Article

Transcription control by long non-coding RNAs

Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.
Transcription 03/2012; 3(2):78-86. DOI: 10.4161/trns.19349
Source: PubMed
ABSTRACT
Non-coding RNAs have been found to regulate many cellular processes and thus expand the functional genetic repertoire contained within the genome. With the recent advent of genomic tools, it is now evident that these RNA molecules play central regulatory roles in many transcriptional programs. Here we discuss how they are targeted to promoters in several cases and how they operate at specific points in the transcription cycle to precisely control gene expression.

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Transcription 3:2, 78-86; March/April 2012; © 2012 Landes Bioscience
POINTOFVIEW
78 Transcription Volume 3 Issue 2
Keywords: chromatin, epigenetic
silencing, large intergenic non-coding
RNAs, long non-coding RNAs, RNA
polymerase II, transcription
Abbreviations: 5C, chromosome con-
formation capture carbon copy; AEBP2,
AE binding protein 2; asRNA, antisense
RNA; CBP, CREB-binding protein;
CCND1, cyclin D1; Cdk9, cyclin-
dependent kinase 9; ChIP, chromatin
immunoprecipitation; ChIRP, chro-
matin isolation by RNA purification;
CoREST, REST co-repressor; CTCF,
CCCTC-binding factor; DDX5, DEAD-
box RNA helicase p68; DHFR, dihy-
drofolate reductase; DNMT3b, DNA
methyl transferase 3b; EED, embryonic
ectoderm development; Ezh2, enhancer
of zeste homolog 2; G9a, euchromatic
histone lysine N-methyltransferase; GTF,
general transcription factor; HAT, his-
tone acetyl transferase; HDAC, histone
deacetylase; HIV, human immunode-
ficiency virus; HMGA1, high-mobility
group A1; hnRNP, heterogeneous nucle-
ar ribonucleoprotein; HOTAIR, HOX
antisense intergenic RNA; HOTTIP,
HOXA transcript at the distal tip; HOX,
homeobox genes; ICR, imprinted control
region; IGF2, insulin-like growth factor
2 (somatomedin A); JARID2, jumonji,
AT rich interactive domain 2; KAT2B,
lysine acetyltransferase 2B; lincRNA,
long intergenic non-coding RNA;
Transcription control by long non-coding RNAs
Tyler Faust,
1
Alan D. Frankel
1,
* and In D’Orso
2,
*
1
Department of Biochemistry and Biophysics; University of California; San Francisco, CA USA;
2
Department of Microbiology; University of Texas Southwestern
Medical Center; Dallas, TX USA
N
on-coding RNAs have been found
to regulate many cellular processes
and thus expand the functional genetic
repertoire contained within the genome.
With the recent advent of genomic tools,
it is now evident that these RNA mol-
ecules play central regulatory roles in
many transcriptional programs. Here we
discuss how they are targeted to promot-
ers in several cases and how they oper-
ate at specific points in the transcription
cycle to precisely control gene expression.
Introduction
The role of RNA in gene expression has
seen a paradigm shift in recent years.
Beyond serving as a passive messenger of
the genetic material, RNA itself can act as
a non-protein coding (ncRNA) regulatory
molecule. In addition to their established
functions in splicing and translation, it is
now clear that ncRNAs are central players
in transcriptional control. Over the past
few years, we have come to understand that
at least half of the mammalian genome is
transcribed, but only about 1–2% encodes
proteins, and that up to 70% of protein-
coding genes are thought to be transcribed
bidirectionally,
1-5
a phenomenon known
as pervasive transcription.
6,7
Previously
regarded as “junk DNA,” many of these
loci generate regulatory ncRNAs, includ-
ing a novel class of long (>150 nt and up
to 100 kb) non-coding RNAs (lncRNAs),
distinct from the well-characterized small
(19–23-nt) siRNAs and microRNAs
involved in RNA interference and post-
transcriptional regulatory pathways.
8-10
Due to their common intergenic location,
many were also referred to as lincRNAs
for large intergenic ncRNAs that regulate
lncRNA, long non-coding RNA; LSD1/
KDM1A, lysine-specific demethylase
1A; Mepce, methylphosphate capping
enzyme; Mistral, (Mira) activates tran-
scription; MLL, mixed lineage leukemia;
NF-Y, nuclear transcription factor Y;
Oct4, octamer-binding transcription fac-
tor 4; PANDA, p21 associated ncRNA
DNA damage activated; PcG, polycomb
group; PCLs, mammalian homologues
of drosophila polycomblike; PICs,
transcription pre-initiation complexes;
P-TEFb, positive transcription elongation
factor b; RNAP II, RNA polymerase II;
pRNA, promoter-associated RNA; PRC,
polycomb repressive complex; PURA,
purine rich element binding protein A;
REST, RE-1 silencing transcription fac-
tor; SINE, short-interspersed element;
SRA, steroid receptor RNA activator;
SUZ12, suppressor of zeste 12 homo-
log (drosophila); TAR, trans-activation
response element; TLS, translocated in
liposarcoma; TSS, transcription start site;
WDR5, WD repeat-containing protein
5; XIST, X-inactive specific transcript
Submitted: 12/13/11
Revised: 01/12/12
Accepted: 01/13/12
http://dx.doi.org/10.4161/trns.19349
*Correspondence to: Alan D. Frankel
and Iván D’Orso; Email: frankel@cgl.ucsf.edu
and ivan.dorso@utsouthwestern.edu
Page 1
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www.landesbioscience.com Transcription 79
POINTOFVIEW
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methyltransferase EZH1/2, SUZ12, EED
and RbAp46/48 (or RBBP7/4), and also
several other polypeptides: AEBP2, PCLs
and JARID2.
