|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 14 January 2009
doi: 10.1242/dev.031328
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research Report |
1 Laboratory of Developmental Genetics and Imprinting, The Babraham Institute,
Cambridge CB22 3AT, UK.
2 Laboratory of Chromatin and Gene Expression, Epigenetics and Chromatin
Programme, The Babraham Institute, Cambridge CB22 3AT, UK.
3 MRC Clinical Sciences Centre, Imperial College London, London W12 0NN,
UK.
4 Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG,
UK.
Author for correspondence (e-mail:
wolf.reik{at}bbsrc.ac.uk)
Accepted 17 December 2008
SUMMARY
Long noncoding RNAs are implicated in a number of regulatory functions in eukaryotic genomes. The paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1 regulates epigenetic gene silencing in an imprinted gene cluster in cis over a distance of 400 kb in the mouse embryo, whereas the silenced region extends over 780 kb in the placenta. Gene silencing by the Kcnq1ot1 RNA involves repressive histone modifications, including H3K9me2 and H3K27me3, which are partly brought about by the G9a and Ezh2 histone methyltransferases. Here, we show that Kcnq1ot1 is transcribed by RNA polymerase II, is unspliced, is relatively stable and is localised in the nucleus. Analysis of conditional Dicer mutants reveals that the RNAi pathway is not involved in gene silencing in the Kcnq1ot1 cluster. Instead, using RNA/DNA FISH we show that the Kcnq1ot1 RNA establishes a nuclear domain within which the genes that are epigenetically inactivated in cis are frequently found, whereas nearby genes that are not regulated by Kcnq1ot1 are localised outside of the domain. The Kcnq1ot1 RNA domain is larger in the placenta than in the embryo, consistent with more genes in the cluster being silenced in the placenta. Our results show for the first time that autosomal long ncRNAs can establish nuclear domains, which might create a repressive environment for epigenetic silencing of adjacent genes. Long ncRNAs in imprinting clusters and the Xist RNA on the inactive X chromosome thus appear to regulate epigenetic gene silencing by similar mechanisms.
Key words: Long noncoding RNA, Imprinting, X inactivation, Epigenetic gene silencing, Kcnq1ot1
INTRODUCTION
Noncoding RNAs (ncRNAs) have key roles in the regulation of complex genome
functions and plasticity in multicellular organisms
(Amaral and Mattick, 2008
).
Several classes of short ncRNAs (such as miRNAs, siRNAs and piRNAs) regulate
not only mRNA stability and translation, but also transcription through
targeting of epigenetic marks, including histone modifications and DNA
methylation (Neilson and Sharp,
2008). Long ncRNAs are less well characterised, but include
antisense and other intergenic transcripts, pervasive genomic transcription
and epigenetic regulators (Umlauf et al.,
2008). The long ncRNA Xist, which is transcribed from the inactive
X chromosome, is a well-studied example of a RNA with a crucial role in
epigenetic gene silencing of a chromosome region in cis. The Xist RNA is
transcribed by RNA polymerase II (Pol II), is spliced and polyadenylated, and
located in the nucleus in a defined three-dimensional (3D) domain into which
the genes along the inactive X chromosome appear to be recruited. X-linked
genes that escape inactivation remain located outside the nuclear domain
defined by the Xist RNA. Coating of the inactive X chromosome by Xist is
associated with exclusion of Pol II from the silencing compartment,
acquisition of repressive histone modifications and CpG island methylation,
thus establishing a stable and heritable repressive chromatin environment
(Heard and Bickmore, 2007;
Payer and Lee, 2008;
Wutz and Gribnau, 2007).
Long ncRNAs are also found associated with epigenetic regulation outside
the X chromosome, namely Kcnq1ot1, Air and Nespas, which are implicated in
epigenetic gene silencing in imprinting clusters
(Peters and Robson, 2008).
These RNAs are paternally expressed and maternally silenced by DNA methylation
of their promoters established in the female germline. By insertion of
premature polyA stop signals, Kcnq1ot1 and Air have been found to be crucial
for inactivation in cis of imprinted genes
(Mancini-Dinardo et al., 2006;
Shin et al., 2008;
Sleutels et al., 2002).
