REvIEWS

Born to run: control of transcription

elongation by RNA polymerase II
Fei Xavier Chen   , Edwin R. Smith and Ali Shilatifard*
Abstract | The dynamic regulation of transcription elongation by RNA polymerase II (Pol II) is an
integral part of the implementation of gene expression programmes during development. In most
metazoans, the majority of transcribed genes exhibit transient pausing of Pol II at promoter-​
proximal regions, and the release of Pol II into gene bodies is controlled by many regulatory
factors that respond to environmental and developmental cues. Misregulation of the elongation
stage of transcription is implicated in cancer and other human diseases, suggesting that
mechanistic understanding of transcription elongation control is therapeutically relevant. In this
Review , we discuss the features, establishment and maintenance of Pol II pausing, the transition
into productive elongation, the control of transcription elongation by enhancers and by factors of
other cellular processes, such as topoisomerases and poly(ADP-​ribose) polymerases (PARPs), and
the potential of therapeutic targeting of the elongation stage of transcription by Pol II.

Super elongation complex

(SEC). A complex containing
the most active form of positive
transcription elongation
factor-b; required for almost
all rapid inductions of
transcription and for release of
paused RNA polymerase II.
Simpson Querrey Center for
Epigenetics and the
Department of Biochemistry
and Molecular Genetics,
Feinberg School of Medicine,
Northwestern University,
Chicago, IL, USA.
*e-​mail: ash@
northwestern.edu
https://doi.org/10.1038/
s41580-018-0010-5
Genome-​wide studies have demonstrated that following
transcription initiation, most metazoan genes undergo
a regulatory step termed promoter-​proximal pausing1,2.
After transcribing 20–120 nucleotides downstream of the
transcription start site (TSS), RNA polymerase II (Pol II)
pauses; its release into productive elongation requires
the activity of positive transcription elongation factor
b (P-​TEFb), which is a kinase composed of the catalytic
subunit cyclin-​dependent kinase 9 (CDK9) and its regu-
latory subunit, cyclin T3–5. The activity of P-​TEFb is highly
regulated, being part of at least three larger complexes:
the super elongation complex (SEC)6,7, bromodomain-​
containing protein 4 (BRD4)-associated P-​T EFb
(BRD4–P-​TEFb)8,9 and 7SK small nuclear ribonucleo­
protein (snRNP)-associated P-​TEFb (7SK–P-​TEFb)10,11.
Pol II that is released to transcribe into gene bodies inter-
acts with additional elongation factors that enhance its
processivity, modify the chromatin landscape and regulate
co-​transcriptional RNA processing12.
Many transcription elongation factors, such as
P-​TEFb, transcription factor S-​II, elongin A and eleven-​
nineteen lysine-​rich leukaemia protein (ELL), were
originally identified in vitro by biochemical methods13.
For example, P-​TEFb was purified from Drosophila
melanogaster cell extracts as a factor sensitive to the
transcription elongation inhibitor 5,6-dichloro-1-β-​
d-ribofuranosylbenzimidazole (DRB), whereas the ELL
component of the SEC was purified from rat liver extracts
as a factor that suppresses transient pausing of Pol II
(refs14,15). Other transcription elongation factors were
identified from genetic screens16, and some of these, such
as the product of the SPT genes, are orthologues of factors
that have also been identified from in vitro studies17.
More recently, transcription elongation factors have
been identified by genome-​wide profiling of factors pre-
viously implicated in other cellular processes, including
topoisomerases and poly(ADP-​ribose) polymerases
(PARPs), thereby uncovering interconnectivity between
DNA replication, DNA damage repair and transcription
elongation control.
In this Review, we discuss recent advances in char-
acterizing the general features of Pol II pausing and the
regulation of early and late transcription elongation
steps. We discuss the progress and remaining chal-
lenges in understanding the molecular mechanisms of
elongation control by comparing the functions of sev-
eral pausing and elongation factors, including negative
elongation factor (NELF), DRB sensitivity-​inducing
factor (DSIF), Pol II-​associated factor 1 (PAF1) and the
P-​TEFb-containing complexes (Table 1). We also discuss
the roles of distal enhancers and factors such as topoi-
somerases and PARPs, which function in other cellular
processes, in the regulation of transcription elongation
by Pol II. On the basis of these findings, we forecast the
potential directions in therapeutic targeting of the regu­
lation of the elongation stage of transcription by Pol II
in cancer and other human diseases.
Features of Pol II pausing control
One of the first genes found to undergo promoter-​
proximal pausing of Pol II was the D. melanogaster heat
shock protein 70 (Hsp70) gene in non-​heat shock con-
ditions18,19. Subsequently, promoter-​proximal pausing in
D. melanogaster was found to occur at highly transcribed

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Table 1 | Pausing-​related factors

Pausing-​related Subunits occupancy Function in pausing refs
factors
NELF • NELF-​A Promoter Stabilizes paused Pol II by 28,88,100,101

• NELF-​B preventing premature promoter-​

• NELF-​C or NELF-​D proximal termination
• NELF-​E
DSIF • SPT4 • Promoter Promotes the recruitment of 88,91,92

• SPT5 • Gene body NELF and capping factors

PAF1C • PAF1 • Enhancer Modulates enhancer activity 25,112,113,269

• CTR9 • Promoter and maintains paused Pol II

• LEO1 • Gene body by hindering its release into
• Parafibromin productive elongation
• WDR61
• RTF1
Gdown1a – Promoter Blocks TFIIF recruitment and 106,109,205

prevents early termination of

promoter-​proximal Pol II
PARP1 – • Enhancer ADP-​ribosylates NELF and 242

• Promoter inhibits its function in pausing

P-​TEFb • CDK9 • Enhancer Phosphorylates the Pol II CTD, 127

• CCNT1 or CCNT2 • Promoter NELF and the SPT5 CTR to

• Gene body promote release from pausing
SEC • AFF1 or AFF4 • Enhancer Most active P-​TEFb-containing 6,7,125,128

• ELL2 • Promoter complex; promotes rapid

• AF9 or ENL • Gene body release of paused Pol II into
• EAF1 or EAF2 productive elongation
• P-​TEFb
BRD4–P-​TEFb • BRD4 • Enhancer Stimulates P-​TEFb activity and 9,132

• P-​TEFb • Promoter promotes pause release

• Gene body
7SK–P-​TEFb • 7SK snRNP Promoter Sequesters P-​TEFb and prevents 10,11,127

• MEPCE pause release

• L ARP7
• HEXIM1 or HEXIM 2
• P-​TEFb
AF9, ALL1-fused gene from chromosome 9 protein; AFF1, AF4/FMR2 family member 1; BRD4, bromodomain-​containing protein 4;
CCNT1, cyclin T1; CDK9, cyclin-​dependent kinase 9; CTD, carboxy-​terminal domain; CTR , carboxy-​terminal region; CTR9, RNA
polymerase-​associated protein CTR9 homologue; DSIF, DRB sensitivity-​inducing factor ; EAF, ELL-​associated factor ELL, eleven-​
nineteen lysine-​rich leukaemia protein; ENL , eleven-​nineteen leukaemia; L ARP7 , La-​related protein 7; LEO1, PAF1/RNA
polymerase II complex component LEO1; MEPCE, methyl phosphate capping enzyme; NELF, negative elongation factor ; PAF1C,
RNA polymerase II-​associated factor 1 complex; PARP1, poly(ADP-​ribose) polymerase 1; P-​TEFb, positive transcription elongation
factor b; RTF1, PAF1/RNA polymerase II complex component RTF1 homologue; SEC, super elongation complex; snRNP, small
nuclear ribonucleoprotein; SPT4, transcription elongation factor SPT4; SPT5, transcription elongation factor SPT5; TFIIF,
transcription factor IIF; WDR61, WD repeat-​containing protein 61. aGdown1 is a subunit of the Pol II complex Pol II(G).