29
The EZH1 and EZH2
enzymatic subunits of PRC2 methyl-
ate H3K27 (H3K27me2/3), which is
essential for long-term gene silencing.
HOTAIR binds EZH2 and is expressed
at the border of an adjacent repressive
chromatin domain, which is enriched for
H3K27me3 and SUZ12. Interestingly,
knockdown of HOTAIR revealed that
it maintains the repressed domain at the
HOXD locus, but not the A, B or C loci
(Fig. 1A). It remains to be determined
if one or all PRC2 subunits are required
for HOTAIR activity, and if gene-specific
complexes are formed at other locations.
30
H3K27 methylation by PRC2 also signals
PRC1 recruitment and H2A monoubiqui-
tination (H2AK119ub), which represses
transcription by interfering with RNAP
II elongation,
31
but it is unclear if this is
a general mechanism since some genes
are targeted by PRC1 and not PRC2. It is
clear, however, that both PRC complexes
are required to maintain gene repression.
More than 3,000 lncRNAs were found
to interact physically with either PRC2 or
another repressive chromatin-modifying
complex termed CoREST.
32
CoREST was
identified initially as a co-repressor for the
silencing factor REST (also called NRSF),
which represses neuronal genes in non-
neuronal cells. The CoREST complex
also contains HDACs 1 and 2, providing a
mechanism by which CoREST can medi-
ate silencing through histone deacety-
lation. Interestingly, PRC2 and CoREST
share 40% of interacting lncRNAs,
including HOTAIR, suggesting that they
may act together to precisely regulate
gene expression at specific genomic loci.
32
HOTAIR possesses extensive secondary
structure
33
and acts as a scaffold for EZH2/
PRC2, which binds to a 5' region of the
RNA and LSD1, an H3K4me2 demethyl-
ase component of CoREST that binds to
a 3' region.
33
Thus, HOTAIR functions
to target these two histone modification
complexes to chromatin to remove an
active H3K4me2 mark, while methylat-
ing H3K27 to favor formation of a repres-
sive environment (Fig. 1A). Interestingly,
recent chromatin isolation by RNA puri-
fication (ChIRP) analyses revealed that
regulatory factors. For example, genes
actively transcribed by RNA polymerase
(RNAP) II are marked by H3 lysine 4
methylation (H3K4me2/3) at their pro-
moters and H3 lysine 36 trimethylation
(H3K36me3) throughout the gene body,
while repressed genes are marked by H3
lysine 27 methylation (H3K27me2/3) or
H3 lysine 9 methylation (H3K9me2/3)
at promoters.
11,24-27
H3K9me2/3 and
H3K27me2/3 marks result in compact
chromatin around the transcription start
site (TSS) of transcriptionally inactive
genes, whereas H3K4me3 marks result in
a more open, transcriptionally active chro-
matin conformation. Different histone
modifications in combination can form
complex networks, which are essential
for regulating gene expression in a spa-
tial and temporal manner. Recent studies
highlight several cases in which lncRNAs
influence the status of chromatin.
HOTAIR
Transcription control is essential during
development because it requires coordi-
nated expression of neighboring genes
through a process termed locus control. In
mice and humans, 39 Hox genes encod-
ing homeo domain transcription fac-
tors are clustered in four loci (A–D) and
expressed in a complex pattern. Seminal
studies by the Chang lab
28
characterized
the transcriptional pattern of the four
human HOX loci revealing that most of
the transcribed region maps to intergenic,
and not exonic, domains. This analysis
led to the identification of 231 lncRNAs,
some as long as 30 kb. The expression
of these HOX lncRNAs demarcates
broad chromosomal domains of dif
-
ferential histone methylation patterns
and RNAP II occupancy. One of these
RNAs transcribed in the HOXC locus
is HOTAIR (HOX Antisense Intergenic
RNA), a 2.2 kb lncRNA that demarcates
active and silent chromatin domains and
represses transcription in trans across
the 40 kb HOXD locus (Fig. 1A). Hox
genes are silenced by the Polycomb group
(PcG) proteins, which regulate chroma-
tin structure, in part through post-trans-
lational histone modifications. The core
Polycomb Repressive Complex 2 (PRC2)
comprises four components: the histone
local chromatin activity.
11
For the pur-
pose of discussion here, the term lncRNA
will be used to describe examples of long
regulatory RNAs and their function in
the transcription cycle irrespective of their
genomic location.