Transcription of Kcnq1ot1 was found to be necessary for the acquisition of
repressive epigenetic markers, such as H3K9me2, H3K27me3 and DNA methylation
(Fitzpatrick et al., 2002;
Lewis et al., 2004;
Umlauf et al., 2004). Indeed,
in mouse mutants of Dnmt1, Eed (part of the polycomb repressive complex 2,
which includes Ezh2) or G9a, loss of imprinting of different sets of genes in
the Kcnq1ot1 cluster has been observed
(Lewis et al., 2004;
Mager et al., 2003;
Wagschal et al., 2008),
consistent with largely separate roles of DNA and histone methylation, but
partly overlapping roles of H3K9 and H3K27 methylation, in epigenetic gene
silencing.
In parallel with X inactivation, the Kcnq1ot1 RNA is first paternally
expressed in two-cell embryos, and inactivation of the cis linked genes is
initiated in preimplantation and early postimplantation embryos
(Lewis et al., 2006;
Green et al., 2007). In
placentae, the silenced domain extends over 780 kb, whereas in the embryo it
is considerably smaller (400 kb; Fig.
1A) (Lewis et al.,
2004; Umlauf et al.,
2004).
The next key step in our understanding of how imprinted long ncRNAs confer
epigenetic gene silencing in cis will come from more precise insights into the
molecular pathways involved. It is important to test whether RNAi contributes
to imprinted silencing, particularly given recent work that has implicated the
RNAi pathway in X inactivation (Ogawa et
al., 2008). Alternatively, or additionally, long ncRNAs in
imprinting clusters may act in the nucleus and establish silencing domains
akin to that established by Xist for X inactivation. Here, we test both of
these models for the Kcnq1ot1 imprinting cluster.
|
Mouse strains and cell lines
All experimental procedures were conducted under licences by the Home
Office (UK) in accordance with the Animals (Scientific Procedures) Act 1986.
We used C57BL6/JOlaHsd or SD7 as wild-type strains. SD7 contains the distal
region of Mus spretus chromosome 7 backcrossed into the F1
(C57BL/6J/CBA/Ca), which provides SNPs to distinguish between parental
alleles. KvDMR1 mice have been described previously
(Fitzpatrick et al., 2002).
Dicerlox/lox mice (Cobb
et al., 2005) were crossed with
MeoxCre/+
(Tallquist and Soriano, 1999)
mice to generate MeoxCre/+;
Dicer
/+ mice. These mice were then crossed with
Dicerlox/lox; SD7/SD7 mice to generate
MeoxCre/+;
Dicer/; SD7/B6 embryos. Yolk sac DNA was
used for genotyping (primer details can be provided on request). Primary mouse
embryonic fibroblasts (MEFs) were obtained from E13.5 C57BL6/JOlaHsd embryos
and cultured in DMEM (Invitrogen) with 10% FBS, 1% penicillin/streptomycin and
2 mM L-glutamine. For RNA stability experiments, NIH3T3 cells were cultured in
medium containing 0.001% saponin±5 µg/ml 
-amanitin, and
analysed as previously described (Seidl et
al., 2006).
RNA isolation, reverse transcription, RACE and Sequenom MassArray
Total RNA was isolated from cells and embryos using Trizol (Applied
Biosystems) or AllPrep DNA/RNA Mini kit (Qiagen). Nuclear and cytoplasmic RNA
was isolated from MEFs using the PARIS kit (Applied Biosystems) and spiked
with rabbit -globin mRNA (Sigma, Dorset, UK) as an internal control.
RNA was reverse transcribed using either Superscript II (Invitrogen) or BD
Sprint PowerScript (BD Biosciences) using either oligo-dT, random primers
(Promega) or specific primers for strand-specific analysis. For miR-16
expression analysis, small RNAs were isolated using the RNeasy MinElute
cleanup kit (Qiagen) and cDNA was made using SnoRNA-202 and miR-16 reverse
transcription kits (Applied Biosystems). 5' and 3' RACE were
carried out using the GeneRacer RACE Ready cDNA Kit (Invitrogen).
Allele-specific expression analysis was performed with iPLEX chemistry on the
SEQUENOM genotyping platform, as per the manufacturer's instructions. Primer
sequence details can be provided on request.
Quantitative PCR (qPCR)
To analyse the length of the Kcnq1ot1 transcript, qPCR was performed using
Platinum SYBR Green qPCR SuperMix-UDG with Rox (Invitrogen) on an ABI Prism
7700 Sequence Detector. cDNA (5 µl, diluted 1:50) were used per reaction
(primer details can be provided on request). Reference RNAs were produced by
in vitro transcription of cloned PCR products and quantified using RiboGreen
(Invitrogen). The respective cDNAs were used to generate a standard curve
allowing quantitative comparison between amplicons. For miR-16 expression
analysis, the SnoRNA-202 and miR-16 qPCR kits (TaqMan) were used on an Mx3005
QPCR System (Stratagene).