Pausing index
An estimate of RNA
polymerase II (Pol II) promoter-​
proximal pausing; the ratio of
Pol II occupancy at the
promoter-​proximal region
compared with the gene body.
Travelling ratio
The ratio of gene-​body RNA
polymerase II (Pol II) to
promoter-​proximal Pol II.
Initiating Pol II
RNA polymerase II that is
recruited by the general
transcription factors and is
involved in promoter melting
before being released for
transcription.
housekeeping genes20. Genome-​wide profiling methods
firmly established that pausing occurred at the major-
ity of Pol II-​transcribed genes1,21. The importance of
understanding promoter-​proximal pausing has long
been recognized, not only because of the importance
of stress response pathways but also because important
developmental genes such as MYC and transcription
of the human immunodeficiency virus (HIV) provirus
were long known to be regulated at this step22. These
and other studies discussed below established sev-
eral features that are common to promoter-​proximal
pausing of Pol II.
The stability and dynamics of pausing. The degree of
pausing is generally estimated from the pausing index
(or stalling index)1,21 or by the inverse travelling ratio
(or Pol II release ratio)23–25. Although instructive in cer-
tain situations, these ratios reflect the net residence of
the initiating Pol II and elongating Pol II during a series
of transcription stages but do not report on the stability
and dynamics of pausing. Recently, the ability to meas-
ure the dynamics of Pol II pausing was made possible
by the discovery of triptolide, a compound extracted
from a traditional Chinese medicine that can inhibit the
ATPase activity of the xeroderma pigmentosum group
B-​complementing protein (XPB), the helicase subunit
of the general transcription factor (GTF) IIH (TFIIH)26.
Triptolide treatment prevents transcription reinitiation
by Pol II and therefore enables the fate of pre-​existing
paused Pol II to be measured at different time points of
treatment. Such studies have shown that genes differ in
the time of residency of Pol II at the promoter-​proximal
region, from a few minutes for most genes to an hour for
a very few stably paused genes27–30. Although the half-​life
of paused Pol II generally correlates with the pausing
index, the former better predicts the steady-​state levels
of mRNA23,29,31, such that genes with the longest half-​life of
paused Pol II rather than the highest pausing index are

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Paused Pol II
RNA polymerase II that has
initiated non-​productive
transcription and is transiently
pausing at the
promoter-​proximal region.
Bisulfite sequencing
Bisulfite deaminates
unmethylated DNA cytosine
into uracil, which can be used
to infer the position of
methylated cytosine through
sequencing.
TATA box
A short AT-​rich motif found in
many RNA polymerase II
promoters; bound by the
general transcription factor
TATA-​binding protein.
G-​quadruplex
(G4). A highly stable nucleic
acid structure comprising two
or more stacked guanine
tetrads. Intramolecular G-​
quadruplexes formed in
nascent RNA can contribute to
RNA polymerase II pausing.
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more likely to be lowly expressed. The lack of correla-
tion between pausing index and pausing stability was
recently demonstrated using methylation footprinting
with whole genome bisulfite sequencing32. Some genes
with a high pausing index and low expression level have
residency times of paused Pol II of just a few minutes,
suggesting that some genes with a high pausing index are
undergoing rounds of initiation and promoter-​proximal
termination32.
A long-​s tanding question is what causes some
genes to exhibit more pausing than other genes and
how gene-​specific pause–release is regulated during
development and in response to environmental cues.
Several studies have examined the specific DNA and
chromatin features associated with promoters of genes
with Pol II pausing. Over 20 years ago, the TATA box
at the MYC promoter was shown to be required for
pausing at this gene22. Later, genome-​wide analyses in
D. melanogaster found that paused promoters that tend
to have high GC content are enriched for a promoter
element called the pause button and for the presence
of the GAGA motif (which recruits transcription factor
GAGA) and tend to lack a TATA box33–35. Although
promoter elements are generally less well defined in
mammalian cells, paused promoters in mammalian
cells also tend to have high GC content and tend to
lack a TATA box2,31.
R-​loops contribute to Pol II pausing. R-​loops are RNA–
DNA hybrids formed when the nascent RNA hybridizes
with the template strand of DNA during transcription
(leaving the non-​template, or coding strand, looping
out)36,37. RNA–DNA hybrids are more stable than double-​
stranded DNA (dsDNA) and thus can block access of
successive Pol II to the DNA template38–40. Interestingly,
the GC skew that is correlated with pausing also favours
the formation of R-​loops owing to the higher thermo-
dynamic stability of RNA–DNA hybrids bearing G-​rich
RNA36,41–43. The capacity of the exposed coding strand to
form stable G-​quadruplex (G4) structures could further
stabilize R-loops44,45. Indeed, G4 motifs correlate with
promoter-​proximal pausing46. However, paused Pol II
mainly localizes 20–120 nucleotides downstream of
the TSS whereas R-​loops can cover wider regions of the
transcription unit, peaking around 1.5 kb downstream
of the TSS37,47. This suggests that R-​loops are more
broadly involved in the control of the processive stage of
transcription elongation.
Pausing and nucleosome features. Nucleosome-​free
regions (NFRs), as determined by micrococcal nucle-
ase sequencing (MNase-​s eq), are found at promot-
ers of actively transcribed genes and correlate with
promoter-​proximal Pol II levels48–50. However, NFRs
are also found by MNase-​seq in Saccharomyces cerevi-
siae, which lacks promoter-​proximal pausing51–54, indi-
cating that the establishment of the NFR is associated
with but not exclusive to paused promoters. GC-​rich
sequences favour intrinsic nucleosome occupancy55,56,
although in vivo, the lowest nucleosome occupancy
can be seen at paused promoters with high GC content,
­perhaps because paused Pol II competes with histones
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for DNA binding to sustain an open chromatin struc-
ture at promoters48,49,56,57. However, one study found
that GC content, independent of transcription or paus-
ing, can explain the position of the NFR58, which could
potentially involve the additional action of chromatin
remodellers59.
One caveat of MNase-​based assays is the sequence
preference of MNase, which is biased towards AT-​
rich sequences, as well as its differential sensitivity to
nucleosomes of different conformations. For exam-
ple, using mainly limited or titrated enzyme digestion,
a class of MNase-​hypersensitive nucleosomes termed
fragile nucleosomes has been reported at NFRs of pro-
moters in yeast and higher eukaryotes60–66. However, it
has also been suggested that these MNase-​hypersensitive
particles at promoters are non-​histone complexes67,68. As
an alternative strategy, chemical mapping of histone
H4 was used to directly map n ­ ucleosome occupancy,
which confirmed the presence of fragile nucleosomes.
Notably, fragile nucleosomes were found in the mouse
and Schizosaccharomyces pombe genomes, but not in
S. cerevisiae69, which coincides with the presence of
paused Pol II in these species52. Indeed, nucleosome levels
measured by chemical mapping appear to correlate with
the degree of pausing, opposing the interpretation of
early stu­dies using MNase. The discrepancy could be
due to the different sensitivities of chemical mapping
and MNase-​based assays in detecting the unique nucleo­
some structure around paused promoters, which exhibit
a looser ­conformation and higher rate of nucleosome
turnover (nucleosome remodelling).
Nucleosome composition can also influence paus-
ing, for example, nucleosomes with the histone variant
H2A.Z are less stable and are suggested to negatively cor-
relate with the establishment of p ­ ausing31,70,71. However,
the exact content and conformation of the nucleosomes
around promoters as well as their i­nterplay with the
dynamics of pausing remain to be fully examined.
The establishment of Pol II pausing
Transcription initiation by Pol II commences with the
assembly of the preinitiation complex (PIC), which
includes the polymerase, GTFs and a large number of
co-​regulators (reviewed in refs72,73) (Fig. 1a). This pro-
cess occurs in a stepwise manner with TFIIH, which is
a complex comprising about ten subunits, and is the last
essential component of the PIC to be recruited. TFIIH
is unique among the GTFs as it is required not only for
transcription initiation but also for subsequent phos-
phorylation of Pol II to ensure the proper establishment
of pausing.
Transient promoter melting by the helicase module
of TFIIH. The TFIIH holoenzyme is organized into
two structurally and functionally distinct subcom-
plexes: the core module and the CDK-​activating kinase
(CAK) module (reviewed in ref.74). The catalytic sub-
unit in the core module is the ATP-​dependent 3′ to 5′
helicase XPB (Ssl2 in budding yeast), which opens the
dsDNA (promoter melting) to allow Pol II access to the
template strand, which is referred to as open complex
formation75 (Fig. 1a). Permanganate can react with the
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a Initiation and pausing establishment

