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Long Noncoding RNAs Xist in Three Dimensions

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Andrew Dimond at University of Oxford
Andrew Dimond
Peter Fraser at Florida State University
Peter Fraser
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The three-dimensional organization of the mammalian genome spatially guides the binding of an RNA to loci for silencing gene expression.
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16 AUGUST 2013 VOL 341 SCIENCE www.sciencemag.org
720
PERSPECTIVES
RNA has traditionally been viewed
as the genomic “messenger” mol-
ecule, carrying the coding informa-
tion from DNA to make proteins. However,
an abundance of short and long noncoding
RNAs also exist, many of which are likely to
have regulatory roles. Little is known about
the mechanisms of action of long noncoding
RNAs, but many appear to act as “guides”
that recruit protein regulatory complexes to
specifi c genomic loci to control gene expres-
sion ( 1). Where these long noncoding RNAs
contact the genome, and how they locate these
regulatory targets, have been open questions.
On page 767 of this issue, Engreitz et al. ( 2)
show that the mouse long noncoding RNA
called X-inactive specifi c transcript (Xist) is
initially targeted to specifi c loci across the X
chromosome using a targeting mechanism
that exploits three-dimensional chromosome
topology.
The Xist RNA orchestrates dosage com-
pensation in female mammalian cells.
Because female mammals have two X chro-
mosomes, inactivating one of them prevents
the expression of twice as many X-chromo-
some genes as in males, which possess only
one X chromosome. In X-inactivation, Xist is
synthesized (17 kilobases in length) from the
X chromosome that will ultimately become
inactivated. It recruits the polycomb repres-
sive complex 2 (PRC2) to switch off gene
expression from one X chromosome in each
female cell ( 3). Eventually, Xist appears to
encapsulate the entire inactive X chromo-
some in a “cloud” (as seen by microscopy),
forming a silencing compartment. The few
genes that escape silencing are seen looping
out of the condensed core ( 4). By develop-
ing and applying a technique to map RNA
interaction sites in the genome, Engreitz et
al. describe these images at a genomic level.
Consistent with microscopic observations,
the authors found that Xist interaction sites
are distributed broadly across the X chromo-
some in mouse fi broblasts, whereas “escape”
genes showed reduced association with Xist.
However, the most interesting questions
relate to how Xist establishes this pattern
of binding during the initiation of X-chro-
mosome inactivation. To investigate these
early events, Engreitz et al. used an induc-
ible system in mouse embryonic stem cells
that allowed them to activate Xist expres-
sion and map Xist interaction sites at defi ned
time points. They observed that Xist does not
spread uniformly outwards along the chro-
mosome sequence, but rather appears to pref-
erentially “jump” from its site of transcription
to certain early binding sites.
One mechanism to explain how Xist could
target specifi c distal loci would be the pres-
ence of high-affi nity binding sites at these
locations. Intriguingly, Engreitz et al. could
not identify any genomic feature to explain
the pattern of Xist early binding. Instead, the
authors considered the three-dimensional
organization of the genome by using pub-
lished data from “Hi-C” ( 5), a high-through-
put “chromosome conformation capture”
technology that allows identifi cation of DNA
sequences in close proximity to each other
within the nucleus. The authors discovered
that the early binding sites for Xist correspond
to loci that are spatially close to the Xist tran-
scription site. This suggested that Xist might
fi nd its targets by binding sequences nearby
in space, rather than those that are close along
the linear sequence. Importantly, the authors
tested their hypothesis by expressing Xist
from a different position on the X chromo-
some and found a new pattern of Xist early
binding sites that corresponded to sites in
close spatial proximity to the new location.
From these early binding sites, Engreitz
et al. show that Xist requires its A-repeats to
spread across and silence active genes. The
A-repeats are required for PRC2 recruitment
( 6) and for repositioning active genes into the
Xist silencing compartment ( 4). A model is
thus proposed whereby the three-dimensional
chromosome conformation is exploited to
extrude Xist onto its early binding site targets
where it then helps to modify and reorganize
the X-chromosome architecture.
Several key questions remain regarding
the secondary spreading of Xist and con-
trol mechanisms to prevent inappropriate
spreading across genes that escape inactiva-
tion or from transferring to other chromo-
somes. Engreitz et al. found sharp transitions
of Xist binding surrounding escape genes,
which may relate to previous work implicat-
ing CCCTC-binding factor (CTCF) in block-
ing Xist spreading ( 7). In addition to tran-
scription, CTCF controls chromatin archi-
tecture by binding together strands of DNA
and forming chromatin loops. CTCF binding
sites that are adjacent to escape genes may
play a role in segregating these regions from
Xist binding. Preventing spread to other chro-
mosomes requires the nuclear matrix protein
heterogeneous nuclear ribonucleoprotein U
(hnRNP U) ( 8), which may facilitate transfer
of Xist from the transcription site to contact-
ing regions. Although hnRNP U is located
throughout the nucleus, differences in con-
Long Noncoding RNAs Xist
in Three Dimensions
MOLECULAR BIOLOGY
Andrew Dimond and Peter Fraser
The three-dimensional organization of the
mammalian genome spatially guides the
binding of an RNA to loci for silencing gene
expression.