Fluorescence in situ hybridisation (FISH) probes
Probes for RNA-FISH (details can be provided on request) were labelled with
digoxigenin by nick-translation (Roche Diagnostics). The Kcnq1 probe is
intronic and located outside of Kcnq1ot1, and so will only detect Kcnq1
transcripts, but the Kcnq1ot1 probe will also detect Kcnq1. However, RNA-FISH
on KvDMR1-knockout embryo sections using the Kcnq1ot1 probe showed only
background levels of fluorescent signals (not shown). The Xist probe is also
capable of detecting both the Xist and Tsix transcripts, but at the
developmental time points in our study Tsix is silent
(Sado et al., 2001). For
DNA-FISH, fosmids (BACPAC Resources; details can be provided on request) were
labelled with biotin by nick-translation (Roche).
RNA-FISH
Frozen E12.5 embryos and placentas were embedded in Cryo-M-Bed (Bright)
prior to 14 µm sectioning with a cryostat (Bright). Sections were collected
on poly-lysine-coated slides and stored at -80°C. Sections were briefly
thawed, permeabilised with 0.5% TX in PBS for 8 minutes on ice, fixed in 4%
formaldehyde/5% acetic acid/0.14 M NaCl for 18 minutes at room temperature,
washed in PBS, dehydrated in 70-100% ethanol series and air-dried. Probes (2.5
µl) were co-precipitated with 1 µg of mouse Cot1 DNA and resuspended in
40 µl of hybridisation mix (Chakalova et
al., 2004). Probe denaturation, hybridisation, washes and probe
detection were performed as described previously
(Chakalova et al., 2004); the
following antibodies were used: sheep anti-digoxigenin (1:1000; Roche)
followed by FITC-conjugated rabbit anti-sheep (1:200; Calbiochem) and
AlexaFluor 488-conjugated donkey anti-rabbit (1:200; Invitrogen).
Combined RNA/DNA-FISH
After RNA-FISH as above, antibodies were fixed with 4% formaldehyde in PBS
for 30 minutes at room temperature. Sections were treated with 0.1 M HCl for
20 minutes, washed in PBS, equilibrated in 70% formamide/2xSSC before
denaturing for 5 minutes at 80°C (in pre-heated 70%
formamide/2xSSC/0.1 M phosphate buffer pH 7.0), and then cooled down in
ice-cold 50% formamide/2xSSC. Probes were precipitated, re-suspended in
hybridisation buffer and denatured as previously described
(Noordermeer et al., 2008),
and hybridised to denatured sections overnight at 37°C. Post-hybridisation
washes were performed as previously described
(Branco and Pombo, 2006).
Biotin was detected using an AlexaFluor 555-conjugated streptavidin
(Invitrogen; 1:500); blocking, antibody dilutions and washes were carried out
in 1% BSA in PSB/0.05% Tween-20.
Image collection and analysis
Optical z-stacks were captured using a Zeiss LSM 510 META
(63x Plan-Apo 1.40 objective) or Olympus FV1000 (63x Plan-superApo
1.40 objective) confocal microscopes.
For analysis of the RNA-FISH signals, Volocity (v4, Improvision) was used for volume rendering of the image z-stacks using the HR opacity renderer. RNA-FISH signal volumes were also measured in Volocity using intensity-based thresholding; signals not touching DAPI stained objects or with a volume less than 0.1 µm3 were excluded.
Distances between DNA-FISH signals and the edge of the Kcnq1ot1 domain were measured in 3D using a custom ImageJ (Wayne Rasband, NIH) macro. Briefly, RNA-FISH signals were segmented using an adaptive threshold algorithm and the 3D coordinates of the centre of mass of the DNA-FISH signals were found; the distance from each centre of mass to the nearest RNA domain edge was automatically measured.
RESULTS AND DISCUSSION
Kcnq1ot1 RNA is nuclear and moderately stable
We determined the size and characteristics of the Kcnq1ot1 transcript by
EST searches, 5' and 3' RACE (rapid amplification of cDNA ends)
and RT-PCR (Fig. 1A,B; see Fig.