CTD Mediator

CDK7 GTFs
TFIIH XPB CTD Ser5 phosphorylation
TFIIF CTD Ser2 phosphorylation
Pol II NELF phosphorylation
DSIF phosphorylation

b Pausing stabilization

Mediator

TFIIF DSIF NELF –1 +1

PAF1C F
Gdown1 NEL

Pol II NELF
Transcript DSIF
SEC

PAF1C

–1 +1
c Release from pausing

DSIF NELF
PAF1C SEC
Gdown1

–1 +1

Fig. 1 | establishment, maintenance and release of rNa polymerase ii pausing. a | General transcription factors (GTFs)
recruit RNA polymerase II (Pol II) to the promoter to form the preinitiation complex. One GTF, TFIIH, has a dual role in
helping to open the double-​stranded DNA through the helicase subunit xeroderma pigmentosum group B-​
complementing protein (XPB) to allow Pol II to initiate transcription, and in establishing pausing by phosphorylating the
Ser5 residues of the Pol II carboxy-​terminal domain (CTD) through its cyclin-​dependent kinase 7 (CDK7) subunit. The
Mediator complex enables the formation of long-​range enhancer–promoter interactions; the GTF TFIIF has dual roles in
promoting transcription initiation and early elongation. b | Paused Pol II is stabilized by interacting with several pausing
factors, such as negative elongation factor (NELF), DRB sensitivity-​inducing factor (DSIF), Pol II-​associated factor 1
complex (PAF1C) and Gdown1. Depletion of NELF (top) results in Pol II destabilization before it passes through the +1
nucleosome downstream of the transcription start site and in promoter-​proximal transcription termination. PAF1C
depletion (bottom) leads to increased recruitment of the super elongation complex (SEC), which results in the release of
paused Pol II into productive elongation. Gdown1 contributes to pausing by blocking recruitment of TFIIF. c | Release from
pausing is driven by the recruitment and activation of positive transcription elongation factor b (P-​TEFb)-containing
complexes, for example, the SEC, which phosphorylates NELF, DSIF and Ser2 residues of the Pol II CTD. Phosphorylated
NELF dissociates from chromatin, whereas PAF1C and phosphorylated DSIF transition from being pausing factors to
being positive elongation factors by promoting Pol II processivity and contributing to co-​transcriptional RNA processing
(not shown).

single-​stranded DNA and has been used to demonstrate
that Pol II rapidly proceeds from the site of open com-
plex formation to the site of pausing1,21,76. The promoter
melting activity of XPB is required for each new round
of transcription initiation76. Treatment of cells with
the XPB inhibitor triptolide blocks new initiation,
thereby allowing the decoupling of the study of
­transcription elongation and Pol II pausing from that
of transcription initiation5,27,28,32.
The kinase module of TFIIH prepares Pol II for pausing.
The CAK module of TFIIH contains CDK7 (Kin28 in
budding yeast), which phosphorylates the Ser5 and
Ser7 residues of the Pol II carboxy-​terminal domain
(CTD)77,78. Ser5 phosphorylation is the much more
studied of the two modifications; it is known to occur
during transcription initiation and to gradually undergo
dephosphorylation as Pol II enters productive elon-
gation79 (Fig.  1). Therefore, Ser5 phosphorylation is

