fully repress enhancers during heat shock, we
also observed defects in pausing of nearby genes
in PAF1 knockdown cells (Fig. 4I). A direct role
of PAF1 at enhancers in this process is suggested by the observed relative increase of PAF1
compared to Pol II at enhancers repressed during
heat shock (fig. S9E), as well as the corresponding decrease in the PAF1–Pol II ratio at enhancers
activated by heat shock (fig. S9F). Together, these
data suggest that pausing and the release from
pausing at these genes are both regulated by the
activity of nearby enhancers in a PAF1-dependent
Acute depletion strategies have recently been
developed as an alternative to multiday knockdown of proteins by RNA interference (RNAi),
and some have led to different conclusions for protein function from the prior RNAi studies (17, 18).
To determine the effect of acute depletion of PAF1
on pause release, we used CRISPR/Cas9 to introduce the auxin-inducible degron (AID) tag at
the C terminus of the endogenous PAF1 locus in
DLD-1 cells expressing the TIR1 protein from
Oryza sativa (fig. S10A). As soon as 60 min after
addition of auxin, PAF1 was largely depleted
from the DLD-1 cells (fig. S10B) and release of
Pol II from promoter-proximal pausing could be
observed (fig. S10, C to I). Therefore, the effects
we report for the role of PAF1 on promoter-proximal pausing appear to be a direct consequence of loss of PAF1 function and not an
indirect effect from several days of knockdown.
A study in Drosophila reported the surprising
finding that promoters, but not enhancers, play a
central role in setting up the paused state of Pol II
(19). Our data from mammalian cells are in agreement with the conclusion that promoters are
sufficient for the establishment of the paused
state. We found that enhancer activation plays
a pivotal role in mediating pause release in a
PAF1-dependent manner. Numerous studies
have used genome-wide analysis of histone marks
and eRNA transcription to classify enhancers
into various states such as inactive, poised, active,
or super-enhancers (20–22). Our finding, that
PAF1 restrains full activation of less active enhancers and consequently hinders the release of
paused Pol II, reveals an additional layer of enhancer regulation that directly connects enhancer
function with the control of gene expression at the
level of transcription elongation.
REFERENCES AND NOTES
1. I. Jonkers, J. T. Lis, Nat. Rev. Mol. Cell Biol. 16, 167–177 (2015).
2. B. Cheng et al., Mol. Cell 45, 38–50 (2012).
3. F. X. Chen et al., Cell 162, 1003–1015 (2015).
4. N. F. Marshall, D. H. Price, J. Biol. Chem. 270, 12335–12338 (1995).
5. C. Lin et al., Mol. Cell 37, 429–437 (2010).
6. C. Lin et al., Genes Dev. 25, 1486–1498 (2011).
7. A. Gardini et al., Mol. Cell 56, 128–139 (2014).
8. P. B. Rahl et al., Cell 141, 432–445 (2010).
9. B. A. Gibson et al., Science 353, 45–50 (2016).
10. R. P. McNamara et al., Mol. Cell 61, 39–53 (2016).
11. M. Yu et al., Science 350, 1383–1386 (2015).
12. N. J. Krogan et al., Mol. Cell 11, 721–729 (2003).
13. B. N. Tomson, K. M. Arndt, Biochim. Biophys. Acta1829, 116–126 (2013).
14. Y. Yang et al., PLOS Genet. 12, e1005794 (2016).
15. H. Fischl, F. S. Howe, A. Furger, J. Mellor, Mol. Cell 65, 685–698.e8 (2017).
16. D. B. Mahat, H. H. Salamanca, F. M. Duarte, C. G. Danko,
J. T. Lis, Mol. Cell 62, 63–78 (2016).
17. E. P. Nora et al., Cell 169, 930–944.e22 (2017).
18. G. E. Winter et al., Mol. Cell 67, 5–18.e19 (2017).
19. M. Lagha et al., Cell 153, 976–987 (2013).
20. N. D. Heintzman et al., Nat. Genet. 39, 311–318 (2007).
21. M. P. Creyghton et al., Proc. Natl. Acad. Sci. U.S.A. 107,
22. W. A. Whyte et al., Cell 153, 307–319 (2013).
We thank all the members of the Shilatifard laboratory, J. Yu,
J. Crispino, J. Wang, and D. Taatjes for helpful discussions during
the course of this work; I. Cheeseman for the gift of the Os TIR1-
expressing DLD-1 cell line; M. Kanemaki for the gift of the pMK286
(mAID-Neo) and pMK287 (mAID-Hygro) plasmids; M. Mendillo and
S. Takagishi for suggestions about CRISPR/Cas9; and L. Shilatifard for
editorial assistance. ChIP-seq, RNA-seq, and 4C-seq data have been
deposited at the Gene Expression Omnibus (GEO) under accession
number GSE97527. Supported by NIH grant MH102616 and Natural
Science Foundation of China grant 31671384 (M.Q.Z.); NIH grant
CA211428 (E.R.S.); NIH grants GM078455 and GM105754 and the
University of Miami Miller School of Medicine, Sylvester Comprehensive
Cancer Center (R.S.); a JSPS Research Fellowship for Young
Scientists (Y.A.); a Eugene McDermott Graduate Fellowship (P.X.); and
a Robert H. Lurie Comprehensive Cancer Center–Lefkofsky Family
Foundation/Liz and Eric Lefkofsky Innovation Research Award (A.S.).
Transcriptional elongation studies in the Shilatifard laboratory are
supported by National Cancer Institute grant CA214035 (A.S.).
Materials and Methods
Figs. S1 to S10
28 March 2017; accepted 22 August 2017
Published online 31 August 2017
Fig. 4. PAF1 is required for the
accumulation of paused Pol II
driven by the heat shock
response. (A) Experimental design.
HCT116 cells were transduced
with NONT or shPAF1 for around
3.5 days and then cross-linked for
ChIP-seq with or without 90 min
of heat shock (HS). (B) Metagene
plot of H3K27ac occupancy in cells
before or after HS for the top
1000 HS-repressed enhancers.
The y axis represents reads per
base per gene. (C) Metagene
plot of Pol II occupancy in cells
before or after HS for genes
within 80 kb of the top 1000
HS-repressed enhancers. (D to G)
Genome browser track examples
of H3K27ac occupancy at
enhancers [(D) and (F)] and
Pol II occupancy at nearby
genes [(E) and (G)] in cells with
or without HS in NONT and PAF1-
depeleted cells. (H) Metagene
plot of H3K27ac occupancy
on HS-repressed enhancers in
cells with or without PAF1
depletion during HS. (I) Empirical
cumulative distribution function
plot of the promoter-release
ratio (PRR) distribution in cells
with or without HS in NONT
and PAF1-depleted cells.