?
?
?
X
i
s
t
X chromosome
Long
noncoding
RNA
Nucleus
Chromosomes
Organizing silence. The Xist long noncoding RNA
binds to sites in close spatial proximity on the X chro-
mosome. A mechanism based on the three-dimen-
sional genome architecture could be a general strat-
egy for targeting long noncoding RNAs.
CREDIT: V. ALTOUNIAN/SCIENCE
Nuclear Dynamics Programme, The Babraham Institute,
Cambridge, UK. E-mail: peter.fraser@babraham.ac.uk
Published by AAAS
on February 6, 2015www.sciencemag.orgDownloaded from on February 6, 2015www.sciencemag.orgDownloaded from
www.sciencemag.org SCIENCE VOL 341 16 AUGUST 2013 721
PERSPECTIVES
The supply, storage, and fl ow of water
under an ice sheet are crucial for its
overall dynamics. The density differ-
ence between ice and water limits subglacial
water pressure to the pressure from overlying
ice. Near this limit, the ice gradually becomes
decoupled from the base (see the figure),
water-filled cavities form, and sediments
become weakened. The velocity of ice mov-
ing over the bedrock increases due to reduced
friction and faster sediment deformation.
Subglacial water pressure is therefore the key
parameter that controls basal motion, ice fl ux,
and the future evolution of an ice sheet. On
page 777 of this issue ( 1), Meierbachtol et al.
present a detailed record of water pressure
variability under one region of the Greenland
ice sheet.
Glacier hydrology has been investigated in
great detail on mountain glaciers, providing
insights into their drainage over the course
of the melt season ( 2– 5). However, similar
measurements have been mostly missing on
the ice sheets. Only very recently, dye-trac-
ing experiments provided information on the
evolution of the hydraulic properties of sub-
glacial drainage ( 6, 7) and their link to surface
velocity variations ( 8). The reason for the
sparse observational data are the diffi culty
of investigating an extended karst-like sys-
tem that can change its characteristics within
days and that is drained by big, violent, and
sediment-laden rivers. Some of the biggest
such rivers are invisible as they emerge from
the ice sheet at great depth in deeply incised
fjords fi lled with icebergs. Directly explor-
ing the under-ice environment is a diffi cult
endeavor because of thick ice, rapidly freez-
ing boreholes, and ice fl ow that can stretch
and rip instrumentation cables.
Meierbachtol et al. explore subglacial con-
ditions on a transect from the Greenland ice
sheet’s margin to its interior, sampling water
pressure at several drill sites. Their main fi nd-
ing is the different characteristics of subgla-
cial water drainage between marginal and
inland areas of the ice sheet. Whereas bore-
holes close to the ice sheet margin showed
large diurnal variations of water pressure, the
inland sites showed persistent high water pres-
sure with small fl uctuations. Their interpreta-
tion is that an effi cient system of high-capac-
ity channels exists under the marginal parts of
Gauging Greenland’s
Subglacial Water
GEOPHYSICS
Martin Lüthi
Subglacial water fl ow regimes differ between
the interior and the margins of the Greenland
Ice Sheet.
CREDIT: MARTIN LÜTHI
Laboratory of Hydraulics, Hydrology and Glaciology (VAW),
ETH Zürich, 8093 Zürich, Switzerland. E-mail: luethi@vaw.
baug.ethz.ch
Freshly exposed, glacially carved bedrock. Sediment-covered areas and solid bedrock form the base of the
Greenland ice sheet. Scratch marks from rocks dragged along this freshly exposed bedrock in West Greenland
illustrate the sliding motion of ice over its base. Meierbachtol et al. provide insights into the mechanisms of
water fl ow, which controls sliding motion.
tact frequency between cis-linked loci and
interchromosomal interactions may inhibit
Xist from establishing a foothold on other
chromosomes. The biggest gaps still to fi ll
are details that explain how Xist eventually
encompasses the entire chromosome. Does
Xist spread from early sites into adjacent
sites by diffusion or transfer across a fairly
static chromosome structure, or do chromo-
some dynamics allow other binding sites to
iteratively contact and collect RNA from the
Xist locus to eventually produce the broad
distribution seen in all cells? Conformational
studies at later time points of X-inactivation
or live-cell studies could potentially provide
a timeline to fi ll these gaps in RNA and chro-
matin dynamics.