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Mediator complex
A large multiprotein complex
that interacts with transcription
factors and RNA polymerase II.
Mediator can promote
enhancer–promoter looping to
activate transcription and can
also recruit transcription
elongation factors.
Auxin-​inducible degradation
In cells expressing a plant
auxin-​inducible E3 ubiquitin
ligase, any protein of interest
can be tagged for rapid
degraded upon addition of
auxin to the medium.
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commonly used as a mark of paused Pol II to distin- to the Pol II CTD, the carboxy-​terminal region (CTR)
guish it from unphosphorylated or non-​transcriptionally of SPT5 has a repeat region (in this case a pentapep-
engaged Pol II. tide repeat) that is phosphorylated by both the kinase
TFIIH recruitment requires CDK8, which is the module of TFIIH and by P-​TEFb and is involved in the
kinase module of the Mediator complex. Once phos- recruitment and activation of the capping complex91–94.
phorylated, TFIIH evicts the Mediator through CDK7- Biochemical and structural studies demonstrated that
mediated phosphorylation of Ser5 of the Pol II CTD and SPT5 makes key contacts with the nascent RNA exiting
thus allows the release of Pol II from the PIC80,81. In bud- Pol II, which could contribute to its role in pausing95–97.
ding yeast, a direct role for Ser5 phosphorylation in this NELF is a four-​subunit complex, in which NELF-​A
process was suggested by replacing wild-​type RNA poly­ and NELF-​C (or its isoform NELF-​D) form the core
merase II subunit 1 (Rpb1) with Rpb1 bearing a CTD-​ sub-​complex, which associates with NELF-​B and NELF-​
S5A mutant that cannot be phosphorylated by CDK7. E98 (Table 1). In the presence of DSIF, NELF is a potent
The CTD-​S5A mutant mimics chemical inhibition of inhibitor of transcription in vitro88. However, deplet-
CDK7, with both the CTD mutant and CDK7 inhibi- ing NELF in cells generally leads to decreased levels of
tion resulting in the accumulation of Mediator at core promoter-​proximal Pol II and reduced transcription at
promoters and impaired escape of Pol II from the PIC82. many paused genes99–101. One way to reconcile the find-
Pol II CTD Ser5 phosphorylation also helps to ings from in vitro transcription studies and from NELF
recruit and activate the capping enzymes83 that carry depletion studies is if NELF stabilizes paused Pol II
out a multi­step modification of the 5′ end of the nas- by preventing premature promoter-​proximal tran-
cent RNA. Sequencing of small capped RNAs indicated scription termination, a process whereby Pol II disso-
that co-​transcriptional capping is important for the stabil- ciates from promoters without passing through the +1
ity of paused Pol II (ref.28). Uncapped RNAs are subject to nucleosomes28,29 (Fig. 1b). All four subunits of NELF can
degradation by nucleases such as 5′-3′ exoribo­nuclease 2 associate with RNA, raising the possibility that NELF
(XRN2), which can mediate the termination of promoter-​ interactions with short nascent RNA could contribute
proximal Pol II pausing, suggesting that decapping factors to the inhibition of premature transcription termina-
are prime regulators of the residency time of paused Pol II tion98,102,103. Studies of NELF function in cells have been
(ref.84). This idea is supported by studies employing CDK7 performed with either depletion or deletion of its sub-
inhibition, otherwise inhibiting Ser5 phosphorylation or units and monitoring of Pol II pausing following a long
blocking nascent RNA capping, which led to defective period of selection, which could include many indirect
establishment of pausing77,85–87. Additionally, phospho- effects of NELF loss. Therefore, future studies of NELF
rylation of Ser5 stimulates the ­catalytic activity of the and its role in pausing should concentrate on the in vivo
histone-​lysine N-​methyltransferase SETD1A and thus role or roles of NELF in pausing immediately following
helps maintain at promoters the levels of trimethylation of NELF loss, as the in vitro conditions that led to current
histone H3 Lys 4 (H3K4me3), which are associated with models of NELF function could be missing many factors
transcription activation86. that regulate NELF activity.
The precise location of pausing downstream of the The Pol II subunit Gdown1 (also known as GRINL1A)
TSS, which can vary between 20 and 120 nucleotides, also appears to regulate pausing through preventing
could be governed in part by the speed of Pol II escap- promoter-​proximal transcription termination. Gdown1
ing from initiation and how quickly pausing factors competes for binding to Pol II with TFIIF, which is a
are recruited to it76. However, extending the distance GTF that boosts both transcription initiation and
between the HSP70 TSS and the normal site of paus- early elongation89,104–109 (Fig. 1b; Table 1). Gdown1 also
ing by inserting a 5 bp linker led to a concomitant shift inhibits transcription termination factor 2 (TTF2).
downstream in the site of Pol II, whereas a 10 bp inser- Therefore, Gdown1 may contribute to pausing by
tion led to a much more distal and less focused site of blocking recruitment of TFIIF and by inhibiting early
pausing, suggesting that additional mechanisms control transcription termination. Further in vivo studies of
the distance between the TSS and the pause site47. the roles of Gdown1 in transcription regulation would
be highly informative.
Maintenance of Pol II pausing
Maintencance of Pol II pausing appears to be controlledMaintaining Pol II pausing by preventing Pol II release.
by different factors that regulate distinct features ofDSIF, NELF and Gdown1 were each implicated in
the transcriptionally engaged Pol II. Current evidence pausing using in  vitro transcription systems, and
indicates that maintaining Pol II pausing requires boththeir depletion in cells leads to decreased transcrip-
factors that prevent promoter-​proximal transcription tion. By contrast, the PAF1 complex (PAF1C), which
termination and factors that prevent the release of Pol II
had been shown to promote transcription elongation
into productive elongation (Fig. 1b). using in vitro transcription systems110,111, was geneti-
cally implicated in antagonizing the release of paused
Maintaining Pol II pausing by preventing promoter-​ Pol II at erythroid genes in zebrafish112. To address
proximal transcription termination. Studies of tran- this discrepancy, genome-​wide studies in mammal­
scription elongation in  vitro have found that two ian cells found that depleting PAF1 by RNAi or by
complexes, DSIF and NELF, are required for transcrip- auxin-​inducible degradation generally leads to the release of
tion inhibition by DRB88–90. DSIF is composed of tran- Pol II into gene bodies and increased levels of mature
scription elongation factors SPT4 and SPT5. Similar transcripts at paused genes, which is in agreement
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with the result of the genetic studies of PAF1C25,113,114 factors at distinct stages of transcription could determine
Acute protein degradation
Experimentally induced
(Fig. 1b). Genes with higher pausing indices were more whether PAF1 functions as a positive or negative regu-
degradation of proteins by likely to exhibit pause release upon PAF1 depletion, but lator of gene expression115. The recent finding that PAF1
targeting them for proteasomal the basis of this gene specificity is not yet fully under- occupies and inhibits the activity of enhancers associated
degradation. stood. Perhaps interactions with particular transcription with genes regulated by PAF1 is consistent with the idea
that transcription factors or other enhancer-​binding fac-
tors function with PAF1 in the regulation of pausing113.
a 7SK–P-TEFb complex CTD Ser5 phosphorylation As discussed below, although PAF1 generally inhibits
CTD Ser2 phosphorylation enhancer activity and the release of promoter-​proximal
P-TEFb Histone acetylaton paused Pol II, PAF1 can function as a positive trans­
HEXIM1 or
cription elongation factor following the release of Pol II
LARP7 HEXIM2
MEPCE from pausing (Fig. 1c).
Together, these studies have revealed that Pol II paus-
7SK RNA
ing is subjected to complex and multilayered regulation
by a cohort of pausing factors. However, a full under-
standing of the detailed interplay among these factors
b BRD4–P-TEFb complex on a genome-​wide scale is still missing. Accordingly,
further investigation is warranted to systematically
CTD compare different pausing factors in the same genetic
P-TEFb and epigenetic backgrounds using not only RNAi but
BRD4 also acute protein degradation to determine their direct
gene targets and their primary roles in maintaining
Pol II pausing.

c SEC and SEC-like complexes Promoting the release of paused Pol II

Target genes of SEC
EAF1 or ELL2
EAF2
AFF1 or AFF4
P-TEFb
AF9 or ENL
Pol II
Target genes of SEC-like complexes
AFF2 or
P-TEFb AFF3
AF9 or ENL
Pol II
Fig. 2 | release of rNa polymerase ii pausing by P-​TeFb-containing complexes.
Three major positive transcription elongation factor b (P-​TEFb)-containing complexes
have been identified in mammals: 7SK small nuclear ribonucleoprotein–P-​TEFb
(7SK–P-TEFb), bromodomain-​containing protein 4–P-​TEFb (BRD4–P-​TEFb) and the super
elongation complex (SEC) or SEC-​like complexes. a | 7SK–P-​TEFb inhibits the activity of
P-​TEFb and thus suppresses the release of paused RNA polymerase II (Pol II). The complex
contains the non-​coding 7SK small nuclear RNA (snRNA), which interacts with and is
stabilized by La-​related protein 7 (L ARP7) and 7SK snRNA methyl phosphate capping
enzyme (MEPCE), and HEXIM1 or HEXIM2, which mediate the inhibition of P-​TEFb by
binding and sequestering it. b | In the BRD4–P-​TEFb complex, BRD4 stimulates the kinase
activity of P-​TEFb, which phosphorylates the Ser2 residues of the Pol II carboxy-​terminal
domain (CTD) and thus promotes the release of paused Pol II. The interaction between
BRD4 and P-​TEFb is mediated by a carboxy-​terminal region of BRD4 (not shown). The
bromodomains of BRD4 recognize acetylated histones and mediate BRD4 recruitment.
c | The SEC seems to be the most active P-​TEFb complex because it is required for the
expression of rapidly induced genes. SEC assembly relies on the scaffold protein
AF4/FMR2 family member 1 (AFF1) or AFF4, which interacts with P-​TEFb and with
ALL1-fused gene from chromosome 9 protein (AF9) or eleven-​nineteen leukaemia (ENL)
and with eleven-​nineteen lysine-​rich leukaemia protein 2 (ELL2). ELL-​associated factor 1
(EAF1) or EAF2 participate in the SEC through binding directly to ELL2. AF9 and ENL
recognize acetylated histones to facilitate SEC recruitment, and ELL2 promotes
transcription elongation by suppressing transient Pol II pausing (not shown). The SEC-​like
complexes are composed of AFF2 or AFF3, P-​TEFb, and AF9 or ENL , but lack ELL2–EAF
modules. The SEC and the SEC-​like complexes might stimulate transcription activation
of different target genes.
The release of Pol II into productive elongation requires
the activity of the Pol II CTD kinase P-​TEFb4. The mech-
anism of release is thought to involve not only phos-
phorylation of the Pol II CTD Ser2 residues but also
phosphorylation of DSIF and NELF. P-​TEFb is part of
macromolecular complexes, including the SEC, which
regulate its activity (Fig. 1c).
P-​TEFb was discovered as a complex that allows Pol II
to efficiently synthesize long transcripts in a DRB-​
sensitive manner14,116. Treatment of cells with DRB or
other P-​TEFb inhibitors leads to a global defect in the
release of paused Pol II and a reduction in steady-​state
levels of transcripts3,5,117, demonstrating the essential role
of P-​TEFb in mediating Pol II release and its require-
ment for transcription of most Pol II-​transcribed genes.
Well-​known substrates of P-​TEFb include NELF, DSIF
and the CTD of Pol II (refs118–121). NELF dissociates from
chromatin upon its phosphorylation by P-​TEFb118,122,
whereas DSIF switches from being a pausing factor
to being a positive elongation factor upon P-​TEFb
phosphorylation of the SPT5 CTR119,123 (Fig. 1c).
Several P-​TEFb complexes control Pol II release from
pausing. Biochemical studies have shown that P-​TEFb is
part of several larger complexes by associating with 7SK
snRNP, BRD4 and the SEC7–11,124,125 (Fig. 2). 7SK snRNP
represses transcription by sequestering and inhibiting a
large portion of cellular P-​TEFb10,11. This inhibition is
mediated through the binding of P-​TEFb by a dimer of
HEXIM1 or HEXIM2 (ref.126) (Fig. 2a). Up to 90% of P-​
TEFb is sequestered with the 7SK-​P-TEFb complex127.
By contrast, the SEC and BRD4 represent active forms
of P-​TEFb that can promote the release of Pol II from
pausing6,9,128 (Fig. 2b,c). During gene activation, especially
rapid transcription induction, P-​TEFb is evicted from
7SK snRNP127 and can incorporate into the SEC, which
leads to the enzymatic activation of P-​TEFb, which then