The wide diversity of these lncRNAs
is commensurate with their diverse roles
in gene regulation, ranging from the
control of metabolic pathways to cell
fate determination and development.
12-14
These lncRNAs serve as molecular scaf-
folds to guide the recruitment or regulate
the activity of RNA-protein complexes
to control transcription circuits. They
can silence or activate gene expression
locally (in cis) by acting on proximally
transcribed protein-coding genes, or glob-
ally (in trans) acting at long-range and
affecting a locus at a distance.
12,15-17
Here
we discuss selected examples of cis- and
trans-acting mammalian lncRNAs that
function at defined steps of the transcrip-
tion cycle, including chromatin modi-
fication and remodeling, DNA looping
and regulation of higher-order structures,
assembly of transcription complexes at
the promoter, transcription initiation and
elongation (Fig. 1). We particularly focus
on chromatin-associated ncRNAs and
what is known about their targeting to
transcription complexes. Other examples
of control by lncRNAs, including ones
that do not act on transcription, such as
imprinting of inactive X chromosomes
by XIST RNA, are described in recent
reviews in reference 13, 14 and 18–23. In
addition to the newly identified lncRNAs
found by genomic methods, other previ-
ously known ncRNAs have now been
shown to regulate transcription (e.g., U1,
Alu, B2 and 7SK) and will be discussed.
Control of Chromatin Modication
and Remodeling
The state of chromatin is a key determi-
nant of transcriptional activity, including
nucleosome structure and the ensemble of
histone modifications that mark regions
of the genome as active or silent. Histone
post-translational modifications regulate
biological processes either by altering
chromatin structure (by loosening or com-
pacting the DNA-histone interactions)
or by contributing to the recruitment of
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80 Transcription Volume 3 Issue 2
each regulates HOX loci through chroma-
tin-remodeling complexes but how they
are specifically recruited to their respective
DNA loci is currently unclear.
Mistral
The mouse HOXA locus expresses a 798-
nt unspliced, polyadenylated lncRNA
between the Hoxa6 and Hoxa7 genes
called Mistral, which was discovered by
RNA-chromatin immunoprecipitation
of MLL1.
35
In vitro analysis revealed a
direct interaction between the MLL1 SET
domain and an RNA hairpin loop in the 3'
region of Mistral. RNAi-mediated knock-
down of Mistral significantly diminished
MLL1 enrichment in the Hoxa6/a7 region
as assessed by ChIP and also inhibited the
Unlike PRC2-mediated recruitment by
HOTAIR, HOTTIP recruits WDR5, a
subunit of the MLL1 H3K4 methylation
complex, to promoters. Chromosome
conformation capture carbon copy (5C)
experiments, which define physical chro-
matin interactions, revealed that actively
expressed HOXA regions have compact
chromosomal loops that facilitate inter-
actions between these loci.
17
HOTTIP
knockdown does not affect this structure
but is required for MLL1 occupancy and
H3K4 methylation. Because HOTTIP
remains close to its site of synthesis, it is
able to transmit information from these
higher order chromatin structures into
epigenetic modifications to coordinate
long-range gene expression.
17
The exam-
ples of HOTTIP and HOTAIR show that
HOTAIR occupancy occurs indepen-
dently of EZH2, suggesting that RNA
can guide PRC2 recruitment and specify
formation and spread of a repressive envi-
ronment.
30
Additionally, sites of HOTAIR
occupancy are significantly enriched for
genes, which become de-repressed upon
endogenous HOTAIR knockdown.
30,33,34
HOTTIP
HOTTIP (HOXA transcript at the distal
tip) is a 3,764-nt lncRNA transcribed
at the 5' end of the HOXA locus, which
coordinates activation of 11 HoxA genes
(
Fig. 1A). HOTTIP appears to act in
cis, based on its proximity to its tar-
get genes and the distance-dependence
of HOXA target gene activation.
17
Figure 1. Schematic of the various steps in the transcription cycle targeted by lncRNAs. (A) A number of newly identied lncRNAs target chromatin-
modifying complexes to DNA to regulate gene expression. Both repressive (PRC2, LSD1) and activating (MLL1) complexes, and factors (TLS) that block
chromatin-modifying complexes are targeted. Me, refers to methyl groups demethylated from H3K4me2 by the action of LSD1. (B) lncRNAs inuence
the higher order, three-dimensional structure of chromatin either by acting as enhancers or by targeting the insulation machinery along with CTCF
and cohesin (e.g., SRA). (C) A number of steps during the initiation phase of transcription are targeted by lncRNAs, including PIC assembly (DHFR
ncRNA
),
formation of ‘open’ complexes (B2/Alu) and abortive initiation (U1). (D) The template-associated 7SK snRNA along with Hexim1 regulate elongation
of the HIV promoter by blocking the P-TEFb kinase. The lncRNAs discussed are either delivered to the DNA in trans (HOTAIR, MISTRAL, Air, Kcnq1ot1,
SRA, pRNA, B2/Alu, U1, 7SK) or are locally synthesized and acting in cis (HOTTIP, CCND1
ncRNAs
, asOct4, DHFR
ncRNA
). How specicity is achieved in trans is a
major question, and it is not clear whether the majority of lncRNAs are passively recruited to the DNA by a protein factor/complex, as is probably the
case for B2/Alu and RNAP II, or whether the lncRNA itself directs the RNP complex to chromatin, as is proposed for HOTAIR.
