiosystems,Foster
City, CA). Primer sequences are available upon request. Values
were normalized to inputs. All values expressed in bar graphs
represent means ? SEM of at least two independent experiments
and three independent ChIP assays.
Northern Blots. Total RNA was extracted by using the hot acid
phenol method. RNA was separated on 1% formaldehyde
agarose gels and blotted onto Nylon membranes. Blots were
hybridized with PCR probes (primer sequences available upon
Uhler et al.PNAS ?
May 8, 2007 ?
vol. 104 ?
no. 19 ?
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GENETICS