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Integrator complex
A large multiprotein complex
found only in metazoans that
can directly interact with RNA
polymerase II; facilitates
transcription and 3′-end
processing of enhancer RNAs
and small nuclear RNAs and
recruitment of the super
elongation complex to
immediate early-​response
genes.
Nucleosome breathing
A property of nucleosomes
whereby DNA at the edges of
the histone core transiently
unwrap and rewrap.
Nature Reviews | Molecular Cell Biology
phosphorylates its targets, including the Ser2 residues of
the Pol II CTD125,127–129. The transition of P-​TEFb from
repressive to active complexes depends on multiple
factors, including KRAB-​associated protein 1 (KAP1;
also known as TRIM28) and the splicing factors serine/
arginine-​rich splicing factor 1 (SRSF1) and SRSF2, which
associate with 7SK snRNP on chromatin130,131.
Although both BRD4–P-​TEFb and the SEC phos-
phorylate Ser2 residues of the Pol II CTD, it remains to
be determined how the two complexes work together
(or separately), to promote transcription elongation.
Intriguingly, a recent study reported that acute deg-
radation of BRD4 resulted in a severe defect in Ser2
phosphorylation and transcription elongation without
obvious change in the genomic occupancy of the P-​TEFb
catalytic subunit CDK9 (ref.132). This suggests that BRD4
is primarily involved in stimulating, but not recruiting,
P-​TEFb or that BRD4 can function independently of P-​
TEFb133. Indeed, it has been reported that BRD4 is also
an (atypical) kinase that can phosphorylate Ser2 of CTD
in vitro134 and can facilitate transcription elongation
independently of P-​TEFb135.
Previous work on HIV activation indicates that
BRD4 maintains basal transcription from the HIV long
terminal repeat (LTR), whereas transactivator of tran-
scription (TAT)-mediated recruitment of the SEC to the
HIV LTR is required for full activation of the provirus
transcription136,137. Interestingly, TAT interacts with P-​
TEFb, specifically comprising the cyclin T1 isoform
through zinc-​dependent binding to a Cys residue that
is not found in cyclin T2 (ref.138). However, the degree to
which cellular genes depend on cyclin T1 versus cyclin
T2 is currently unknown.
SEC subunits are frequently found with transloca-
tion partners of the histone H3 Lys4 methyltransferase
myeloid/lymphoid or mixed-lineage leukaemia (MLL;
also known as KMT2A) in cancer139. Although mecha-
nisms have been proposed for SEC recruitment to MLL
target genes through its interactions with MLL fusion
proteins in cancer and for the co-​option of the SEC by
TAT for HIV transcription139,140, it is less clear how the
SEC is recruited to chromatin in normal physiological
conditions. One possibility is that tissue-​specific tran-
scription factors, such as the Notch pathway effector
HES1, bind and recruit the SEC to activate their tran-
scription programmes141. At epidermal growth factor tar-
get genes such as FOS and JUN, the SEC is recruited by
the Integrator complex142. Another, nonexclusive possibil-
ity is that the SEC recognizes certain chromatin profiles.
For instance, the YEATS (YAF9, ENL, AF9, TAF14 and
SAS5) domains of the SEC subunits eleven-​nineteen leu-
kaemia (ENL) and ALL1-fused gene from chromosome 9
protein (AF9), which are frequent translocation part-
ners of MLL, can bind acetylated H3 and are required
for recruiting the SEC to chromatin and driving high
levels of transcription in acute myeloid leukaemia and of
HIV provirus143–146.
Metazoan-​specific Mediator complex subunit 26
(MED26) has been suggested to contribute to SEC recruit­
ment through its interaction with the SEC subunits ELL-​
associated factor 1 (EAF1) and EAF2 (refs147,148). However,
EAF1 and EAF2 are also part of a distinct transcription
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews
elongation complex named the little elong­ation com-
plex (LEC), which regulates Pol II transcription of small
nuclear RNA (snRNA) genes, suggesting that additional
factors could specify the recruitment of LEC to snRNA
genes and of the SEC to protein-​coding and long non-​
coding RNA genes147,149–151. The use of auxin-​inducible
degradation of MED26 and other Mediator subunits
should allow further dissection of their suggested con-
tribution to SEC recruitment and other possible roles in
transcription elongation control. Notably, SEC recruit-
ment would be present at most genes actively transcribed
by Pol II. In mammals, AF4/FMR2 family member 1
(AFF1) and AFF4, which are the scaffolding proteins of
the canonical SEC, have two additional paralogues, AFF2
and AFF3. AFF2 and AFF3 can each be assembled into
active P-​TEFb-containing and ENL-​containing SEC-​like
complexes, and affect the expression of distinct groups
of genes compared with those affected by the full SEC6
(Fig. 2c). This suggests that generic recruitment to actively
transcribed genes is not sufficient to explain the unique
function of the SEC.
Productive transcription elongation
Productive elongation by Pol II affects and is also
affected by chromatin structure through both nucleo­
some turnover and co-​transcriptional modification of
histones and DNA. A chromatin structure that allows
polymerases to move forward while preventing cryptic
intragenic transcription initiation requires factors that
balance the disassembly and reassembly of nucleosomes
(reviewed in refs152–154). At the 3′ end of genes, elon-
gating Pol II transitions into transcription termination,
which involves RNA cleavage and polyadenylation
(Box 1).
Nucleosome turnover during transcription elong­
ation. The dynamic turnover of nucleosomes during
elongation is mainly driven by the activity of chromatin
remodellers and histone chaperones, which facilitate
the movement of Pol II through nucleosomes and aid in
their reassembly. The histone octamer of a nucleosome
has a tripartite arrangement of one H3–H4 tetramer and
two H2A–H2B dimers155. During transcription by Pol II,
one H2A–H2B dimer is removed from the nucleo­some,
leaving a subnucleosomal particle called the h ­ exasome156.
This process can be enhanced by facilitates chroma-
tin transcription (FACT), which travels with Pol II,
interacts directly with the H2A–H2B dimer and regu­
lates transcription-​couple disassembly and reassem-
bly of nucleosomes157–159. Dislodgement of H2A–H2B
dimers by FACT can enhance nucleosome breathing to
facilitate the passage of Pol II (refs160,161). The dynamic
regulation of nucleosomes during transcription elong­
ation by FACT can be further facilitated by other
­histone chaperones and chromatin remodellers, such as
SPT6, chromodomain-​helicase-DNA-​binding protein 1
(CHD1) and imitation SWI (ISWI)162,163.
Factors implicated in the regulation of pausing can
also facilitate transcription elongation through chroma-
tin. For example, after release of Pol II into productive
elongation, PAF1C travels with the elongating Pol II and
acts as an organizing platform for co-​transcriptional
Reviews