Page 3
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www.landesbioscience.com Transcription 81
epigenetic remodeling complexes to par-
ticular loci.
Control of Enhancer Activity
and Higher-order
Chromosome Structures
Some lncRNAs are located further than
1 kb from known protein-coding genes
and may function as enhancers in cis to
regulate neighboring genes.
15,16,43
Such
lncRNAs are marked with chromatin
signatures characteristic of transcribed
genes, such as H3K4me3 at the 5'-end and
H3K36me3 downstream, and not marks
that characterize enhancer elements,
such as H3K4 monomethylation.
11,44,45
Knockout of these lncRNAs decreases
neighboring gene expression,
15
where they
may function as co-activators to recruit
positive-acting factors or form higher
order chromatin structures through DNA
looping.
46-48
SRA
Chromatin insulators are DNA elements
that can protect a gene from outside influ-
ences, which might lead to either inappro-
priate activation or silencing of the gene.
CTCF (CCCTC-binding factor) is a
zinc-finger protein required for transcrip-
tional insulation and accomplishes this
via long-range physical interactions with
CTCF sites and cohesin.
49
CTCF-binding
sites are widely distributed throughout
the genome and function via CTCF and
cohesin to either separate or bring together
distant regulatory elements.
24,49,50
For
example, cohesin enables CTCF to insu-
late promoters from distant enhancers and
control transcription at the IGF2/H19
(insulin-like growth factor 2) imprinted
control region (ICR) (Fig. 1B). A recent
report shows a role for a ~700-nt lncRNA,
SRA (steroid receptor RNA activator), in
CTCF-mediated insulation.
51
SRA, which
is reported to act as both a transcriptional
co-activator and co-repressor,
52-54
is a chro-
matin-associated lncRNA found in a com-
plex with the DEAD-box RNA helicase
p68 (DDX5) and CTCF (Fig. 1B). p68/
DDX5 was detected at CTCF sites in the
IGF2/H19 locus, and depletion of p68 or
SRA reduced CTCF-mediated insulator
activity which ultimately increased levels
additional examples.
37-39
Air, a 108 kb
lncRNA, regulates genomic imprinting
of a cluster of autosomal genes on mouse
chromosome 17, and is required for allele-
specific silencing of the cis-linked Slc22a3,
Slc22a2 and Igf2r genes in mouse placenta.
Mechanistically, Air represses Slc22a3
transcription by interacting with its pro-
moter and recruiting the H3K9 histone
methyltransferase G9a to dictate local-
ized H3K9 methylation (H3K9me3).
38
Likewise, Kcnq1ot1, a 91 kb lncRNA that
controls bidirectional silencing of genes in
the Kcnq1 domain in placenta, also inter-
acts with G9a to direct H3K9 methylation
and, in addition, interacts with PRC2 to
direct H3K27 methylation.
39
Similarly to
HOTAIR (Fig. 1A), Kcnq1ot1 scaffolds
two distinct chromatin-modifying com-
plexes to direct transcriptional repression
across a locus. Again, the basis for target
specificity remains to be determined.
Antisense Oct4 lncRNAs
Oct4 is a transcription factor essential for
maintaining the pluripotent state, and
the epigenetic silencing of its gene is an
important step in differentiation. In par-
ticular, the methylation state of H3K9
at the Oct4 promoter, mediated by G9a,
is a driving factor in differentiation and
reprogramming. A recent report describes
an antisense non-polyadenylated Oct4
lncRNA, named asOct4, which origi-
nates from a pseudogene.
40
The model
proposes that asOct4 functions in cis by
associating with the Oct4 promoter and
recruiting the regulatory complexes PRC2
(EZH2 subunit) and G9a to prevent Oct4
transcription, thus pointing to a role in
regulating pluripotency. The specificity
of recruitment of this antisense lncRNA
remains unknown but it is possible that it
forms a DNA-RNA hybrid with the Oct4
promoter. Interestingly, this repressive
mechanism is counteracted by the nucleic
acid-binding protein PURA (purine rich
element binding protein A), which inter-
acts with asOct4 and prevents its promoter
localization, ultimately upregulating Oct4
gene expression. Because many lncRNAs
are generated antisense to protein-cod-
ing genes,
1,2,41,42
it will be intriguing to
determine whether they, too, function to
regulate gene transcription by directing
transcription of Mistral, Hoxa6 and Hoxa7
but no other genes in the HOXA locus.
Thus, it appears that localized recruit-
ment of Mistral activates transcription of
its adjacent genes through the recruitment
of the same histone methyltransferase
complex as HOTTIP (
Fig. 1A) and pre-
sumably through changes in chromatin
conformation. Like the previous cases, it
remains unclear how Mistral is specifically
targeted to its DNA loci.
