Regulating the regulators: the pervasive
effects of Pol II pausing on stimulus-
responsive gene networks
Daniel A. Gilchrist,1George Fromm,1Gilberto dos Santos,1,3Linh N. Pham,1Ivy E. McDaniel,1,4
Adam Burkholder,2David C. Fargo,2and Karen Adelman1,5
1Laboratory of Molecular Carcinogenesis,
National Institutes of Health, Research Triangle Park, North Carolina 27709, USA
2Integrated Bioinformatics, National Institute of Environmental Health Sciences,
The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that
has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive
pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether
this represents the major function of pausing in these pathways remains to be determined. To address this
question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that
paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades
than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains
basal expression of critical network hubs, including the key NF-kB transcription factor that triggers gene
activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly
attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is
similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the
MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation
and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks.
[Keywords: gene expression; transcription elongation; polymerase pausing; gene networks]
Supplemental material is available for this article.
Received January 26, 2012; revised version accepted March 23, 2012.
All organisms have evolved strategies to facilitate rapid
and balanced responses to environmental and develop-
mental cues. One mechanism for achieving robust up-
regulation of transcription in response to the external
environment is exemplified by the Drosophila heat-
shock (Hsp) genes, which possess preloaded RNA poly-
merase II (Pol II) on their promoters prior to induction (Lis
1998). This Pol II is engaged in early elongation and
remains paused promoter-proximally, associated with a
20- to 60-nucleotide (nt) nascent RNA. Heat shock triggers
nearly immediate release of paused Pol II into the Hsp
genes, permitting the scaffold of general transcription
factors left at the promoter to be reused by additional Pol
II molecules that generate a dramatic induction of RNA
levels withinminutes ofheat shock (Lis 1998; Zobeck et al.
Pol II pausing has recently been identified as a wide-
spread mechanism of transcriptional regulation in higher
eukaryotes. Genome-wide localization of Pol II in Dro-
sophila, mouse, and human cells showed that thousands
of genes display an accumulation of Pol II just down-
stream from their promoters (Guenther et al. 2007; Muse
et al. 2007; Zeitlinger et al. 2007; Core et al. 2008; Lee
et al. 2008; Gilchrist et al. 2010; Rahl et al. 2010), and
analyses of RNA confirm that this polymerase is pre-
dominantly in a transcriptionally engaged, but paused,
state (Core et al. 2008; Nechaev et al. 2010; Min et al.
2011). Notably, in all cell types investigated, pausing is
found to be highly enriched among genes in stimulus-
responsive networks, such as those that sense environmen-
tal and developmental cues (Muse et al. 2007; Zeitlinger
et al. 2007; Hendrix et al. 2008; Gilchrist et al. 2010; Min
et al. 2011). Based on these findings, Pol II pausing has been
proposed to play a regulatory role in these critical signaling
pathways; however, the mechanistic basis of this role
By analogy with the heat-shock system, the presence of
a preloaded and paused Pol II has been proposed to ‘‘poise’’
the downstream targets of these pathways for efficient
Harvard University, Cambridge, MA 02138, USA;4Department of Molec-
ular and Cellular Biology, University of California at Berkeley, Berkeley,
CA, 94720, USA.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.187781.112.
3Department of Molecular and Cellular Biology,
GENES & DEVELOPMENT 26:933–944 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org933
gene activation (Lis 1998; Nechaev and Adelman 2008;
Levine 2011). In support of this hypothesis, investigations
of individual stimulus- and developmentally responsive
genes suggest that the presence of paused Pol II correlates
with rapid and synchronous induction (Adelman et al.
2009; Boettiger and Levine 2009). However, recent geno-
mic studies of steroid hormone-mediated transcription
clearly demonstrate that rapidly activated genes are not
uniformly occupied by Pol II prior to induction (Hah et al.
2011; Lin et al. 2011). Similarly, a number of key targets of
developmental signaling were found not to harbor paused
Pol II in murine embryonic stem (ES) cells (Min et al. 2011).
As a result, the functional role of paused Pol II in inducible
networks is a matter of current debate, and identifying the
physiologically relevant targets of pausing has emerged as
a subject of significant interest.
Importantly, the vast majority of paused genes are
actively transcribed in resting cells (Core et al. 2008;
Gilchrist et al. 2010; Rahl et al. 2010; Min et al. 2011).
factors that establish and release paused Pol II suggest that
pausing could modulate basal gene expression (Gilchrist
et al. 2010; Rahl et al. 2010). Establishment of paused
polymerase requires the negative elongation factor NELF
(Yamaguchi et al. 1999; Narita et al. 2003; Wu et al. 2003;
Cheng and Price 2007). NELF interacts with Pol II in
association with the DSIF complex, and DSIF and NELF
are sufficient to inhibit early transcription elongation in
vitro (Cheng and Price 2007). Pause release is triggered by
recruitment of the P-TEFb kinase, which phosphorylates
the Pol II C-terminal domain (CTD), DSIF, and likely
NELF (Peterlin and Price 2006; Cheng and Price 2007).