Box 1 | ending transcription elongation by termination

the transition from transcription elongation to
transcription termination requires expulsion of Polyadenylation CTD R1810me2s
signal CTD Ser2 phosphorylation
elongating rNa polymerase ii (Pol ii) from chromatin
at the end of the gene. this process is coordinated DNA
with rNa cleavage and polyadenylation, which are
mediated by various protein complexes, including the XRN2
cleavage and polyadenylation specificity factor (CPsF) SMN
Senataxin DSIF
and the cleavage stimulation factor (CstF) (reviewed AAU
CTD PAF1C
A A A
in refs 259,260
). two different but not mutually exclusive Transcript R-loop
models for Pol ii transcription termination have been
CPSF Pol II
debated for decades, namely, the torpedo model and
CSTF
the allosteric model. the torpedo model posits that
cleavage of the nascent transcript allows access to 5′-3′ exoribonuclease 2 (XrN2), which chases down and promotes the
eviction of Pol ii. the allosteric model posits that a poly(a) signal-​dependent conformational change of Pol ii at the end of
genes triggers termination and dislodges Pol ii from chromatin. two recent studies, one advocating for the torpedo
model261 and the other for the allosteric model262, reported decreased, but not abolished, termination by XrN2
inhibition261,262, implying that rNa degradation following cleavage might contribute to efficient transcription termination
but that it is not absolutely required for termination at most genes263.
transcription termination of many genes is associated with Pol ii pausing downstream of the poly(a) site (reviewed in
refs259,260), which is thought to favour the cleavage of transcripts and degradation of the downstream cleaved rNa.
indeed, reduction of 3′-end Pol ii pausing by inhibition of positive transcription elongation factor b (P-​teFb) leads to
decreased cleavage and termination264,265. similar to the proposed role of r-​loops in promoter-​proximal pausing
regulation, pausing at the 3′ end could be enhanced by r-​loop formation downstream of the poly(a) site (see the figure)
and could be further enhanced by rNai-​induced formation of repressive chromatin36,266. r-​loops at the 3′ end of
genes are resolved by senataxin, which facilitates degradation of transcripts downstream of poly(a) by XrN2 (ref.267)
(see the figure). a recent study suggests that senataxin is recruited to termination sites by its interaction with survival
of motor neuron protein (sMN), which recognizes symmetrical dimethylation of arg1810 (r1810me2s) on the
carboxy-​terminal domain (CtD) of Pol ii (ref.268).
DsiF, DrB sensitivity-​inducing factor; PaF1C, Pol ii-​associated factor 1 complex.

Pol II clamp
Part of the RNA polymerase II
(Pol II) complex comprising the
amino-​terminal regions of RNA
Pol II subunit 1 (Rpb1) and
Rpb2, and the carboxy-​
terminal region of Rpb6, which
swings over the Pol II active-​site
cleft to encase the DNA
template and nascent RNA.
processes by recruiting a variety of nucleosome remod-
ellers (CHD1 (ref. 164) ), histone chaperones (SPT6
(ref.165) and FACT166,167) and histone modifiers (com-
plex proteins associated with SET1 (COMPASS)168, E3
ubiquitin-​protein ligase BRE1169,170 and histone-​lysine
N-​methyltransferase, H3K79 specific (also known as
DOT1L)168,169. Therefore, defects in productive elong­
ation owing to PAF1C ablation could result from the
deregulation of any number of these factors. As another
example, DSIF transits from being a pausing factor to
an elongation factor upon phosphorylation by P-​TEFb.
Upon the release of Pol II pausing, DSIF promotes
productive elongation by interacting with elongating
Pol II and reinforcing the closed conformation of the
Pol II clamp to facilitate the passage of the template DNA
through the central cleft of the polymerase92,171–173.
The chromatin landscape of productive elong­ation.
A variety of post-​translational modifications are asso-
ciated with distinct stages of transcription. Active
promoters possess high levels of H3K4me3 and mul-
tiple acetylated H3 and H4 Lys residues, whereas gene
bodies of transcribed genes are enriched with mono-​
ubiquitylated H2B (H2Bub), H3K36me3, H3K79me2
and H3K79me3 (refs174–176) (Fig. 3). However, owing
to the high correlation of all these histone modi-
fications with gene expression and their complex
­crosstalk177,178, it has been challenging to discriminate
the direct involvement of each modification in tran-
scription regulation. In budding and fission yeasts, the
deposition of H2Bub by the E3 ubiquitin ligase Bre1
is regulated by chromatin remodelling factor Chd1,
but perturbation of its deposition has a minor effect
on gene expression179–181. Studies in mammalian cells
have identified a role for regulators of H2B ubiqui-
tylation in regulating transcription at both enhanc-
ers and promoters, but no direct role in promoting
transcription elongation has been identified182,183.
Histone-​lysine N-​methyltransferase SETD2 associ-
ates with the phosphorylated Pol II CTD and deposits
H3K36me3 in gene bodies, where it can affect RNA
splicing and prevent cryptic transcription154,184 (Fig. 3c).
This regulation is mediated by a variety of factors that
can recognize H3K36me3, such as histone deacetylases
and nucleosome remodellers185,186. In mammals, SETD2-
catalysed H3K36me3 is recognized by the PWWP
domain of DNA (cytosine-5)-methyltransferase 3B,
which methylates DNA in gene bodies to help prevent
cryptic transcription187,188.
Histone H3K79me2 and H3K79me3 are found
in gene bodies of actively transcribed genes, but the
function of these modifications is not well under-
stood (Fig. 3c). The methylation of H3K79 is mediated
by DOT1L with the assistance of PAF1 and BRE1
(refs168,189–192). In metazoans, DOT1L associates with
the SEC proteins AF9 and ENL, suggesting that DOT1L
and the SEC share some degree of co-​regulation193. The
catalytic domain of DOT1 is structurally unrelated to
the SET domain found in other Lys methyltransferases,
and the Lys that it modifies is located in the globular
domain of H3, not in its tail194. No domains that recog-
nize H3K79me or any demethylases for these residues
have been identified, further hampering investigation of
the role of H3K79me in transcription elongation.