Other long noncoding RNAs ( 9– 11) have
been described that exert their regulatory
effects by exploiting the three-dimensional
folding of the genome. The similarities with
Xist suggest that this may be a general tar-
geting mechanism (see the figure). It will
be interesting to see if other, trans-acting
noncoding RNAs (both long and short) also
exploit three-dimensional genome organiza-
tion to target loci on other chromosomes ( 12,
13). With tools to map RNA binding sites and
describe genome conformation now at hand,
answers to these questions are within reach.
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10.1126/science.1243257
Published by AAAS
DOI: 10.1126/science.1243257
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  • Apr 2012
  • NATURE
The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term 'topological domains', as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.
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Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs
Article
Full-text available
  • Apr 2012
  • NUCLEIC ACIDS RES
The recent discovery that the human and other mammalian genomes produce thousands of long non-coding RNAs (lncRNAs) raises many fascinating questions. These mRNA-like molecules, which lack significant protein-coding capacity, have been implicated in a wide range of biological functions through diverse and as yet poorly understood molecular mechanisms. Despite some recent insights into how lncRNAs function in such diverse cellular processes as regulation of gene expression and assembly of cellular structures, by and large, the key questions regarding lncRNA mechanisms remain to be answered. In this review, we discuss recent advances in understanding the biology of lncRNAs and propose avenues of investigation that may lead to fundamental new insights into their functions and mechanisms of action. Finally, as numerous lncRNAs are dysregulated in human diseases and disorders, we also discuss potential roles for these molecules in human health.
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LincRNAs act in the circuitry controlling pluripotency and differentiation
Article
Full-text available
  • Aug 2011
  • NATURE
Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.
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A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression
Article
Full-text available
  • Mar 2011
  • NATURE
The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.
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Polycomb Proteins Targeted by a Short Repeat RNA to the Mouse X Chromosome
Article
Full-text available
  • Nov 2008
  • SCIENCE
To equalize X-chromosome dosages between the sexes, the female mammal inactivates one of her two X chromosomes. X-chromosome inactivation (XCI) is initiated by expression of Xist, a 17-kb noncoding RNA (ncRNA) that accumulates on the X in cis. Because interacting factors have not been isolated, the mechanism by which Xist induces silencing remains unknown. We discovered a 1.6-kilobase ncRNA (RepA) within Xist and identified the Polycomb complex, PRC2, as its direct target. PRC2 is initially recruited to the X by RepA RNA, with Ezh2 serving as the RNA binding subunit. The antisense Tsix RNA inhibits this interaction. RepA depletion abolishes full-length Xist induction and trimethylation on lysine 27 of histone H3 of the X. Likewise, PRC2 deficiency compromises Xist up-regulation. Therefore, RepA, together with PRC2, is required for the initiation and spread of XCI. We conclude that a ncRNA cofactor recruits Polycomb complexes to their target locus.
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Gracefully ageing at 50, X-chromosome inactivation becomes a paradigm for RNA and chromatin control
Article
  • Nov 2011
  • NAT REV MOL CELL BIO
The discovery of X-chromosome inactivation (XCI) celebrated its golden anniversary this year. Originally offered as an explanation for the establishment of genetic equality between males and females, 50 years on, XCI presents more than a curious gender-based phenomenon that causes silencing of sex chromosomes. How have the mysteries of XCI unfolded? And what general lessons can be extracted? Several of the cell biological mechanisms that are used to establish the inactive X chromosome, including regulatory networks of non-coding RNAs and unusual nuclear dynamics, are now suspected to hold true for processes occurring on a genome-wide scale.
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The Matrix Protein hnRNP U Is Required for Chromosomal Localization of Xist RNA
Article
  • Sep 2010
In XX female mammals, one of the two X chromosomes is epigenetically inactivated to equalize gene expression with XY males. The formation of the inactive X chromosome (Xi) is regulated by an X-linked long noncoding RNA Xist, which accumulates on the entire length of the chromosome in cis and induces heterochromatin formation. However, the mechanism by which Xist RNA "paints" the Xi has long remained elusive. Here, we show that a matrix protein hnRNP U/SP120/SAF-A is required for the accumulation of Xist RNA on the Xi. Xist RNA and hnRNP U interact and upon depletion of hnRNP U, Xist RNA is detached from the Xi and diffusely localized into the nucleoplasm. In addition, ES cells lacking hnRNP U expression fail to form the Xi. We propose that the association with matrix proteins is an essential step in the epigenetic regulation of gene expression by Xist RNA.
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