P-TEFb activity leads to dissociation of NELF, conversion of
DSIF to a positive elongation factor, and escape of paused
polymerase into the gene. Global studies of NELF and
DSIF distribution have revealed that they are enriched
near the promoters of active genes (Gilchrist et al. 2010;
Rahl et al. 2010), confirming that pausing and high-level
expression are not mutually exclusive. Further support-
ing the idea that regulated pause release impacts the
expression of active genes, disruption of P-TEFb activity
using the specific kinase inhibitor flavopiridol broadly
blocked release of polymerase into productive elongation
and reduced gene expression (Rahl et al. 2010).
Although pausing slows the release of polymerase into
productive RNA synthesis, the presence of paused Pol II
has been shown to have both positive and negative effects
on gene expression. In particular, loss of Pol II pausing
following depletion of NELF leads to decreased expres-
sion of many genes in Drosophila, mouse, and human cells
(Narita et al. 2007; Gilchrist et al. 2008; Amleh et al. 2009;
Sun and Li 2010). This positive role of paused polymerase in
gene activity has been most thoroughly studied in Dro-
sophila,wheretheprolongedresidence timeof paused PolII
was found to protect promoters against the assembly of
nucleosomes (Gilchrist et al. 2008, 2010). Thus, by main-
taining Pol II and a scaffold of general transcription factors
associated with the promoter, genes can be held in an
accessible chromatin state that facilitates RNA synthesis.
These findings raise the possibility that by regulating
the duration of pausing, a cell could not only poise in-
ducible genes for efficient activation, but also fine-tune
basal gene expression. To address this possibility, we
analyzed the targets and consequences of NELF-mediated
pausing within Drosophila stimulus-responsive net-
works. We report that pausing is neither limited to rapidly
activated genes nor required for efficient activation. In-
stead, we found that pausing plays a previously unappre-
ciated role in maintaining the basal expression of signaling
proteins and regulators of critical stress- and immune-
mediated pathways. Disruption of pausing perturbs mul-
and gene activation. In support of this being a conserved
functionofpausing, we found thatpausedPol II isenriched
in murine ES cells at genes encoding signaling molecules
and regulators of self-renewal and pluripotency. Thus, we
propose that the critical role of paused Pol II in environ-
mentally and developmentally responsive networks is to
regulate the expression and activities of signal transduc-
tion machineries in resting cells, thereby defining their
responsiveness to external cues.
Highly induced immunity genes are only modestly
enriched in paused Pol II
We used the Drosophila innate immune network as a
model system to investigate the role of pausing within
stimulus-responsive pathways. Our previous analyses of
gene expression in Drosophila S2 cells revealed that the
defense and immune responses were the most significantly
affected gene ontology (GO) categories among genes down-
regulated following NELF depletion (Gilchrist et al. 2008),
suggesting that pausing might positively influence these
responses. Furthermore, our genomic chromatin immu-
noprecipitation (ChIP)–chip assays of Pol II distribution in
S2 cells showed that the immune pathways were dramat-
ically enriched among highly paused genes (Gilchrist et al.
this inducible gene network.
Innate immune pathways in the fly are very similar to
their mammalian counterparts; invading pathogens are
recognized by pattern recognition receptors and circulat-
ing cytokines, which in turn activate specific signal
transduction cascades (Lemaitre and Hoffmann 2007).
The two central immune pathways in Drosophila, Toll
and immune deficiency (Imd), both converge on transcrip-
tion factors of the NF-kB family that enter the nucleus to
activate transcription of genes encoding antimicrobial pep-
tides (AMPs) and other cyto-protective proteins. Thus,
current models would predict that pausing is prevalent at
the AMP genes and other targets of these pathways, where
it could facilitate their rapid and robust activation. To test
this idea, we investigated Pol II distribution at genes that
are highly induced following challenge with bacterial
lipopolysaccharide (LPS) (see the Materials and Methods)
using expression microarrays and ChIP–chip data from S2
cells (Boutros et al. 2002; Gilchrist et al. 2010).
Gilchrist et al.
934GENES & DEVELOPMENT
RNA was reverse-transcribed, and qPCR was performed as
above. mRNA levels are plotted relative to each gene’s value in
mock-treated GFP(?) cells.
ChIP in ES cells and analysis of ChIP-seq data
The C2 line of ES cells was grown in KOSR+2i medium as
described (Gertsenstein et al. 2010) except that insulin was
omitted. Cells were harvested using trypsin and cross-linked
for 10 min prior to cell lysis and sonication in a Bioruptor. ChIP
was performed using 50 mL of an antibody targeting the N
as control, using 7.5 3 106cells per immunoprecipitation.
We thank the Adelman laboratory members for their helpful
suggestions on this manuscript, and Neal Silverman and Svenja
Stoven for generously providing expression constructs. This
research was supported by the Intramural Research Program of
the NIH, National Institute of Environmental Health Sciences
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