S1 in the supplementary material). The RACE experiments provided four
transcription start sites within 20 bp and a polyadenylation site 121 kb
downstream of the main start site, showing that the Kcnq1ot1 RNA has both a
5' methyl cap and a polyadenylated tail. All ESTs were contiguous with
the genomic sequence, providing no evidence for splicing. We found by
quantitative RT-PCR (qRT-PCR) that the main region of transcription extends to
about 80 kb from the start site and is dependent on the Kcnq1ot1
promoter (Fig. 1B), but
strand-specific RT-PCR showed that transcription from this promoter can still
be detected up to 120 kb into the gene (see Fig. S1 in the supplementary
material). The decrease in the level of Kcnq1ot1 RNA along the length of the
transcription unit might be the result of several unmapped polyadenylation
sites within the transcript; alternatively, transcription might be impeded by
a non-permissive chromatin structure. By comparison, the Air RNA is 108 kb
long and largely unspliced (Lyle et al.,
2000; Seidl et al.,
2006), whereas the Xist RNA is spliced from 23 kb to 17.9 kb, and
this splicing may be needed for Xist-mediated epigenetic silencing
(Ciaudo et al., 2006).
Quantification of nuclear and cytoplasmic RNA from primary MEFs showed that
Kcnq1ot1 RNA is significantly enriched in the nucleus relative to the
cytoplasm (Fig. 1C). To
determine the half-life of the Kcnq1ot1 RNA, NIH 3T3 cells were treated with
the fungal toxin -amanitin, at a concentration known to inhibit Pol II
but not Pols I or III. Kcnq1ot1 RNA was found to have a half-life of 3.4
hours, whereas the mRNA of the protein-coding gene Kcnq1 has a
half-life of over 20 hours (see Fig. S2 in the supplementary material). The
stability of Kcnq1ot1 is similar to that of the ncRNAs Air and Xist, which
have half-lives of 2.1 and 4.6 hours, respectively
(Seidl et al., 2006). The
stability of the Xist RNA is regulated by the NMD pathway
(Ciaudo et al., 2006); whether
this is also the case for Kcnq1ot1 or Air is not known. In summary, Kcnq1ot1
is transcribed by Pol II, localised in the nucleus and moderately stable, thus
resembling other long ncRNAs with epigenetic roles
(Umlauf et al., 2008).
Imprinting in the Kcnq1ot1 domain is not regulated by the RNAi pathway
Our observation that Kcnq1ot1 is a nuclear unspliced transcript suggests
that the RNA itself may have regulatory roles in epigenetic gene silencing.
Recently, it has been suggested that the RNAi pathway plays a role in X
inactivation through the nuclear noncoding transcript Xist
(Ogawa et al., 2008). We
therefore tested genetically whether the RNAi pathway was involved in
regulating imprinting at the Kcnq1ot1 cluster by using mouse mutants for the
crucial RNAi enzyme Dicer (Bernstein et
al., 2001).
Dicer knockout embryos die at around E7.5
(Bernstein et al., 2003), thus
hampering a comprehensive analysis of imprinting across the locus. Instead, we
made use of a conditional knockout of Dicer
(Cobb et al., 2005), which we
specifically deleted in the epiblast (from E5.5) using a Cre
transgene driven by the Meox promoter
(Tallquist and Soriano, 1999).
Deletion of Dicer in the embryonic lineage resulted in lethality at
around E11, with characteristic malformations of the embryonic vasculature and
oedema (E.R.P., B.S.C. and Wendy Dean, unpublished). The expression levels of
miR-16 were substantially reduced in Dicer-null embryos when compared
with heterozygous control littermates, showing that Dicer function had been
effectively ablated in these knockout embryos
(Fig. 2A). We also attempted to
delete Dicer in the placental lineage with a Cyp19Cre transgene
(Wenzel and Leone, 2007), but
this did not result in significant deletion of the floxed Dicer
allele (not shown). If the RNAi pathway is involved in maintaining repression
of the maternal Kcnq1ot1 promoter, we would expect maternal Kcnq1ot1
expression in the Dicer knockout, and perhaps downregulation of the
adjacent imprinted genes on the maternal allele. Conversely, if Dicer is
needed for silencing via the Kcnq1ot1 RNA of the imprinted genes in the
cluster on the paternal chromosome, we would expect elevated transcription of
these genes from their paternal alleles in the knockout.