CCND1 lncRNAs
In an attempt to uncover cellular factors
that modulate the histone acetyltrans-
ferase (HAT) activity of CREB-binding
protein (CBP)/p300/KAT2B, the RNA-
binding protein TLS (translocated in lipo-
sarcoma) was found associated with CBP
by mass spectrometry and shown to be an
RNA-dependent HAT inhibitor.
36
The
N-terminal portion of TLS, but not the
full-length protein, is a potent inhibitor.
Interestingly, addition of RNA oligonucle-
otides stimulated inhibition by full-length
TLS and also enhanced its proteolysis,
suggesting that RNA allosterically acti-
vates the HAT inhibitory activity. One
specific target gene of TLS-mediated
inhibition of CBP is the cyclin D1 gene
(CCND1), which has several 200–330 nt
RNAs transcribed upstream, referred to
as CCND1 lncRNAs (Fig. 1A). These
regulatory RNAs were found to bind
TLS and all are targeted to the CCND1
promoter. The direct requirement of
these lncRNAs at the CCND1 locus was
confirmed by knockdown experiments,
showing decreased TLS occupancy at the
promoter and increased levels of H3 acet-
ylation (H3K9-K14Ac), a target of CBP/
p300, and of CCND1 transcripts. Thus,
it appears that these locally synthesized
lncRNAs are induced in response to DNA
damage and are tethered to 5' regulatory
regions of the CCND1 promoter to recruit
TLS, which controls CBP/p300 HAT
activity to cause gene-specific silencing.
36
Air and Kcnq1ot1
The localized recruitment of lncRNAs to
promoters along with chromatin-mod-
ifying complexes is emerging as a com-
mon theme. Air and Kcnq1ot1 are two
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82 Transcription Volume 3 Issue 2
model posits that these lncRNAs medi-
ate transcriptional repression on the DNA
template, and presumably are targeted to
promoters as a consequence of RNAP II
recruitment, but the mechanistic basis
remains to be established.
U1 RNA
In searching for ncRNAs that regulate
transcription initiation, the ~160-nt U1
snRNA, a core splicing component, was
found to associate with TFIIH.
66
Stem-
loop 2 of U1 RNA, and not the entire
U1 snRNP, mediates an interaction with
the cyclin H subunit of TFIIH.
67
In con-
trast to the repressive action of the B2
and Alu RNAs, U1 enhances TFIIH-
dependent transcription initiation in vitro
from a RNAP II promoter (Fig. 1C).
Reconstituted transcription in vitro dem-
onstrates an increase in the rate of forma-
tion of the first phosphodiester bond by
RNAP II in the presence of U1 RNA,
suggesting that it regulates transcription
in addition to its well-established role in
RNA processing. While the mechanism
is not yet clear, one model posits that U1
RNA is a component of a re-initiation scaf-
fold anchored by proximal 5' splice sites,
potentially linking its roles in transcription
control and splicing. It remains to be deter-
mined if U1 RNA binding to TFIIH and
its subsequent effect on transcription is a
regulated or constitutive process and which
are the in vivo targets of this lncRNA.
Control of Transcription
Elongation
Until recently, it was widely believed that
recruitment of RNAP II and the early
steps of initiation were rate-limiting for
transcription at most promoters. This
notion has been challenged by the finding
that many cellular genes exhibit RNAP II
occupancy downstream of the TSS, a phe-
nomenon known as transcriptional paus-
ing in which RNAP II is unable to escape
into productive elongation.
68-72
P-TEFb,
the positive transcription elongation fac-
tor b (composed of cyclin T1 and Cdk9
subunits), has been studied extensively
due to its role in facilitating RNAP II
escape from this paused state globally.
73,74
When recruited to promoters, P-TEFb
suggesting a model whereby the DHFR
lncRNA represses transcription in cis
by preventing PIC formation (Fig. 1C).
Interestingly, DHFR lncRNA can inhibit
transcription only when its sequence
extends into the major promoter sequence,
where it has been shown to form a triplex
DNA-DNA-RNA structure in vitro. This
mechanism is similar to the formation of
a DNA:RNA triplex by pRNA (promoter-
associated RNA) at the rDNA promoter
which signals the recruitment of the DNA
methyltransferase DNMT3b to silence the
transcription of rRNA genes.
59
The perva-
sive transcription observed near annotated
genes
7
might act similarly to generate
lncRNAs locally that regulate the assem-
bly of nearby transcription complexes.
B2 and Alu ncRNAs
A number of short-interspersed elements
(SINEs) are transcriptionally upregulated
during heat shock, such as the 178-nt
mouse B2 RNA and the ~350-nt human
Alu RNA. The majority of RNAP II
transcription is coordinately downregu-
lated during heat shock, and thus it was
postulated that these RNAP III-derived
SINE transcripts might be responsible for
broadly repressing transcription.