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 Reviews

Cornelia de Lange syndrome

Enhancers regulate elongation transcription elongation. Heterozygous mutations in
A developmental syndrome Enhancer–promoter interactions are essential for fine- cohesin subunits cause Cornelia de Lange syndrome, prob-
caused by heterozygous tuning transcription, including transcription elong­ ably owing to defective enhancer–promoter communica-
germline loss-​of-function ation22,195 (reviewed in refs196,197). Although the topo- tion and not the chromosome cohesion role of cohesin203.
mutations in the cohesin loading
logical organization of long-​range enhancer–promoter Mutations that stabilize the SEC core component AFF4
protein nipped-​B-like protein or
in structural components of the interactions is not fully understood, the cohesin and were found in patients with CHOPS syndrome, which shares
cohesin ring. Mediator complexes have important roles in enabling many phenotypic properties with Cornelia de Lange syn-
these interactions198–200. Cohesin and Mediator physi­ drome204. The similarities in phenotypes between loss of
CHOPS syndrome cally interact with each other and co-​o ccupy both function of cohesin and gain of function of the SEC raise
A developmental syndrome with
a similar phenotype to Cornelia
active enhancers and promoters199,200. Cohesin forms a the possibility that cohesin-​dependent enhancer activity
de Lange syndrome that is ring-​like structure that is thought to hold together the modulates the role of the SEC in transcription elongation.
caused by germline gain-​ enhancer and promoter198 (Fig. 4). However, cohesin There are at least two lines of evidence that link
of-function mutations in the depletion predominantly affects the release of paused Mediator to transcription elongation control in addition
super elongation complex
Pol II rather than the establishment of pausing201,202, sug- to its interaction with cohesin80. First, Mediator allevi-
scaffolding protein AF4/FMR2
family member 4. gesting that enhancer–promoter looping directly governs ates the Gdown1-dependent stabilization of Pol II paus-
ing106,205, and second, Mediator appears to recruit the SEC
and BRD4 to promoter regions and thus stimulates Pol II
release147,206,207. Owing to the transient association of
Enhancer TSS TTS Mediator with PIC before the full establishment of pausing
as discussed above, the SEC and BRD4, which are recruited
a
TTotal Pol II early during that short time frame by Mediator, must be
Ser5P retained on chromatin through additional mechanisms,
Ser2P for example, through their binding to acetylated histones.
Active enhancers are enriched in H3K4me1 and in
acetylated H3K27 (H3K27ac) (Fig. 3), which are largely
catalysed by the COMPASS family members myeloid/
lymphoid or mixed-​lineage leukaemia protein 3 (MLL3;
also known as KMT2C) and MLL4 (also known as
KMT2D)208,209 and by p300 and CREB-​binding pro-
b
H3K4me1 tein (CREBBP)210,211, respectively (Fig. 4). In contrast to
H3K4me2 H3K4me1, which is present at both active and poised
H3K4me3 enhancers, the presence of H3K27ac correlates with
enhancer activity139,211,212. Enhancer RNAs were suggested
to interact with and facilitate the eviction of NELF from
promoters to activate the release of paused Pol II (ref.103),
providing a potential direct link between enhancer acti-
vation and transcription elongation. Recent work has sug-
c gested that components of all three P-​TEFb-containing
H3K27ac complexes bind to enhancers and regulate their activity:
H2Bub the snRNA in the inactive 7SK–P-​TEFb complex inter-
H3K36me3 acts with a chromatin remodelling complex to inhibit
H3K79me3
enhancer RNA transcription 213, whereas BRD4–P-​
TEFb and the SEC promote enhancer activation135,214–216
(Table 1). PAF1 occupies enhancers and can restrict the
hyperactivation of a subset of them113. Increased enhancer
activity upon PAF1 depletion correlates with release from
pausing of nearby genes. Furthermore, deletion of PAF1-
Fig. 3 | Post-​translational modification of rNa polymerase ii and histones at active
genes with pausing and at active enhancers. a | Average chromatin occupancy profiles
regulated enhancers resulted in loss of pause–release at
of total RNA polymerase II (Pol II), Pol II with carboxy-​terminal domain (CTD) their cognate genes without greatly affecting transcription
phosphorylated at Ser5 (Ser5P) and at Ser2 (Ser2P) on active genes that exhibit Pol II initiation113. The concordant change of enhancer activa-
pausing and at active enhancers. In general, the occupancy of Pol II is higher at promoters tion and Pol II release without increasing rates of initiation
than at enhancers. Ser5P levels are enriched at promoter regions, whereas Ser2P levels reinforces the notion that enhancers contribute directly to
increase along the gene bodies. b | Average profiles of histone H3 Lys4 (H3K4) the elongation process.
methylation on active genes and enhancers. On active genes, trimethylation of H3K4
(H3K4me3) localizes around the transcription start site (TSS) and is surrounded by Multitasking factors control elongation
H3K4me2; H3K4me1 extends furthest into gene bodies. Enhancers are generally Recent studies identified various regulators of transcrip-
enriched in H3K4me1. c | Average profiles of acetylation of H3K27 (H3K27ac), mono-​
tion elongation that have known roles in other cellular
ubiquitylation of H2B (H2Bub), H3K36me3 and H3K79me3 on active genes and
enhancers. H3K27ac peaks at active enhancers and promoters of active genes. By processes, including factors previously implicated in
contrast, H2Bub, H3K36me3 and H3K79me3 levels are highest in gene bodies. The level DNA replication and the DNA damage response. These
of H3K79me3 is relatively higher towards the 5′ end of gene bodies, and H3K36me3 is factors modulate signal-​induced transcription or directly
enriched towards the 3′ end of genes. H2Bub is more evenly distributed across gene perturb the interplay between the transcription machin-
bodies. TTS, transcription termination site. ery and chromatin. Here, we discuss recent progress in

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Reviews

Lowly active or
during transcription introduces overwound DNA ahead
PAF1C of the polymerase and underwound DNA behind it, with
inactive enhancers
the former impeding the processivity of the polymer-
Pol II MLL3–COMPASS ase218. Notably, the formation of supercoiled structures
NURD or MLL4–COMPASS around promoter regions could stabilize paused Pol II,
and the activity of DNA topoisomerase IIβ (TOP2β)
can promote the release of pausing219,220. TOP2β activ-
ity can introduce DNA double strand breaks (DSBs) at
promoters of a subset of paused, neuronal-​stimulation
DSIF
NELF
early-​response genes and induces Pol II release and pro-
Gdown1 CTD Ser5 phosphorylation ductive elongation221. Upon the relaxation of supercoils,
CTD Ser2 phosphorylation TOP2β ligates the DSBs; this is facilitated by tyrosyl-​
DSIF phosphorylation DNA phosphodiesterase 2 (TDP2), which hydrolyses the
H3K4me1
–1 Transcript +1 phosphotyrosyl bond between trapped topoisomerases
H3K4me3
Promoter-proximal Signal-induced H3K27ac and DNA222,223.
pausing gene expression H4 acetylation In contrast to TOP2β, which is thought to be active
through enhancer around promoter regions, a recent study suggested
activation
that TOP1 is inactive at promoters but becomes fully
Highly active
enhancers activated by Pol II CTD phosphorylation and relieves
torsional stress to promote efficient transcription
SEC MLL3–COMPASS elongation at gene bodies224. Intriguingly, only CTD
BRD4 or MLL4–COMPASS phosphorylation induced by BRD4, but not P-​TEFb or
eRNA
p300 TFIIH, is capable of stimulating the catalytic activity of
and CBP TOP1 in vitro, suggesting that TOP1 activation by CTD
Cohesin
Mediator phosphorylation is context-​dependent or conformation-​
dependent224. The activity of TOP1 can also be regu-
lated through sumoylation by the protein inhibitor of
activated STAT protein 1 (PIAS1) E3 ligase complex225.
Consistent with a role of TOP1 in promoting produc-
tive elongation, previous work has shown that poisoning
–1 +1
or depletion of TOP1 leads to more severe defects in
transcription at longer genes226,227.
Release of Pol II from
promoter-proximal pausing Underwound DNA behind paused Pol II favours
the opening of dsDNA and thus the formation of R-​
Fig. 4 | regulation of transcription elongation by enhancers. Lowly active or inactive loops 228,229, which can promote Pol II pausing and
enhancers are marked by the presence of monomethylated histone H3 Lys4 (H3K4me1), inhibit productive elongation. Resolution of R-​loops to
which is catalysed by myeloid/lymphoid or mixed-​lineage leukaemia protein 3 promote productive elongation has been described for
(MLL3)-containing or MLL4-containing complex of proteins associated with SET1 ATP-​dependent DNA helicase Q5 (RECQ5), RNase H
(COMPASS), with or without low levels of acetylated H3K27 (H3K27ac), which is catalysed and the helicases senataxin and ATP-​dependent RNA
by p300 or CREB-​binding protein (CBP). RNA polymerase II (Pol II)-associated factor 1
helicase A (DHX9)230,231. RECQ5 is a general elongation
complex (PAF1C) and the nucleosome remodelling and deacetylase (NURD) complex can
bind less active enhancers and prevent their activation and transcription of enhancer RNAs factor that attenuates the movement of Pol II to mini-
(eRNAs). Genes neighbouring the less active enhancers tend to exhibit higher levels of mize co-​transcriptional DNA instability232. Furthermore,
pausing. Following signalling cues, p300, CBP and H4 acetyltransferases (not shown) are RECQ5 induces PIAS1-catalysed sumoylation of TOP1
recruited to enhancers by transcription factors (not shown) to catalyse histone acetylation and thus enhances the recruitment of RNA splicing fac-
at enhancers and promoters. Activation of enhancers is often associated with the tors to actively transcribed genes225. TOP3β is the only
production of eRNAs. The physical interaction between enhancers and promoters requires known RNA topoisomerase in humans, but it is also
cohesin and Mediator complexes. The increased level of histone acetylation contributes to active towards DNA233,234 and consequently can con-
the binding of bromodomain-​containing protein 4 (BRD4) and the super elongation tribute to the resolution of R-​loops235. The recruitment
complex (SEC) to chromatin through their bromodomains and YEATS (YAF9, ENL , AF9, of TOP3β to chromatin requires its interaction with
TAF14 and SAS5) domains, respectively. BRD4-containing and SEC-​containing positive
Tudor domain-​containing protein 3 (TDRD3), which
transcription elongation factor b (P-​TEFb) phosphorylate negative elongation factor (NELF),
DRB sensitivity-​inducing factor (DSIF) and Ser2 residues of the Pol II carboxy-​terminal recognizes methylated histone H3 Arg235. Additionally,
domain (CTD), which releases the paused Pol II into productive transcription elongation. binding to TDRD3 facilitates substrate recognition by
TOP3β and thus stimulates its activity in preventing
R-​loop formation236,237.
understanding how topoisomerases and PARPs regulate
transcription elongation. PARP1 ADP-​ribosylates transcription elongation
factors. PARPs are a group of enzymes that catalyse
The role of topoisomerases in transcription elongation. the transfer of ADP-​ribose derived from NAD+ to tar-
Topoisomerases are enzymes capable of creating tran- get proteins. Aside from the well-​known function of
sient DNA breaks to alleviate DNA supercoiling and thus PARPs in promoting DNA repair and thus maintaining
are essential for cellular processes that require access to genome stability238, numerous studies have implicated
chromatin (reviewed in ref.217). The progression of Pol II PARPs, especially PARP1, in regulating transcription

