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Supported by the NSF Graduate Research Fellowship Program
(J.A.L.); the Center for Brains, Minds and Machines, funded by NSF
STC award CCF-1231216 (L.E.S.); and the Simons Center for
the Social Brain. We thank the Boston Children’s Museum;
participating families; K. Richardson and S. Robinson for help
coding the data;
R. Saxe, J. Tenenbaum, K. Scott, R. Magid, and J. Jara-Ettinger
for helpful comments on the draft; and members of the Early
Childhood Cognition Lab for helpful comments and discussion. All
data and analysis code can be found at https://osf.io/xjz8e/.
Materials and Methods
15 March 2017; accepted 23 August 2017
PAF1 regulation of promoter-proximal
pause release via enhancer activation
Fei Xavier Chen,1 Peng Xie,2 Clayton K. Collings,1 Kaixiang Cao,1 Yuki Aoi,1
Stacy A. Marshall,1 Emily J. Rendleman,1 Michal Ugarenko,1 Patrick A. Ozark,1
Anda Zhang,3 Ramin Shiekhattar,3 Edwin R. Smith,1
Michael Q. Zhang,2,4 Ali Shilatifard1,5*
Gene expression in metazoans is regulated by RNA polymerase II (Pol II) promoter-proximal
pausing and its release. Previously, we showed that Pol II–associated factor 1 (PAF1) modulates
the release of paused Pol II into productive elongation. Here, we found that PAF1 occupies
transcriptional enhancers and restrains hyperactivation of a subset of these enhancers.
Enhancer activation as the result of PAF1 loss releases Pol II from paused promoters of nearby
PAF1 target genes. Knockout of PAF1-regulated enhancers attenuates the release of paused
Pol II on PAF1 target genes without major interference in the establishment of pausing at their
cognate promoters. Thus, a subset of enhancers can primarily modulate gene expression by
controlling the release of paused Pol II in a PAF1-dependent manner.
Promoter-proximal pausing by RNA Pol II is found at the majority of actively tran- scribed and developmentallyregulated genes in metazoans. The most-studied example has been the induction of HSP70 gene ex-
pression during heat shock, but even highly tran-
scribed genes exhibit some degree of pausing (1).
The direct regulation of pausing relies on fac-
tors physically associated with Pol II, including
negative elongation factor (NELF), DRB sensitivity–
inducible factor (DSIF), Gdown1, and PAF1 (1–3).
Release from pausing requires positive transcrip-
tion elongation factor b (P-TEFb) and P-TEFb–
containing complexes such as the super elongation
complex (SEC), which physically and function-
ally associates with the Integrator complex (4–7).
In addition, transcription factors such as Myc,
PARP1, and KAP1 can act as hinges between
signaling pathways and gene expression by
communicating with the direct regulators of
Previously, we found that PAF1 depletion leads
to a substantial release of paused Pol II into
productive elongation, which suggests that PAF1
functions in the maintenance of the paused state
(3). To further explore the relationship between
PAF1 and paused Pol II genome-wide, we conducted PAF1 chromatin immunoprecipitation
sequencing (ChIP-seq) and compared its distribution to Pol II and several histone modifications. Unexpectedly, we found that PAF1 occupies
not only active promoters marked by a high level
of H3 Lys4 trimethylation (H3K4me3) but also
can be found at active enhancers marked by the
presence of histone H3 Lys27 acetylation (H3K27ac)
and H3 Lys4 monomethylation (H3K4me1) (Fig. 1A).
Global analysis reveals a widespread distribution
of PAF1 at both active promoters and enhancers
(Fig. 1B). The relative occupancy of Pol II is much
greater at active promoters than at active enhancers, whereas the occupancy of PAF1 at active
enhancers is similar to its occupancy at active
promoters (Fig. 1, A and B, and fig. S1A).
The higher ratio of PAF1 to Pol II at enhancers
than at promoters suggests that PAF1 could also
have a role in regulating enhancer activity (Fig.
1C). To test this hypothesis, we performed PAF1
knockdown and H3K27ac ChIP-seq. Knockdown
of PAF1 leads to decreased protein levels of other
PAF1 subunits (fig. S1B). Increased levels of
H3K27ac were seen at active enhancers (Fig. 1D)
but not at active promoters upon PAF1 depletion
(fig. S1C). Enhancers with significantly increased
H3K27ac (~35% of active enhancers) tended to
exhibit a corresponding increase in Pol II occu-
pancy and enhancer RNA (eRNA) transcription
(Fig. 1, E, F, and H, and fig. S1D). The decreased
occupancy of PAF1 at enhancers after PAF1
knockdown was confirmed by ChIP–quantitative
polymerase chain reaction (qPCR) (fig. S1F). Ac-
tive enhancers without a significant change of
H3K27ac (~54% of active enhancers) had higher
levels of Pol II occupancy and eRNA transcription
before knockdown, which suggests that these
enhancers were already fully activated (Fig. 1,
G and I, and fig. S1E). Together, these findings
indicate that PAF1 could repress the activity of a
subset of enhancers.
To examine the relationship between the activation of enhancers and their nearby genes in
response to PAF1 depletion, we first divided the
intergenic active enhancers (abbreviated as enhancers hereafter) into “activated enhancers,”
which exhibit increased eRNA in addition to increased H3K27ac, and “stable enhancers,” which
are unchanged for eRNA and H3K27ac levels
upon PAF1 knockdown. We found that the relative occupancy of PAF1 is much higher on activated enhancers (fig. S2A), suggesting a direct
role in attenuation of enhancer activity by PAF1.
To examine expression changes of nearby genes,
we purified total RNA, performed ribosomal RNA
depletion, and then separated polyadenylate
[poly(A)]–depleted and poly(A)-enriched fractions using oligo(d T) beads, representing mostly
nascent RNAs and mature RNAs, respectively.
Genes within 80 kb of activated enhancers, but
not genes farther than 100 kb, showed the greatest
up-regulation of the nascent RNA-enriched fraction (Fig. 2A). In contrast, genes near or distant
from stable enhancers were relatively unchanged
(Fig. 2B). Analysis of the mature mRNA-enriched
fraction also revealed that genes within 80 kb of
activated enhancers are more likely to be up-regulated (Fig. 2, C and D). To further validate this
analysis, we separated genes into three groups
according to their distance from enhancers and
confirmed that genes within 80 kb of activated
enhancers show the strongest increase in both
poly(A)-depleted RNA (Fig. 2E and fig. S2B) and
poly(A)-enriched RNA (Fig. 2F and fig. S2C);
1294 22 SEPTEMBER 2017 • VOL 357 ISSUE 6357 sciencemag.org SCIENCE
1Department of Biochemistry and Molecular Genetics,
Feinberg School of Medicine, Northwestern University,
Chicago, IL 60611, USA. 2Department of Biological Sciences,
Center for Systems Biology, University of Texas at Dallas,
Richardson, TX 75080, USA. 3Sylvester Comprehensive
Cancer Center, Department of Human Genetics, University of
Miami Miller School of Medicine, Miami, FL 33136, USA.
4MOE Key Laboratory of Bioinformatics, Bioinformatics
Division and Center for Synthetic & Systems Biology,
TNLIST, Tsinghua University, Beijing 100084, China. 5Robert
H. Lurie Comprehensive Cancer Center, Feinberg School of
Medicine, Northwestern University, Chicago, IL 60611, USA.
*Corresponding author. Email: email@example.com