60
Upon
heat shock, B2 and Alu RNAs were found
to specifically occupy the promoters of
repressed genes in vivo along with RNAP
II and GTFs (Fig. 1C).
60
After binding to
DNA, RNAP II switches from a closed to
an open complex and this change involves
the separation of the DNA strands to form
an unwound section of DNA of approxi-
mately 13 bp, referred to as the transcrip-
tion bubble. Both Alu and B2 RNAs were
found to tightly bind RNAP II in vitro,
and biochemical experiments showed that
they prevent RNAP II from establishing
contacts with the promoter both upstream
and downstream of the TATA box during
open complex formation (
Fig. 1C). The
results are consistent with a model in which
their repression domains bind in the DNA
cleft of RNAP II
61,62
and repress TFIIH-
mediated phosphorylation of RNAP
II.
60,63,64
A 50-nt fragment of B2 RNA (nts
81–131) is sufficient for its activity and
includes an 18-nt single-stranded region
flanked by two hairpins, both of which are
required for repression.
62,65
Altogether the
of IGF2.
51
Although p68 and SRA deple-
tion does not affect CTCF recruitment
to its genomic sites, it does reduce cohe-
sin binding, implicating the p68/SRA
protein-RNA complex in stabilizing the
interaction of cohesin with CTCF. This
stabilization may function as a regulated
step in transcription insulation. Thus,
lncRNAs are also required for proper
insulator function to shield a locus from
the effects of flanking chromatin domains.
Transcription Initiation Control
In eukaryotes, the general transcription
factors (GTFs) TFIIA, TFIIB, TFIID,
TFIIE, TFIIF and TFIIH, and RNAP II
assemble at promoters into pre-initiation
complexes (PICs) to specify the TSS.
55
PIC formation usually begins with TFIID
binding to the TATA box, initiator or
downstream promoter element found in
most core promoters, followed by the entry
of other GTFs and RNAP II through
either sequential assembly or a preassem-
bled RNAP II holoenzyme pathway. For
activator-dependent (or regulated) tran-
scription, additional cofactors typically
are required to transmit regulatory signals
between gene-specific activators and the
general transcription machinery.
55,56
In
addition to protein components, a number
of lncRNAs are known to influence these
early steps in transcription.
DHFR lncRNA
Alternative promoters for the same gene
are a common phenomenon in gene regu-
lation. The human DHFR gene contains
two promoters, minor and major, with
the major promoter producing 99% of the
transcribed RNA. Seminal studies have
shown that the transcript generated from
the upstream minor promoter can impede
the formation of PICs on the major pro-
moter in vitro (Fig. 1C).
57
In quiescent
cells, the cell cycle-regulated DHFR
gene is repressed in a manner that cor-
relates with the expression of an ~400-nt
lncRNA, referred to as DHFR lncRNA,
from the upstream minor promoter, which
shuts off transcription from the major pro-
moter. This lncRNA was found to directly
bind TFIIB in vitro and reduce its occu-
pancy on the major promoter in vivo,
58
Page 5
© 2012 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Transcription 83
shock domain protein A, which has an
established RNA-binding activity, and
an ~120 kDa protein of unknown func-
tion annotated as C9orf10.
83,92-94
7SK
can also function cooperatively with the
chromatin regulator HMGA1 to control
transcription in both P-TEFb-dependent
and -independent modes.
95,96
These cases
exemplify a vast range of new mechanistic
possibilities by which a regulatory RNA
may control transcription by interacting
with different protein complexes. It is
possible that 7SK dynamically switches
between chromatin-bound and -unbound
states to regulate the genesis or termina-
tion of elongation programs. This would
differentiate 7SK from other stably chro-
matin-bound lncRNAs such as HOTAIR
that direct the establishment of silent
environments by spreading repressive
chromatin marks. Since P-TEFb globally
regulates transcription elongation, it is
reasonable to envisage that 7SK snRNA
also functions to shut off transcription
elongation programs genome-wide.
Summary
While our understanding of the mecha-
nistic details is still at an early stage, it
seems that lncRNAs can already be con-
sidered another class of transcription
factor, along with chromatin modifiers,
DNA-binding regulators and other cofac-
tors. Even though the model of lncRNAs
acting as molecular scaffolds is tempting,
only a few examples have been described.
Additionally, the step of the transcription
cycle targeted by certain lncRNAs is cur-
rently unclear. An example is the recent
discovery of lincRNA-p21 and PANDA
which function by assembling with
hnRNPK and the heterotrimeric NF-Y
factor, respectively, to regulate p53-depen-
dent transcriptional responses.
44,97
A
common theme among the lncRNAs
discussed here is that they function as
chromatin-bound regulators. There are
many examples of cis-acting lncRNAs,
although recent discoveries point to others
that act in trans, suggesting both local and
global regulatory roles. However, little is
known about how lncRNAs are targeted
in trans to their loci. Many mechanisms
are possible including targeting to specific
sequences by Watson-Crick base pairing,
directly occupy the promoter surrounding
the TSS, suggesting that inactive P-TEFb
complexes are assembled on the DNA
template (Fig. 1D).