We analysed the relative allelic expression of four imprinted genes in the
cluster, including Kcnq1ot1, by RT-PCR followed by Sequenom
MassArray, comparing control with mutant embryos
(Fig. 2B). Additionally, we
also analysed six biallelically expressed genes (not shown). The profile of
relative allelic expression determined by MassArray corresponded closely to
that determined previously by allele-specific RT-PCR
(Lewis et al., 2004). No
differences were found between control and mutant embryos, showing that
Dicer-dependent siRNA pathways are not involved in maintaining epigenetic gene
silencing in the Kcnq1ot1 cluster (Fig.
2B; data not shown). However, given that Dicer is expressed in the
conditional knockout embryos before E5.5, we cannot exclude the possibility
that the siRNA pathway is necessary for the establishment of silencing, after
which a Dicer-independent maintenance mechanism would take over. Dicer has
been implicated in Xist-dependent X inactivation in one study
(Ogawa et al., 2008), whereas
another study has found no evidence of an involvement of the siRNA pathway in
X inactivation (Nesterova et al.,
2008).
The Kcnq1ot1 RNA establishes a lineage-specific nuclear domain for silenced genes
The Xist RNA has been shown by RNA FISH to produce a large nuclear signal
that overlaps with most of the inactive X chromosome, and is thus thought to
be coating the chromosome that it inactivates
(Clemson et al., 1996;
Chaumeil et al., 2006). By
contrast, the primary transcripts of protein-coding genes show a small nuclear
signal at the site of transcription
(Osborne et al., 2004). As we
found that Kcnq1ot1 RNA is localised in the nucleus, and apparently does not
work through the RNAi pathway, we tested the alternative hypothesis that the
RNA might physically coat the chromatin that it silences.
|
|
2 test;
Fig. 3B). As expected, the
Kcnq1 probe produced small signals characteristic of primary transcription
(Osborne et al., 2004),
whereas the Xist probe produced a large signal, which is known to be covering
the majority of the inactive X chromosome
(Clemson et al., 1996).
Interestingly, the Kcnq1ot1 probe produced signals of intermediate size
between Kcnq1 and Xist. Indeed, only 5.6% of the Kcnq1 probe signal volumes
were greater than 0.4 µm3, whereas 34.4% of the Kcnq1ot1 probe
signals and 35.7% of the Xist probe signals exceeded a volume of 0.4
µm3. In addition, 14.6% of the Xist probe signals were greater
than 2.0 µm3 compared with 1.6% of the Kcnq1ot1 probe signals.
As the Kcnq1ot1 silenced domain is 780 kb in the placenta and 400 kb in the
embryo, we compared the signal volumes of the RNA between the two tissues
(Fig. 3C). Strikingly, the
volumes occupied by the Kcnq1ot1 RNA in the embryo were significantly smaller
than those in the placenta (P<0.0001, 2 test),
consistent with a model in which the RNA establishes a physical compartment in
the nucleus associated with the chromosomal domain that is silenced.
In order to test this model more directly, we carried out combined
RNA/DNA-FISH experiments on E12.5 embryo and placental sections, in which the
Kcnq1ot1 RNA domain was visualised together with gene loci regulated by
Kcnq1ot1 within the cluster (Cdkn1c), at the edge of the cluster
(Ascl2) and at a locus not regulated by Kcnq1ot1 located just outside
the cluster (Igf2) (Fig.
4A). The distance of each of the three DNA probes (
40 kb in
size) to the edge of the RNA domain was measured in 3D
(Fig. 4B). Strikingly, both in
embryo and placenta the RNA signal frequently overlapped the position of
Cdkn1c, which was found mostly inside of the domain, whereas
Igf2 was predominantly located outside
(Fig. 4). In the placenta, the
positions of all three genes were significantly different from each other,
with Ascl2 assuming a position close to the edge of the domain.
Although we did not find a significant difference in Ascl2 position
between the placenta (where it is imprinted) and the embryo (where it is not
imprinted), interestingly, in the latter, the position of Ascl2 did
not differ significantly from that of the adjacent Igf2 gene, which
is not regulated by Kcnq1ot1. This suggests that the local chromatin structure
is more compact in the embryo, and that the proximity between Ascl2
and the Kcnq1ot1 RNA domain in the embryo may not be functionally relevant.