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 Reviews

regulation and the control of promoter-​proximal paus-

MLL translocation ing, it will be important to investigate enhancer versus
in paediatric leukaemia promoter-​proximal roles of PARP1 in the regulation of
Pause MYC overexpressed Go transcription elongation.
in solid tumours
Concluding remarks
Understanding the full extent of the regulation of tran-
Fig. 5 | cancer addiction into transcription elongation. The precise regulation of scription elongation at the level of both pause release and
transcription elongation in physiological conditions is mediated by a cohort of processive elongation has been challenging owing to the
elongation factors that modulate different steps of elongation through cooperative and intertwined processes during gene expression, including
competitive mechanisms. Misregulated transcription elongation can be used to support
transcription initiation, splicing and transcription ter-
oncogenesis, as observed in paediatric leukaemia resulting from myeloid/lymphoid or
mination. Although disrupting factors by depletion or
mixed-​lineage leukaemia (MLL) translocations or in solid tumours driven by MYC
overexpression. Skewing the balance of transcription elongation control to higher rates deletion has been used as the major strategy to interro­
of Pol II pause–release and increased productive elongation leads to altered gene gate the function of proteins or protein complexes in
expression and altered splicing, which is essential for oncogenic gene expression controlling transcription elongation, dissecting the
programmes. The regulation of this process can in turn provide therapeutic targets for primary function of a factor in transcription regulation
human diseases that are caused by defects in transcription elongation control. will be greatly aided by the additional development of
specific small molecule inhibitors of different steps
of the transcription process. Also, the use of the auxin-​
elongation (Table 1). PARP1 can regulate transcription by inducible degradation system on factors involved in the
decompacting chromatin through the ADP-​ribosylation process will allow the study of transcription immediately
of histones and chromatin-​binding proteins, including following factor removal, which could distinguish indi-
transcription factors and remodellers in response to rect effects. Small molecule inhibitors are already avail-
heat shock or signal transduction pathways (reviewed able for CDK7, CDK8 and CDK9, which enables the
in refs239,240). ADP-​ribose on chromatin could also be selective targeting of the kinase modules of the TFIIH,
converted into ATP and serve as a local source of energy Mediator and P-​TEFb complexes, respectively253–256.
for chromatin remodelling to promote transcription241. More recently, protein depletion by auxin-​inducible
A recent study using a chemical genetic approach degradation and phthalimide-​based acute degradation
identified the NELF complex as one of the hundreds of has provided new insight into transcription elongation
potential PARP1 substrates242,243. Consistent with PARP1 mechanisms113,132,144,173.
regulation of transcription elongation, small molecule Disambiguating the various roles of multitasking
inhibition of PARP1 or depletion of PARP1 increased the factors in transcription elongation from the other cel-
levels of paused Pol II at a subset of genes242. Moreover, lular processes in which they participate could be rele-
PARP1 can be recruited by TOP2β to become activated vant for understanding human diseases. A recent study
by TOP2β-​introduced DNA breaks to promote tran- used an in vivo functional screening strategy and iden-
scription activation220,244. In addition, PARP1 can ADP-​ tified numerous pausing and elongation factors that are
ribosylate the FACT complex to promote its dissociation required for glioblastoma cell survival257. Mutations that
from chromatin245,246. PARP1 also ADP-​r ibosylates stabilize the SEC, which is already known to be a cofactor
enhancer-​binding factors247,248. For example, PARP1- of HIV TAT136,140 and the oncogenic MLL chimaeras139,
catalysed ADP-​ribosylation of lysine-​specific demethyl­ lead to the developmental disorder CHOPS syndrome204.
ase 5B (KDM5B), which demethylates H3K4me3 at both Therefore, progress in elucidating the molecular mecha-
enhancers and promoters249, prevents the interaction nisms underlying the regulation of transcription elong­
of KDM5B with chromatin and thus could affect the ation will not only contribute to our understanding of
enhancer chromatin landscape248. the intricate control of gene expression programmes but
Independent of its catalytic activity, PARP1 can pro- could also provide new therapeutic strategies for these
mote binding of the pioneer transcription factor and and other diseases linked to transcription elongation
pluripotency factor SOX2 to a subset of enhancers in factors. Given that devastating diseases, such as paedi-
embryonic stem cells, whereas ADP-​ribosylation of atric blood cancers resulting from MLL translocations
SOX2 promotes its degradation during differentia- or solid tumours driven by MYC overexpression, appear
tion247,250. ADP-​ribosylation by PARP1 at enhancers to be diseases of uncontrolled transcription elongation,
can serve as a platform to facilitate the recruitment of such molecular and biochemical studies could have a
chromatin factors such as the nucleosome remodelling major therapeutic impact23,258 (Fig. 5).
and deacetylase (NURD) complex during DNA dam-
Published online xx xx xxxx
age repair251,252. Given the relationship between enhancer

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Author contributions
F.X.C., E.R.S. and A.S. researched data for the article, made
substantial contributions to the discussion of content, wrote
the article and reviewed and edited the manuscript before
submission.
Competing interests
The authors declare no competing interests.
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