91
The enrichment of
the snRNP proteins is lost downstream
of the TSS in a Tat- and TAR-dependent
manner, suggesting a model in which the
protein-RNA interaction fully displaces
the inhibitory 7SK snRNP, thereby tim-
ing the transition into elongation. It is
currently unclear if the prevailing mech-
anism of P-TEFb recruitment is in its
7SK snRNP-inhibited form or in its pre-
activated state, or whether P-TEFb is also
recruited without 7SK as part of another
complex. Also, it is unknown if there is a
hierarchical order of 7SK snRNP remod-
eling by Tat or if multiple independent
Tat complexes exist with P-TEFb or with
the 7SK snRNA, and how this relates to
TAR binding. Different pools of Tat could
extract P-TEFb from the pre-formed 7SK
snRNP complex to transfer it to TAR,
and could also bind to 7SK RNA to pre-
vent transcription elongation shut off and
favor multiple rounds of transcription
elongation.
7SK snRNA
and Cellular Promoters
7SK snRNA is enriched in nuclear speck-
les, a subnuclear domain rich in pre-
mRNA processing factors, from which it
can be recruited to sites of active transcrip-
tion. In situ hybridization experiments
revealed that 7SK transiently associates
with a stably integrated reporter gene
within minutes of inducing transcriptional
repression and displaces P-TEFb from the
locus. An interesting model is that the
7SK snRNP is dynamically recruited from
nuclear speckles to cellular promoters,
where it can sequester or inhibit P-TEFb
and thereby induce transcriptional elonga-
tion shut off. These results raise the pos-
sibility that 7SK snRNA may be either
bound to a subset of cellular genes along
with the transcription machinery or be
rapidly recruited to promoters to shut
off transcription in response to environ-
mental cues. Recent reports suggest that
7SK snRNA may also be associated with
other factors that in some cases are found
at promoters, including a diverse set of
hnRNP proteins (A1, R, Q and K), cold
phosphorylates the C-terminal domain
(CTD) of RNAP II allowing escape into
productive elongation.
74,75
P-TEFb cata-
lytic activity is tightly controlled in cells
by regulating the equilibrium between
two states: the active P-TEFb form, which
can be recruited to chromatin by interact-
ing with Brd4 and other factors, and an
inactive ribonucleoprotein form, referred
to as 7SK snRNP, containing a 331-nt
RNA known as 7SK snRNA.
76,77
RNase
footprinting and mutagenesis indicate that
7SK contains a high degree of secondary
structure, with stem-loops at both the 5'
and 3' ends.
78-80
5' stem-loop binds P-TEFb
as well as the Hexim1 protein, which acts
to inhibit the kinase activity, while the 3'
stem-loop binds the Larp7/PIP7S protein,
which, in addition to a methylphosphate
capping enzyme (Mepce), stabilizes the
RNA.
76,79,81-86
Even though 7SK does not
seem to tightly bind chromatin, recent
findings link this RNA with the transcrip-
tion machinery and localized regulation at
promoters, implying a more dynamic role
in its recruitment to target genes.
7SK snRNA and the HIV Promoter
The HIV promoter is well known to rely
on P-TEFb for transcription elongation.
The virally encoded Tat protein, which
binds to a 5' RNA stem-loop in HIV tran-
scripts known as TAR (trans-activation
response element) recruits P-TEFb to the
viral promoter to activate the switch from
transcription initiation to elongation.
Another function of Tat is to dismantle
the 7SK snRNP to increase the pool of
P-TEFb and does so by binding to cyclin
T1 and competing with Hexim1.
87
Tat
also binds to the 5' stem-loop of 7SK,
apparently triggering a conformational
change in the RNA, but it is unclear if
RNA-binding is required for P-TEFb
displacement or if it occurs after P-TEFb
and Hexim1 are removed from the 7SK
sn R NP.
83,88-91
A prominent model is that
Tat captures active P-TEFb from a large
pool of inactive 7SK-bound P-TEFb com-
plexes via this competition mechanism and
then delivers it to the HIV promoter by
binding to TAR.
87
Recent ChIP analyses
on an integrated HIV promoter indicate
that the 7SK snRNP proteins Larp7 and
Hexim1, along with cyclin T1 and Cdk9,
Page 6
© 2012 Landes Bioscience.
Do not distribute.
84 Transcription Volume 3 Issue 2
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the formation of DNA-DNA-RNA triple
helical structures as in the case of DHFR
and pRNA, or by contacting factors
already bound to chromatin. HOTAIR
exemplifies that the RNA itself could
recruit and spread the chromatin-remod-
eling complexes to establish the epigenetic
state at its target loci. A systematic and
comprehensive definition of the assembly
of protein factors on selected lncRNAs
will be required to define whether these
RNAs can form alternative complexes on
or off their genomic targets to regulate
gene expression programs. These early
glimpses reveal that lncRNAs operate
throughout the transcription cycle. Even
more, they demonstrate ways in which
RNA and protein can be functionally
interchangeable and how the RNA world
remains with us today.