Our data suggest that Kcnq1ot1 forms a domain where genes located inside have
the potential to be epigenetically inactivated, whereas genes located outside
of this domain do not. Importantly, work in human fibroblasts
(Murakami et al., 2007) and
mouse E14.5 placenta (Pandey et al.,
2008) provides evidence that the Kcnq1ot1 RNA associates with
chromatin rather than forming an independent RNA cluster.
|
;
Nesterova et al., 2008;
Nagano et al., 2008) (this
study). The Kcnq1ot1 RNA domain may interact with chromatin and also with G9a
(Pandey et al., 2008) and
Ezh2, resulting in cluster-wide repressive histone marks, gene silencing and
DNA methylation of CpG islands in some promoters (e.g. Cdkn1c), with
methylation being more pronounced in the embryo than in the placenta. Paternal
expression of Kcnq1ot1 and Xist begins in the two-cell embryo, and the
dynamics of inactivation of individual genes is developmentally regulated and
differs between the embryonic and extra-embryonic lineages [most dramatically
for the X chromosome, where erasure of imprinted X inactivation in ICM cells
in the blastocyst is followed by random inactivation in the epiblast
(Mak et al., 2004;
Okamoto et al., 2004)],
consistent in the case of Kcnq1ot1 with the lineage-specific differences in
the organisation of its nuclear silencing domain. Our observations, together
with those of others (Pandey et al.,
2008; Terranova et al.,
2008; Nagano et al.,
2008), establish that the epigenetic mechanisms regulating X
chromosome inactivation by Xist and clustered genomic imprinting by long
ncRNAs share fundamental similarities.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/525/DC1
Footnotes
We thank Matthew Turley, Wendy Dean and Guillaume Smits for help and advice. This work was funded by the BBSRC, MRC and the EU NoE The Epigenome. M.R.B. is funded by FCT (Portugal) and C.K. by EMBO and the DFG. Deposited in PMC for release after 6 months.
* Present address: CRUK London Research Institute, London WC2A 3PX, UK 
Present address: The Royal Veterinary College, London NW1 0TU, UK
REFERENCES
Amaral, P. P. and Mattick, J. S. (2008).
Noncoding RNA in development. Mamm. Genome
19,454
-492.[CrossRef][Medline]
Bernstein, E., Caudy, A. A., Hammond, S. M. and Hannon, G.
J. (2001). Role for a bidentate ribonuclease in the
initiation step of RNA interference. Nature
409,363
-366.[CrossRef][Medline]
Bernstein, E., Kim, S. Y., Carmell, M. A., Murchison, E. P.,
Alcorn, H., Li, M. Z., Mills, A. A., Elledge, S. J., Anderson, K. V. and
Hannon, G. J. (2003). Dicer is essential for mouse
development. Nat. Genet.
35,215
-217.[CrossRef][Medline]
Branco, M. R. and Pombo, A. (2006).
Intermingling of chromosome territories in interphase suggests a role in
translocations and transcription-dependent associations. PLoS
Biol. 4,e138
.[CrossRef][Medline]
Chakalova, L., Carter, D. and Fraser, P.
(2004). RNA fluorescence in situ hybridization tagging and
recovery of associated proteins to analyze in vivo chromatin interactions.
Methods Enzymol. 375,479
-493.[Medline]
Chaumeil, J., Le Baccon, P., Wutz, A. and Heard, E.
(2006). A novel role for Xist RNA in the formation of a
repressive nuclear compartment into which genes are recruited when silenced.
Genes Dev. 20,2223
-2237.
Ciaudo, C., Bourdet, A., Cohen-Tannoudji, M., Dietz, H. C.,
Rougeulle, C. and Avner, P. (2006). Nuclear mRNA degradation
pathway(s) are implicated in Xist regulation and X chromosome inactivation.
PLoS Genet. 2,e94
.[CrossRef][Medline]
Clemson, C. M., McNeil, J. A., Willard, H. F. and Lawrence, J.
B. (1996). XIST RNA paints the inactive X chromosome at
interphase: evidence for a novel RNA involved in nuclear/chromosome structure.
J. Cell Biol. 132,259
-275.
Cobb, B. S., Nesterova, T. B., Thompson, E., Hertweck, A.,
O'Connor, E., Godwin, J., Wilson, C. B., Brockdorff, N., Fisher, A. G., Smale,
S. T. and Merkenschlager, M. (2005). T cell lineage choice
and differentiation in the absence of the RNase III enzyme Dicer.
J. Exp. Med. 201,1367
-1373.
Fitzpatrick, G. V., Soloway, P. D. and Higgins, M. J.