Acknowledgments
We thank Cheng-Ming Chiang and
Nicholas Conrad for their invaluable
comments and suggestions. We apolo-
gize for the non-comprehensive nature of
this review and to colleagues whose work
could not be cited due to space constraints.
This work was supported by NIH grants
R00AI083087 (I.D.) and GM082250
(A.D.F.).
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  • Source
    • "Altered patterns of epigenetic changes such as DNA methylation and histone modifications are central to many common human diseases including cancer. In recent years, accumulating molecular evidence has revealed that lncRNAs directly bind to the following: polycomb repressive complex 1 (PRC1) and 2 (PRC2) [110], chromatin-modifying proteins such as corepressor of RE1 silencing transcription factor (CoREST) and jumonji AT-rich interaction domain 1C (JARID1C, also known as SMCX) [111]. They may also bind to trithorax chromatin-activating complexes, which guide the site specificity of chromatin-modifying complexes to effect epigenetic changes (Fig. 3) [112]. "
    [Show abstract] [Hide abstract] ABSTRACT: The human genome contains a large number of nonprotein-coding sequences. Recently, new discoveries in the functions of nonprotein-coding sequences have demonstrated that the "Dark Genome" significantly contributes to human diseases, especially with regard to cancer. Of particular interest in this review are long noncoding RNAs (lncRNAs), which comprise a class of nonprotein-coding transcripts that are longer than 200 nucleotides. Accumulating evidence indicates that a large number of lncRNAs exhibit genetic associations with tumorigenesis, tumor progression, and metastasis. Our current understanding of the molecular bases of these lncRNAs that are associated with cancer indicate that they play critical roles in gene transcription, translation, and chromatin modification. Therapeutic strategies based on the targeting of lncRNAs to disrupt their expression or their functions are being developed. In this review, we briefly summarize and discuss the genetic associations and the aberrant expression of lncRNAs in cancer, with a particular focus on studies that have revealed the molecular mechanisms of lncRNAs in tumorigenesis. In addition, we also discuss different therapeutic strategies that involve the targeting of lncRNAs.
    Full-text · Article · Nov 2015 · Tumor Biology
  • Source
    • "LncRNAs can impact genes in the same chromosomal locus or in other chromosomes; however, this review focuses on antisense lncRNAs that modify the expression of neighboring genes. Antisense lncRNAs have been found to act at nearly every level of gene regulation [22,31,55]: pretranscriptionally (Figure 2A), as guides of proteins into specific parts of the genome, as decoys keeping proteins away from chromatin and through epigenetic changes by histone modifications or DNA cytosine methylation [46]; transcriptionally (Figure 2B), conferring modulatory effects in the transcriptional process [56]; and posttranscriptionally (Figure 2C,D), through RNA-RNA interactions that alter mRNA structure or cellular compartmentalization, either in the nucleus or the cytoplasm [31,48]. The versatile regulatory functions of lncRNAs fall into different categories, depending on the interacting partner (Table 1), as follows: lncRNA-DNA, lncRNA–RNA and lncRNA–protein interactions [12]. "
    [Show abstract] [Hide abstract] ABSTRACT: Antisense transcription, considered until recently as transcriptional noise, is a very common phenomenon in human and eukaryotic transcriptomes, operating in two ways based on whether the antisense RNA acts in cis or in trans. This process can generate long non-coding RNAs (lncRNAs), one of the most diverse classes of cellular transcripts, which have demonstrated multifunctional roles in fundamental biological processes, including embryonic pluripotency, differentiation and development. Antisense lncRNAs have been shown to control nearly every level of gene regulation-pretranscriptional, transcriptional and posttranscriptional-through DNA-RNA, RNA-RNA or protein-RNA interactions. This review is centered on functional studies of antisense lncRNA-mediated regulation of neighboring gene expression. Specifically, it addresses how these transcripts interact with other biological molecules, nucleic acids and proteins, to regulate gene expression through chromatin remodeling at the pretranscriptional level and modulation of transcriptional and post-transcriptional processes by altering the sense mRNA structure or the cellular compartmental distribution, either in the nucleus or the cytoplasm.
    Full-text · Article · Feb 2015 · International Journal of Molecular Sciences
  • Source
    • "In vivo, P-TEFb is present in two states: an active P-TEFb form, associated with Brd4 and other factors, and in an inactive ribonucleoprotein from, referred to as 7SK snRNP, containing a 331-nt non-coding RNA known as 7SK snRNA. RNase footprinting and mutagenesis experiments have indicated that 7SK contains a high degree of secondary structure, with stem-loops at both the 5' and 3' ends [96,148149150. The 5' stem loop binds P-TEFb as well as the Hexim1 protein, which acts to inhibit the kinase activity, while the 3' stem-loop binds the Larp7/PIP7S protein, which, in addition to a methylphosphate capping enzyme (Mepce), stabilizes the RNA9596979899151,152]. "
    [Show abstract] [Hide abstract] ABSTRACT: Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs) are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and trans-regulatory roles. Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2) with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research.
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