(2002). Regional loss of imprinting and growth deficiency in mice
with a targeted deletion of KvDMR1. Nat. Genet.
32,426
-431.[CrossRef][Medline]
Green, K., Lewis, A., Dawson, C., Dean, W., Reinhart, B.,
Chaillet, J. R. and Reik, W. (2007). A developmental window
of opportunity for imprinted gene silencing mediated by DNA methylation and
the Kcnq1ot1 noncoding RNA. Mamm. Genome
18, 32-42.[CrossRef][Medline]
Heard, E. and Bickmore, W. (2007). The ins and
outs of gene regulation and chromosome territory organisation.
Curr. Opin. Cell Biol.
19,311
-316.[CrossRef][Medline]
Lewis, A., Mitsuya, K., Umlauf, D., Smith, P., Walter, J.,
Higgins, M., Feil, R. and Reik, W. (2004). Imprinting on
distal chromosome 7 in the placenta involves repressive histone methylation
independent of DNA methylation. Nat. Genet.
36,1291
-1295.[CrossRef][Medline]
Lewis, A., Green, K., Dawson, C., Redrup, L., Huynh, K. D., Lee,
J. T., Hermberger, M. and Reik, W. (2006). Epigenetic
dynamics of the Kcnq1 imrpinted domain in the early embryo.
Development 133,4203
-4210.
Lyle, R., Watanabe, D., te, Vruchte, D., Lerchner, W., Smrzka,
O. W., Wutz, A., Schageman, J., Hahner, L., Davies, C. and Barlow, D. P.
(2000). The imprinted antisense RNA at the Igf2r locus overlaps
but does not imprint Mas1. Nat. Genet.
25, 19-21.[CrossRef][Medline]
Mager, J., Montgomery, N. D., de Villena, F. P. and Magnuson,
T. (2003). Genome imprinting regulated by the mouse Polycomb
group protein Eed. Nat. Genet.
33,502
-507.[CrossRef][Medline]
Mak, W., Nesterova, T. B., de Napoles, M., Appanah, R.,
Yamanaka, S., Otte, A. P. and Brockdorff, N. (2004).
Reactivation of the paternal X chromosome in early mouse embryos.
Science 303,666
-669.
Mancini-Dinardo, D., Steele, S. J., Levorse, J. M., Ingram, R.
S. and Tilghman, S. M. (2006). Elongation of the Kcnq1ot1
transcript is required for genomic imprinting of neighboring genes.
Genes Dev. 20,1268
-1282.
Murakami, K., Oshimura, M. and Kugoh, H.
(2007). Suggestive evidence for chromosomal localization of
non-coding RNA from imprinted LIT1. J. Hum. Genet.
52,926
-933.[CrossRef][Medline]
Nagano, T., Mitchell, J. A., Sanz, L. A., Pauler, F. M.,
Ferguson-Smith, A., Feil, R. and Fraser, P. (2008). The Air
noncoding RNA epigenetically silences transcription by targeting G9a to
chromatin. Science 322,1717
-1720.
Neilson, J. R. and Sharp, P. A. (2008). Small
RNA regulators of gene expression. Cell
134,899
-902.[CrossRef][Medline]
Nesterova, T. B., Popova, B. C., Cobb, B. S., Norton, S.,
Senner, C. E., Tang, Y. A., Spruce, T., Rodriguez, T. A., Sado, T.,
Merkenschlager, M. and Brockdorff, N. (2008). Dicer regulates
Xist promoter methylation in ES cells indirectly through transcriptional
control of Dnmt3a. Epigenet. Chrom.
1, 2.[CrossRef]
Noordermeer, D., Branco, M. R., Splinter, E., Klous, P., van
Ijcken,W., Swagemakers, S., Koutsourakis, M., van der Spek, P., Pombo, A. and
de Laat, W. (2008). Transcription and chromatin organization
of a housekeeping gene cluster containing an integrated beta-globin locus
control region. PLoS Genet.
4,e1000016
.[CrossRef][Medline]
Ogawa, Y., Sun, B. K. and Lee, J. T. (2008).
Intersection of the RNA interference and X-inactivation pathways.
Science 320,1336
-1341.
Okamoto, I., Otte, A. P., Allis, C. D., Reinberg, D. and Heard,
E. (2004). Epigenetic dynamics of imprinted X inactivation
during early mouse development. Science
303,644
-649.
Osborne, C. S., Chakalova, L., Brown, K. E., Carter, D., Horton,
A., Debrand, E., Goyenechea, B., Mitchell, J. A., Lopes, S., Reik, W. and
Fraser, P. (2004). Active genes dynamically colocalize to
shared sites of ongoing transcription. Nat. Genet.
36,1065
-1071.[CrossRef][Medline]
Pandey, R. R., Mondal, T., Mohammad, F., Enroth, S., Komorowski,
J., Mancini-DiNardo, D. and Kanduri, C. (2008). Kcnq1ot1
antisense noncoding RNA mediates lineage-specific transcriptional silencing
through chromatin-level regulation. Mol. Cell
32,232
-246.[CrossRef][Medline]
Payer, B. and Lee, J. T. (2008). X chromosome
dosage compensation: how mammals keep the balance. Annu. Rev.
Genet. 42,733
-772.[CrossRef][Medline]
Peters, J. and Robson, J. E. (2008). Imprinted
noncoding RNAs. Mamm. Genome
19,493
-502.[CrossRef][Medline]
Sado, T., Wang, Z., Sasaki, H. and Li, E.
(2001). Regulation of imprinted X-chromosome inactivation in mice
by Tsix. Development
128,1275
-1286.[Abstract]
Seidl, C. I., Stricker, S. H. and Barlow, D. P.
(2006). The imprinted Air ncRNA is an atypical RNAPII transcript
that evades splicing and escapes nuclear export. EMBO
J. 25,3565
-3575.[CrossRef][Medline]
Shin, J. Y., Fitzpatrick, G. V. and Higgins, M. J.
(2008). Two distinct mechanisms of silencing by the KvDMR1
imprinting control region. EMBO J.
27,168
-178.[CrossRef][Medline]
Sleutels, F., Zwart, R. and Barlow, D. P.
(2002). The non-coding Air RNA is required for silencing
autosomal imprinted genes. Nature
415,810
-813.[Medline]
Tallquist, D. and Soriano, P. (1999).
Epiblast-restricted Cre expression in MORE mice. A tool to distinguish
embryonic vs. extra-embryonic gene function. Genesis
26,113
-115.[CrossRef]
Terranova, R., Yokobayashi, S., Stadler, M. B., Otte, A. P., van
Lohuizen, M., Orkin, S. H. and Peters, A. H. (2008). Polycomb
group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted
repression in early mouse embryos. Dev. Cell
15,668
-679.[CrossRef][Medline]
Umlauf, D., Goto, Y., Cao, R., Cerqueira, F., Wagschal, A.,
Zhang, Y. and Feil, R. (2004). Imprinting along the Kcnq1
domain on mouse chromosome 7 involves repressive histone methylation and
recruitment of Polycomb group complexes. Nat. Genet.
36,1296
-1300.[CrossRef][Medline]
Umlauf, D., Fraser, P. and Nagano, T. (2008).
The role of non-coding RNAs in chromatin structure and gene regulation:
variations on a theme. Biol. Chem.
389.323
-331.[CrossRef][Medline]
Wagschal, A., Sutherland, H. G., Woodfine, K., Henckel, A.,
Chebli, K., Schulz, R., Oakey, R. J., Bickmore, W. A. and Feil, R.
(2008). G9a histone methyltransferase contributes to imprinting
in the mouse placenta. Mol. Cell. Biol.
28,1104
-1113.
Wenzel, P. L. and Leone, G. (2007). Expression
of Cre recombinase in early diploid trophoblast cells of the mouse placenta.
Genesis 45,129
-134.[CrossRef][Medline]
Wutz, A. and Gribnau, J. (2007). X inactivation
Xplained. Curr. Opin. Genet. Dev.
17,387
-393.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  M. E. Dinger, P. P. Amaral, T. R. Mercer, and J. S. Mattick Pervasive transcription of the eukaryotic genome: functional indices and conceptual implications Brief Funct Genomic Proteomic, November 1, 2009; 8(6): 407 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Bartolomei Genomic imprinting: employing and avoiding epigenetic processes Genes & Dev., September 15, 2009; 23(18): 2124 - 2133. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
P<0.0001. Error bars represent s.e.m.
(C) qRT-PCR analysis of nuclear and cytoplasmic fractions from primary
mouse embryonic fibroblasts shows that Kcnq1ot1 is mainly located in the
nucleus. The 45S and 18S rRNAs are included as controls for predominantly
nuclear and cytoplasmic RNAs, respectively. Error bars represent s.e.m.
